CA2272843C - Broad-spectrum delta-endotoxins - Google Patents

Abstract

Disclosed are novel synthetically-modified B. thuringiensis chimeric crystal proteins having improved insecticidal activity against coleopteran, dipteran and lepidopteran insects. Also disclosed are the nucleic acid segments encoding these novel peptides. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention.

Description

I

(1ta('1tIP'1'ION
13ROAD-SPECTRIJM DtrL.TA-1::NDOTOXiNS

1.1 FIELD OFTI1E. 1NVE:N'1'ION
T'hc Present invention provides neX%. genes cncodin,' lluwllrrs thrrringic=rasis crystal proteins toxic to coleorteran, ciirtrran, anei lrhidopteran inwcts. Also provided are protein conlposttiotis contprisrn,; chimcric crystal proteins have enhanced insccticidal activity and increased insecticidal specificity.

,5 1.2 1)ts ctilr=I'ION OF Ittu.~~ rt=:u ART

1.?.1 B. TJII/RIN(:/r=.N.S/S CItI'tiT,1L I4K(Yrh:I4s I'he Gram-positive soil bactrriunt B. rluu-ittgierrsis V, wc1l known fiir its production ol' proteinaccott, parasporal crystals, 01- 8-endxOxins, that are tuxic t(v a varictv o1' lepidopteran, coleopteran, and dipteran Ic+rvaw. R. thurinr=it=n.,=i.c pr(xlurcS rIN.,t:ll PrOtrins cluring spotulation wltich are specifically toxic to ccrtain tihccies of insect~. Mctn\ dit'fcrrnt strains of B.
tluu-in,~*ieitsis have been shown to produce insecticidal rrvstctl proteins, and compositions conlprising B. t/rrtrirtgien.vis strains which rrodttce hrotcin; havinz insccticid.tl activity ,..ve been uscd commercially as cnvirt)iimetuully-acccptablc insecticides because of their toxicity to the specific targct insect, antl non-toxicity to plants an(i other rnon-targitecl orl:;tnisms.
Comnicrcial fomtulatitms of naturallv (1CClln-!nL P. t/rrui,rt;irt:sis isolates have long been used for the biological cr,ritrol crf aLriculttual insect re,t;. In commercial production, the spores and crystals obtained froni the fermentation process are c(+nccntrated :uid formulated for foliar application according tti conventional agricultural rractices.

1.2.2 NOMENCLATURE OF CRYSTAL PROTEINS

A review by 1-Iofte et al., (1989) describes the general state of the art with respect to the majority of insecticidal B. thuringiensis strains that have been identified which are active against insects of the Order Lepidoptera, i.e., caterpillar insects. This treatise also describes B.

thuringiensis strains having insecticidal activity against insects of the Orders Diptera (i.e. flies and mosquitoes) and Coleoptera (i.e. beetles). A number of genes encoding crystal proteins have been cloned from several strains of B. thuringiensis. H6fte et al. (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B.

thuringiensis genes and proteins. cry] genes encode lepidopteran-toxic Cryl proteins. cry2 genes encode Cry2 proteins that are toxic to both lepidopterans and dipterans.
cry3 genes encode coleopteran-toxic Cry3 proteins, while cry4 genes encode dipteran-toxic Cry4 proteins, etc.

Recently a new nomenclature has been proposed which systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. This classification scheme is summarized in Table 1.

REVISED B. THURINCIENSIS S-ENDOTOXIN NOMENCLATUREA

New Old GenBank Accession #
CrylAa CryIA(a) M I 1250 CrylAb CryIA(b) M13898 Cry 1 Ac CrylA(c) M 11068 CrylAd CryIA(d) M73250 Cry lAe CryIA(e) M65252 CrylBa CryIB X06711 CrylBb ET5 L32020 Cry 1 Bc PEG5 Z46442 Cry 1 Bd CryEl U70726 Cry 1 Ca CryIC X07518 Cry l Cb CryIC(b) M97880 New Old GenBank Accession #
Cry 1 Da Cryl D X54160 Cry 1 Db PrtB Z22511 CrylEa CrylE X53985 Cry l Eb CryIE(b) M73253 CrylFa CryIF M63897 CrylFb PrtD Z22512 CrylGa PrtA Z22510 Cry I Gb CryH2 U70725 Cry I Ha PrtC Z22513 Cry 1Hb U35780 Cryl Ia CryV X62821 Cryl Ib CryV U07642 Cryl Ja ET4 L32019 CrylJb ETI U31527 CrylK U28801 Cry2Aa CryllA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIiB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry5B U19725 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIlIC M64478 New Old GenBank Accession #
Cry7Ab CryIIICb tJ04367 Cry8A CrylIIF tJ04364 Cry8B CryIIIG tJ04365 Cry8C CrylIIF tJ04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry l 0A CryIVC M12662 Cryl lA CryIVD M31737 Cry 11 B Jeg80 X86902 Cry 12A CryVB L07027 Cryl3A CryVC L07023 Cryl4A CryVD U13955 Cry 15A 34kDa M76442 Cry 16A cbm7l X94146 Cry 17A cbm7l X99478 Cry 18A CryBP 1 X99049 Cry 19A Jeg65 Y08920 CytlAa CytA X03182 Cyt 1 Ab CytM X98793 Cyt1 B U37196 Cyt2A CytB Z14147 Cyt2B CytB U52043 aAdapted from: http://epunix.biols.susx.ac.uk/Home/Neil CrickmoreBt/index.html 1.2.3 MODE OF CRYSTAL PROTEIN TOXICITY

All 6-endotoxin crystals are toxic to insect larvae by ingestion.
Solubilization of the crystal in the midgut of the insect releases the protoxin form of the 8-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease.
The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Knight et crl., 1995; Gill et crl., 1995; Masson et al., 1995). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining 5 the midgut epithelium, and eventual larvae mortality.
1.2.4 MOLECULAR BIOLOGY OF S-ENDOTOXINS

With the advent of molecular genetic techniques, various 8-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered B. thuringiensis products that have been approved for commercial use. Recent developments have seen new 8-endotoxin delivery systems developed, including plants that contain and express genetically engineered S-endotoxin genes.
The cloning and sequencing of a number of S-endotoxin genes from a variety of Bacillus thuringiensis strains have been described and are summarized by Hofte and Whiteley, 1989. Plasmid shuttle vectors designed for the cloning and expression of S-endotoxin genes in E. coli or B. thuringiensis are described by Gawron-Burke and Baum (1991). U.
S. Patent No.
5,441,884 discloses a site-specific recombination system for constructing recombinant B.
thuringiensis strains containing 8-endotoxin genes that are free of DNA not native to B.
thuringiensis.

The Cry1 family of crystal proteins, which are primarily active against lepidopteraii pests, are the best studied class of S-endotoxins. The pro-toxin form of Cryl S-endotoxins consist of two approximately equal sized segments. The carboxyl-half, or pro-toxin segment, is not toxic and is thought to be important for crystal formation (Arvidson et al., 1989). The amino-half of the protoxin comprises the active-toxin segment of the Cry1 molecule and may be further divided into three structural domains as determined by the recently described crystallographic structure for the active toxin segment of the Cry1 Aa 8-endotoxin (Grochulski et al., 1995). Domain 1 occupies the first third of the active toxin and is essential for channel formation (Thompson et al., 1995). Domain 2 and domain 3 occupy the middle and last third of the active toxin, respectively. Both domains 2 and 3 have been implicated in receptor binding and insect specificity, depending on the insect and b-endotoxin being examined (Thompson et al., 1995).

1.2.5 CHIMERIC CRYSTAL PROTEINS

In recent years, researchers have focused effort on the construction of hybrid Fi-endotoxins with the hope of producing proteins with enhanced activity or improved properties.
Advances in the art of molecular genetics over the past decade have facilitated a logical and orderly approach to engineering proteins with improved properties. Site-specific and random mutagenesis methods, the advent of polymerase chain reaction (PCRT"t) methodologies, and the development of recombinant methods for generating gene fusions and constructing chimeric proteins have facilitated an assortment of methods for changing amino acid sequences of proteins, fusing portions of two or more proteins together in a single recombinant protein, and altering genetic sequences that encode proteins of commercial interest.

Unfortunately, for crystal proteins, these techniques have only been exploited in limited fashion. The likelihood of arbitrarily creating a chimeric protein with enhanced properties from portions of the numerous native proteins which have been identified is remote given the complex nature of protein structure, folding, oligomerization, activation, and correct processing of the chimeric protoxin to an active moiety. Only by careful selection of specific target regions within each protein, and subsequent protein engineering can toxins be synthesized which have improved insecticidal activity.

Some success in the area, however, has been reported in the literature. For example, the construction of a few hybrid S-endotoxins is reported in the following related art: Intl. Pat.
Appl. Publ. No. WO 95/30753 discloses the construction of hybrid B.
thuringiensis 8-endotoxins for production in Pseudomonas fluorescens in which the non-toxic protoxin fragment of CrylF has been replaced by the non-toxic protoxin fragment from the Cry 1 Ac/Cry 1 Ab that is disclosed in U. S. Patent 5,128,130.

U. S. Patent 5,128,130 discloses the construction of hybrid B. thuringiensis S-endotoxins for production in P. fluorescens in which a portion of the non-toxic protoxin segment of CrylAc is replaced with the corresponding non-toxic protoxin fragment of CrylAb. U. S. Patent 5,055,294 discloses the construction of a specific hybrid 6-endotoxin between CrylAc (amino acid residues 1-466) and CrylAb (amino acid residues 466-1155) for production in P..fluorescens. Although the aforementioned patent discloses the construction of a hybrid toxin within the active toxin segment, no specifics are presented in regard to the hybrid toxin's insecticidal activity. Intl. Pat. Appl. Publ. No. WO 95/30752 discloses the construction of hybrid B. thuringiensis S-endotoxins for production in P..fluorescens in which the non-toxic protoxin segment of Cry1C is replaced by the non-toxic protoxin segment froni Cry 1 Ab. The aforementioned application further discloses that the activity against Spodoptera exigua for the hybrid 8-endotoxin is improved over that of the parent active toxin, Cry 1 C.

Intl. Pat. Appl. Publ. No. WO 95/06730 discloses the construction of a hybrid B.
thuringiensis S-endotoxin consisting of domains 1 and 2 of Cry 1 E coupled to domain 3 and the non-toxic protoxin segment of Cry1 C. Insect bioassays performed against Manduca sexta (sensitive to Cry1 C and Cry1 E), Spodoptera exigua (sensitive to Cry1 C), and Mamestra brassicae (sensitive to Cry1 C) show that the hybrid Cry1 E/Cry1 C hybrid toxin is active against M. sexta, S. exigua, and M. brassicae. The bioassay results were expressed as ECSo values (toxin concentration giving a 50% growth reduction) rather than LC50 values (toxin concentration giving 50% mortality). Although the S-endotoxins used for bioassay were produced in B. thurrngiensis, only artificially-generated active segments of the 8-endotoxins were used, not the naturally-produced crystals typically produced by B.
thuringiensis that are present in commercial B. thuringiensis formulations. Bioassay results indicated that the LC5o values for the hybrid Cry1 E/Cry1 C crystal against S. frugiperda were 1.5 to 1.7 fold lower (more active) than for native Cry1 C. This art also discloses the construction of a hybrid B.
thuringiensis 6-endotoxin between CrylAb (domains 1 and 2) and Cry 1 C(domain 3 and the non-toxic protoxin segment), although no data are given regarding the hybrid toxin's activity or usefulness.

Lee et al. (1995) report the construction of hybrid B. thuringiensis S-endotoxins between CrylAc and CrylAa within the active toxin segment. Artificially generated active segments of the hybrid toxins were used to examine protein interactions in susceptible insect brush border membranes vesicles (BBMV). The bioactivity of the hybrid toxins was not reported.

Honee et al. (1991) report the construction of hybrid S-endotoxins between Cry1 C
(domain 1) and CrylAb (domains 2 and 3) and the reciprocal hybrid between CrylAb (domain 1) and CrylC (domains 2 and 3). These hybrids failed to show any significant increase in activity against susceptible insects. Furthermore, the Cry1 C(domain 1)/CrylAb (domains 2 and 3) hybrid toxin was found to be hypersensitive to protease degradation. A
report by Schnepf et al. (1990) discloses the construction of Cry l Ac hybrid toxin in which a small portion of domain 2 was replaced by the corresponding region of CrylAa, although no significant increase in activity against susceptible insect larvae was observed.

1.3 DEFICIENCIES IN THE PRIOR ART

The limited successes in producing chimeric crystal proteins which have improved activity have negatively impacted the field by thwarting efforts to produce recombinantly-engineered crystal protein for commercial development, and to extend the toxic properties and host specificities of the known endotoxins. Therefore, what is lacking in the prior art are reliable methods and compositions comprising recombinantly-engineered crystal proteins which have improved insecticidal activity, broad-host-range specificities, and which are suitable for commercial production in B. thuringiensis.

2.0 SUMMARY OF THE INVENTION
The present invention overcomes these and other limitations in the prior art by providing novel chimeric 6-endotoxins which have improved insecticidal properties, and broad-range specificities.

Disclosed are methods for the construction of B. thuringiensis hybrid 6-endotoxins comprising amino acid sequences from native CrylAc and Cry1 F crystal proteins. These hybrid proteins, in which all or a portion of Cry l Ac domain 2, all or a portion of Cryl Ac domain 3, and all or a portion of the CrylAc protoxin segment is replaced by the corresponding portions of Cry1F, possess not only the insecticidal characteristics of the parent 8-endotoxins, but also have the unexpected and remarkable properties of enhanced broad-range specificity which is not proficiently displayed by either of the native 6-endotoxins from which the chimeric proteins were engineered.

Specifically, the present invention discloses and claims genetically-engineered hybrid 6-endotoxins which comprise a portion of a CrylAc crystal protein fused to a portion of a Cry1 F crystal protein. These chimeric endotoxins have broad-range specificity for the insect pests described herein.

In a further embodiment, the present invention also discloses and claims recombinant B. thuringiensis hybrid 6-endotoxins which comprise a portion of CrylAb, CryIF, and CrylAc in which all or a portion of Cry1 Ab domain 2 or all or a portion of Cry 1 Ab domain 3 is replaced by the corresponding portions of Cry1 F and all or a portion of the CrylAb protoxin segment is replaced by the corresponding portions of CrylAc. Exemplary hybrid S-endotoxins between Cry 1 Ab and Cry 1 F are identified in SEQ ID NO:13 and SEQ ID NO:14.

One aspect of the present invention demonstrates the unexpected result that certain hybrid 6-endotoxins derived from CrylAc and Cry1F proteins exhibit not only the insecticidal characteristics of the parent S-endotoxins, but also possess insecticidal activity which is not proficiently displayed by either of the parent 8-endotoxins.

Another aspect of the invention further demonstrates the unexpected result that certain chimeric Cry 1 Ab/Cry 1 F proteins maintain not only the insecticidal characteristics of the parent 8-endotoxins, but also exhibit insecticidal activity which is not displayed by either the native Cry 1 Ab or Cry 1 F endotoxins.

The present invention also encompasses Cry1 Ac/Cry1 F and Cry1 Ab/Cry1 F
hybrid S-endotoxins that maintain the desirable characteristics needed for commercial production in B.
thuringiensis. Specifically, the hybrid 6-endotoxins identified in SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:34 can efficiently form proteinaceous parasporal inclusions in B.
thuringiensis and have the favorable characteristics of solubility, protease susceptibility, and insecticidal activity of the parent S-endotoxins.

In a further embodiment, the present invention also discloses and claims recombinant B. thuringiensis hybrid S-endotoxins which comprise a portion of Cry 1Ac and Cry 1C in which all or a portion of Cry 1 Ac domain 3 is replaced by the corresponding portions of Cry 1 C and all or a portion of the CrylAc protoxin segment is replaced by the corresponding portion of Cry1 C. Exemplary hybrid 8-endotoxins between CrylAc and Cry1 C are identified in SEQ ID
NO:29 and SEQ ID NO:30.

One aspect of the present invention demonstrates the unexpected result that, although neither Cry1 Ac nor Cry1 C possess S. frugiperda activity, the Cry1 Ac/Cry1 C
hybrid 8-endotoxin identified by SEQ ID NO:29 and SEQ ID NO:30 has significant activity against S.
frugiperda. Furthermore, the Cry1 Ac/Cry1 C hybrid S-endotoxin identified by SEQ ID NO:29 and SEQ ID NO:30 has significantly better activity against S. exigua than the Cry1 C parental S-endotoxin.

The impetus for constructing these and other liybrid 8-endotoxins is to create novel toxins with improved insecticidal activity, increased host-range specificity, and improved production characteristics. The DNA sequences listed in 'I'able 8 define the exchange points for the hybrid 6-endotoxins pertinent to the present invention and as oligonucleotide primers, 5 may be used to identify like or similar hybrid 8-endotoxins by Southern or colony hybridization under conditions of moderate to high stringency. Researchers skilled in the art will recognize the importance of the exchange site chosen between two or more 6-endotoxins can be achieved using a number of in vivo or in vitro molecular genetic techniques. Small variations in the exchange region between two or more 6-endotoxins may yield similar results 10 or, as demonstrated for EG11062 and EG11063, adversely affect desirable traits. Similarly, large variations in the exchange region between two or more 6-endotoxins may have no effect on desired traits, as demonstrated by EG 11063 and EG 11074, or may adversely affect desirable traits, as demonstrated by EG 11060 and EG 11063.
Favorable traits with regard to improved insecticidal activity, increased host range, and improved production characteristics may be achieved by other such hybrid 8-endotoxins including, but not limited to, the cry], cry2, cry3, cry4, cry5, cry6, cry7, cry8, cry9, cry10, cry]], cry12, cry13, cry14, cry15 class of 6-endotoxin genes and the B.
thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to Cry l Aa, Cry l Ab, Cry l Ac, Cry l Ad, Cry l Ae, Cry 1 Ba, Cry l Bb, Cry 1 Ca, Cry1 Cb, Cryl Da, Cry1 Db, CrylEa, CrylEb, CrylFa, CrylFb, CrylGa, Cryl Ha, Cry2a, Cry2b, CrylJa, CrylKa, Cry11Aa, Cry11Ab, Cryl2Aa, Cry3Ba, Cry3Bb, Cry3C, Cry4a, Cry4Ba, Cry5a, Cry5Ab, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa, Cry8Ba, CryBCa, Cry9Aa, Cry9Ba, Cry9Ca, CrylOAa, Cry1lAa, Cry12Aa, Cry13Aa, Cryl4Aa, Cryl5Aa, CytlAa, and Cyt2Aa. Related hybrid S-endotoxins would consist of the amino portion of one of the aforementioned 6-endotoxins, including all or part of domain 1 or domain 2, fused to all or part of domain 3 from another of the aforementioned S-endotoxins. The non-active protoxin fragment of such hybrid S-endotoxins may consist of the protoxin fragment from any of the aforementioned 6-endotoxins which may act to stabilize the hybrid 6-endotoxin as demonstrated by EG11087 and EG11091 (see e.g., Table 5). Hybrid S-endotoxins possessing similar traits as those described in the present invention could be constructed by conservative, or "similar" replacements of amino acids within hybrid S-endotoxins. Such substitutions would mimic the biochemical and biophysical properties of the native amino acid at any position in the protein. Amino acids considered similar include for example, but are not limited to:

Ala, Ser, and Thr;
Asp and Glu;
Asn and Gln;

Lys and Arg;

Ile, Leu, Met, and Val; and Phe, Tyr, and Trp.

Researchers skilled in the art will recognize that improved insecticidal activity, increased host range, and improved production characteristics imparted upon hybrid S-endotoxins may be further improved by altering the genetic code for one or more amino acid positions in the hybrid S-endotoxin such that the position, or positions, is replaced by any other amino acid. This may be accomplished by targeting a region or regions of the protein for mutagenesis by any number of established mutagenic techniques, including those procedures relevant to the present invention. Such techniques include site-specific mutagenesis (Kunkel, 1985; Kunkel et al., 1987), DNA shuffling (Stemmer, 1994), and PCRTM overlap extension (Horton et al., 1989). Since amino acids situated at or near the surface of a protein are likely responsible for its interaction with other proteinaceous or non-proteinaceous moieties, they may serve as "target" regions for mutagenesis. Such surface exposed regions may consist of, but not be limited to, surface exposed amino acid residues within the active toxin fragment of the protein and include the inter-a-helical or inter-p-strand "loop" -regions of S-endotoxins that separate (x-helices within domain 1 and (3-strands within domain 2 and domain 3. Such procedures may favorably change the protein's biochemical and biophysical characteristics or its mode of action as outlined in the Section 1. These include, but are not limited to: 1) improved crystal formation, 2) improved protein stability or reduced protease degradation, 3) improved insect membrane receptor recognition and binding, 4) improved oligomerization or channel formation in the insect midgut endothelium, and 5) improved insecticidal activity or insecticidal specificity due to any or all of the reasons stated above.

The present invention provides an isolated nucleic acid segment encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. Preferably the nucleic acid segment encodes a polypeptide having insecticidal activity against Spodoplera frugiperda, Spodoptera exigua, Heliothis virescens, Helicoverpazea or Ostrinia nubilalis.
Such nucleic acid segments are isolatable from Bacillus thuringiensis NRR.I, B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, NRRL B-21780, or NRRL B-21781 cells, respectively.
In preferred embodiments, these nucleic acid segments specifically hybridize to a nucleic acid segment having the sequence of SEQ ID NO:9, SEQ ID NO:I 1, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33, or a complement thereof, and in highly preferred embodiments these nucleic acid segments comprise the nucleic acid sequences which are disclosed in SEQ ID NO:9, SEQ ID NO:I 1, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:33, respectively.

Such a nucleic acid segment may be operably linked to a promoter to express the nucleic acid segment in a host cell. In such embodiments, the nucleic acid segment may be comprised within a recombinant vector such as a plasmid, cosmid, phage, phagemid, viral, baculovirus, bacterial artificial chromosome, or yeast artificial chromsome. Exemplary plasmid vectors include the vectors pEG1068, pEG1077, pEG11092, pEG1093, pEG365, pEG378, and pEG381.
A further aspect of the invention involves the use of the nucleic acid segments described herein in the preparation of recombinant polypeptide compositions, in the generation of a vector for use in producing an transformed host cell, and the generation of an insect resistant transgenic plant.
Host cells also represent an important aspect of the invention. Such host cells generally comprise one or more of the nucleic acid segments as described above.
Preferred host cells include bacterial cells such as E. coli, B. thuringiensis, B. subtilis, B.
megaterium, and Pseudomonas spp. cells, with B. thuringiensis EG 11060, EG 11062, EG 11063, EGl 1071, EG11073, EG11074, EG11090, EG11091, EG11092, EGl 1735, EGl 1751, EG11768, NRRL
B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, NRRL B-21780, and NRRI. B-21781 cells being particularly preferred. Preferred host cells may also include eukaryotic cells, such as plant and animal cells. Preferred plant cells include grain, tree, vegetable, fruit, berry, nut, grass, cactus, succulent, and ornamental plant cells, with commercial crops such as corn, rice, tobacco, potato, tomato, flax, canola, sunflower, cotton, wheat, oat, barley, and rye being particularly preferred.

In one embodiment, the host cell may be coniprised within a transgenic plant, or may be used in the preparation of a transgenic plant, or in the generation of pluripotent plant cells.
Alternatively, the host cell may be used in the recombinant expression of a crystal protein, or in the preparation of an insecticidal polypeptide formulation comprising one or more of the toxins disclosed herein. Such a composition preferably comprises one or more isolated polypeptides habing the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. Such a composition has particular use in killing an insect cell, in the preparation of an insecticidal formulation, and in the fornlulation of a plant protective spray.
The invention also provides a method for preparing a B. thuringiensis crystal protein.
Such a method generally involves culturing a B. thuringiensis NRRL B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, NRRL B-21780, NRRL B-21781, EG11768, EG 11090, EG11063, EG11074, EG11092, EG 1173 5, or EG 11751 cell under conditions effective to produce a B. thuringiensis crystal protein, and then obtaining said B.
thuringiensis crystal protein from the cell. Such methods are quite useful in recombinant production of the crystal proteins disclosed herein.

The invention also discloses and claims a method of killing an insect cell.
The method generally involves providing to an insect cell an insecticidally-effectiveamount of one or more of the insecticidal compositions disclosed herein. Such cells may be isolated cells, or alternatively, may be comprised within an insect itself. Typically the composition will be provided to the insect either by directly spraying the insects, or alternatively, by the insect ingesting the composition either directly, or by ingesting a plant which has been either coated with the composition, or alternatively, by ingesting part of a transgenic plant which expresses one or more of the insecticidal compositions.

A further aspect of the invention is a purified antibody that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. Such antibodies may be operatively attached to a detectable label, provided in an immunodetection kit or used in a method for detecting an insecticidal polypeptide in a biological sample by contacting a biological sample suspected of containing said insecticidal polypeptide witll such an antibodyunder conditions effective to allow the formation of immunecomplexes, and then detecting the immunecomplexeswhich are formed.

Another important aspect of the invention concerns a transgenic plant having incorporated into its genome a transgene that encodes a polypeptide comprising the amino sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:30, or SEQ ID NO:34. Preferably, such transgenic plants will comprise one or more of the nucleic acid sequences which are disclosed in SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:33. Progeny and seeds from such transgenic plants and their offspring or descendants are also important aspects of the invention which are described in detail elsewhere herein.

2.1 CRYSTAL PROTEIN TRANSGENES AND TRANSGENIC PLANTS

In yet another aspect, the present invention provides methods for producing a transgenic plant which expresses a nucleic acid segment encoding the novel chimeric crystal proteins of the present invention. The process of producing transgenic plants is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA
segment which contains a promoter operatively linked to a coding region that encodes a B.
thuringiensis Cry1 Ac-1 F or Cry1 Ab-1 F, Cry1 Ac-1 C, or a Cry1 Ab-1 Ac-1 F
chimeric crystal protein. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo.
Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant crystal protein expressed in a particular transgenic cell, the invention also provides for the expression of crystal protein antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises a transgenic plant which express a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein.
As used herein, the term "transgenic plant" is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, 1)NA sequences n<-t ncrr-nallv transcrihecl intu RNA or tr.ri-slatcci intu a protein ("expressed"), or any other genes or 1)N A ticyuences which unr ctesires to i--lrucluce into the non-transformecl plant, such as Rencs which mav norniullv be rreticnt in the nun-transtiirnicei plant hut which one clesires to eithcr genetically eneinc=rr or to havc ,,iterecl rxrression.
The construction and expression of synthetic B. tluu=in,r~icn.cis genes in plants ha, heen clescrihed in detail in U. S.
l'atents 5,500,365 and 5,380,83 1.
It is contemplateci that in solnc instances the oenctnic (if a transgenic plant of the present invention will havc been augntcnted ttirciugh the stable intrcxlucticin c-t'one or more cvylAe-IF, crvlAb-Il; crvlAe=-IC', or rrrl:fh-1;Ic=-I/'iranst;ettes, cithet- native, synthetically-modified, ot' 1U further niutated. In some instanceti, niore than one transgene \vill he incorporated into the genonic of the transtornieci host plant cell. Such is the case when niore Ihan one crystal protein-encodinp DNA scgnicnt is incorporated into ttie grnonw ol' such a plant. In certain situations, it may be desirable to have one, two, thrce, laur, or even mote 11. thuringiensis crystal proteins (either native or reconihinantly-enpineere(l) incorroratecl and stably expressed in the transformed transgenic plant.
A preferrect getie, such as those disclosed in SE(,) Il) NO:9, SEQ ID NO:11, SEQ ID NO:13, SI:Q (1) NO:25. SEQ 11) NO:27. SEQ II) NO):29, and SI:Q ID NO:33 which may be introduced includes, fi-r example, a crystal protein-encociing a DNA
sequence from bacterial origin, and particularly onc or niorc of those described herein which are obtained fcom llucilltt.c spp. lliglily preferreci nuclcic acid ucluencrs :tre those obtained from B.
tlruritri!0rsis, or any of those sequcnces which have been genetically engineered to decrease or increase the insecticidal activity ol'the crystal protein in such a transformed host cell.
Means for transtortning a plant cell and the preparation of a transgenic cell line are well-known in the art, and are discussed herein. Vectors, plasmids, costnids, yeast artificial chromosomes (YACs) and nucleic acid segments lor use in transformin~ such cells will, of course, generally coniprise either the operons, genes, or genc-cierived sequences of the present invention, either native, or synthetically-derivecl, and particularly those encoding the disclosed crystal proteins. 'I'hese DNA constructs can lurther include structures such as promoters, enliancers, polylint:ers, or even gene sequenees which have positivelv- or nepatively-regulating activity upoti the particular penes of interest as desirecl. 'I hc l)NA
segment or gene may encode either :t natlve, or nwclitied crystal protein, which will be expressed in the resultant reeunibinant cells, and/ur whicli will imrart an improvccl hhenotvhc to the r-egenerated plant.
Nucleic aciel s tuences optimized fi-r exhre,sion in pl:urt; hervc been tiisclosed in Intl. Pat.
Appl. 1'ubl. No. Wc ) ()3/07278, Sucir tran,genic plants may be desirablc fi)r incrcatiinv thc insceticitlal resistance ot'a monocotyledonous or dicutyledonous plant, by incorporating into such a plant, a transgenic DNA segment enccrding C ry I Ac-11: and/or Cry 1 Ac- l(', antiior ('rv I Ab- I
F and/or Cryl Ab-lAc-ll~ crystal protein(s) which possess hmad-insrct specilicity. Particularly preferred plattts such as grains, including but not limited to curn, wheat, oat,, rice, maize, and barley; cotton;
soybeans and uther legumes; trees, incluLling but not Iimite(i to ornamentals, shrubs, fruits, nuts; vegetablcs, turl' and pasture prasses, berries, citrus, ancl oilier crops of commercial intcrest; such as garden crops and/or huusrrlants, succulents, cacti, and fluwering species.
In a related aspect, tl)c present invention also enconnpasses a seed produced by the transfi)rmcd plant, a progeny f1oni such seed, and a sce(l rrodtrc.eci bv the progeny of tiie original transgenic plant, produced in accordance with ttu above process. Sueh progeny and seeds will have a stably ct-vstal protein trattsE!ene stably incorporated into its genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian 1'ashi.on. All such transgenic plants having incc)rporate(i into their genome transgenic DNA septncnts encoding one or tnore ciiimeric crystal proteins or polypeptides are aspects of this invention.

2.2 CRYST'AL PROTEIN SCREL:NIN(; ANI) IMMUNOnI=:'1'E(TtON K17'S
"I'he present invention contemplates methods and kits f'-r screening samples suspected of containing crystal protein polypepti(leti or crystal prutciri-related polypeptides, or cells producing such polypcptidcs. Exemplary proteins include those (lisclosed in SEQ ID NO: 10, SEQ 11) NO:12, SEQ ID NO:14, SEQ IC) NO:26, SEQ t 11) NO:38, SEQ ID NO:30, and SEQ lE) NO:34. Said kit can contain a nucleic acid segment or an antibody of the present invention. '1'he kit can contain reagents for detecting an interaction between a sample and a nucleic acid or antibody uf the present invetition. The provided reagent can be radio-, fluorescently- or enzymatically-labeled. '1'he kit can contain a known radiolabeled agent capable of binding or interactin6 with a nucleic acid or antibody of'the present invention.

The reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the crystal proteins or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect crystal proteins or crystal protein-related epitope-containing peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like. Generally, immunocomplex formation.

will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any sample suspected of comprising either a crystal protein or peptide or a crystal protein-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of crystal proteins or related peptides and/or antibodies in a sample. Samples may include cells, cell supematants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions t8 suspected of containing crystal proteins or peptides. Generally speaking, kits in accordance with the present invention will include a suitable crystal protein, peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.

The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

2.3 ELISAS AND IMMUNOPRECIPITATION

ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incorporating crystal protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tweeri . These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from about 2 to about 4 hours, at temperatures preferably on the order of about 25 to about 27 C.
Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween", or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of iminunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tweeri ).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2, 2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H202, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

The anti-crystal protein antibodies of the present invention are particularly useful for the isolation of other crystal protein antigens by immunoprecipitation.
Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells, must be solubilized into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. enzyme-substrate pairs.

2.4 WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-peptide antibodies niay be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support nlatrix, such as nitrocellulose, 5 nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with 10 the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
Imniunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.

2.5 EPITOPIC CORE SEQUENCES

The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-crystal protein antibodies.

In particular, the invention concelns epitopic core sequences derived from Cry proteins or peptides.
As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-crystal protein antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a crystal protein or polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the crystal protein or polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

The iclentification c-I' Cry immuu0(l--min;-nt ehiture,~. and/or their functional equivalents, suitable lor usc in vaccinc,~ is a relutivel). strcri~-htt'Orw:rrd matter. For examhlc, one niay cmploy the tuethoclti uf I lopp, as taught in 11. S. Patent 4,554.101.

which teaches the identification and preparation of epitopes trom amino acid sequcnces on the basis of hvdrophilicitv. The rncthocis dcscrihrd in several other papers, and software programs based tliereun, can also hc use(i to identitN' cl)itopic core sequences (see, for example. Jirmeson and Wc-II; I988; Wulf e1 ul., I9M- U. ti. Patent. 4.55-1.101). The amino acid sequence of these "ehitopic corr sequences" may thrn be readily incorporated into peptides, either through the application ot'peptide synthetiis or recombinant technology.

Prelerrcd lu:ptides fur use in accordance with the present inventiun will generally be on the order of about K to about 20 amino acids in length, and nturc prcfi:rahly about 8 to about 15 anlino acids in lenl;th. It is pruposed that shcrrter antigenic crystal protein-derived peptides will provide advantages in certain circumst,tnces, fi-r examplC, in the preparation of immunologic detection assays. l:xemplarv advantages include the case c-t'rreparation an(l purification, the relatively low cost anci itnproved reproducibility of production, and advantageous biodistribtttion.
It is proposed that particular advantages c-f' the present 1nvCntlOn may be realized through the preparation ol' synthetic peptides which incluLic moditieci and/or extended epitopic/immunot!enic core sequences whie:ii result in a"univcrs:rl" epitopic peptide directed to crystal proteins, and i.n particular Crv and Cry-related se(lucnces. These epitopic core sequences are idcntified herein in particutar aspeets as hvclruhhilic rcgions of the particular polypcptide antigett. It is proposed that thesc repiuns rcprescnt those which are most likely to promote T-cell or [3-cell stimulatlon, and, hcnce, elicit specific antibody production.
An epitopic core seeluencc. as uscil hcrein, is a relativrly shc-t-t stretch of amino acids that is "complementary" to, and therefi,-re will bind, antigen hincling sites on the crystal protein-directed antibodies disclosed herein. Additionally or alternativelv, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide conipositions of the present invention. It will be understood that in the context of the present disclosure, thc term "complementary" refers to aminc- acids or peptides that exhibit an attractive force towards each otlier. "Il-us, certain epitope core scyuenccs of the present invention niay be c-perationally detine(i in tenns of thcir ability to ci-rnpete with or perhaps dishlacc the hintlint, ol' thc clcsircd pr-otein antigen with tlre corre,ronc.iino, rrotcin-dircctecl antisera.

In general, the tiiic uf the hulyhehtidc ^ntWlen is not hclirvcil to hc particularly crucial, so long as it is at least large enough to carry the itlentilic(l io~rc ;ccluencc or sequences. The sntallest useful core scqucnce anticipatecl by ihc rresent (lisrlcisurc wuulcl generally be on the order of' about 8 aminc- aciels in Iens!th, with tiequenccs r111 thC urLicr 0[
10 to 20 being more prclcrrc(l. Thus, this sir.c will 'Llenerally correspond to thc smalk=st hrP(icle antis;cns prepared in accordance with the invcntic-n. liowcvcr, the sir.c of the antiecn ma), he larger where desired, so long as it contains a basic epitopic core scquence.

Tlte identi.lication of epitopic et -c secluenccs is known to thc-se of skill in the art, for example, as described in lI. S. 1'atent 4,554,101, which teaches the identification and preparation uf cpitopes l'rcmi antinci acid uquences on the basis of hydrophilieity. Morcovcr, nutncrous cc-mputer rrogr:rmti are available lior use in predicting antigenic portiotis of Proteins (sec e.g., Janieson and W'olt", 1988; Wolf et al., 1988).

gratns (e.g.. f)Nrltitar(NI suttware, DNAStar, Inc., Computerized peptide seduencc analysis prot Madison, WI) niav also be usefiil in tIc,il!ning synthetic rel)tides in accordance with the present disclosure.
Syntheses of'epitoric scquenccs, or peptides xvhich includc an antigenic epitope witliin their sequence, are rcaelily achieved ttsin>; conventional svnthetic tecliniqtres such as the solid phase niethod (e.g., through the use of conlmcrcially available pcpticie synthesizer such as an Applied Biosystenis Modcl 430A Peptide Synthesizer). I'cptitle antigens svnthesized in this manner may then be aliquotted in predetcrmincd amounts and ,torecl in conventional manners, such as in aqueous solutions ur, even morc nrctcr.rbly, in a powder or Iyophilired state pending use.
In general, due to the relative stability of pcpticlc,. thev may he readily stored in aqueous solutions for fairly long periods of titne if desired, t=. i~., up to six nionths or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers sucii as 'fris or pho5phate huClers to maintain a pH of about 7.0 to about 7.5. Moreover, it may he desirablc to include agents which will inhibit microbial growtlt, sttch as sodium azide or Merthiolate. For extcnclctl storage in an aqueous state it will be desirable to store the solutions at about 4 C, or more preferably, frozen.
Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

2.6 NUCLEIC ACID SEGMENTS ENCODING CRYSTAL PROTEIN CHIMERAS

The present invention also concerns DNA segments, both native, synthetic, and mutagenized, that can be synthesized, or isolated from virtually any source, that are free from total genomic DNA and that encode the novel chimeric peptides disclosed herein. DNA

segments encoding these peptide species may prove to encode proteins, polypeptides, subunits, functional domains, and the like of crystal protein-related or other non-related gene products.
In addition these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art.

As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA
segment encoding a crystal protein or peptide refers to a DNA segment that contains crystal protein coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained, which in the instant case is the genome of the Gram-positive bacterial genus, Bacillus, and in particular, the species of Bacillus known as B. thuringiensis. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystal protein-encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, operon sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding a bacteria] crystal protein, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA
segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a Cry peptide species that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34.

The term "a sequence essentially as set forth in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34" means that the sequence substantially corresponds to a portion of the sequence of either SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34 will be sequences that are "essentially as set forth in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34."

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

I'he nucleic acid segments of the present invention, regardless of the length of the 5 coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended 10 recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch encoding either of the peptide sequences disclosed in SEQ ID NO:10, SEQ ID NrJ:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, or that are identical to or complementary to DNA
sequences which encode any of the peptides disclosed in SEQ ID NO:10, SEQ ID
NO:12 15 SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, and particularly those DNA segments disclosed in SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. For example, DNA
sequences such as about 14 nucleotides, and that are up to about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 base 20 pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.;
21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to 25 and including sequences of about 10,000 nucleotides and the like.

It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequences of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, including those DNA sequences which are particularly disclosed in SEQ ID NO:9, SEQ ID NO:11 SEQ ID NO:13, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33. Recombinant vectors and isolated DNA

segnlents may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologically-funetional, equivalent peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention.
Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.

2.7 RECOMBINANT VECTORS AND PROTEIN EXPRESSION

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, "' 7 promoter. I1s usccl hcrcin, a rccunihrnvu or Iietcnrltvgtitts pnomutcr is intcndcd to refer to a protnc-trr that is ncit nurniallY asu-ciatCd with a !)Nr1 scemcnt rncoding a crystal protcin or penticle in it. natural envirunmrnt. tittch prcmtctter, tnav includc promoters normally associatcd with uthcr tencs, and/or promoters isc-latccl front any bacterial, viral, eukaryotic, or plant cell. Naturally, it xvill he iniluortant tu employ a promoter that cflcctively directs the csrressic-n c-f the 1)NA segment in thr cell tvpc, cirianisnt, or cvcn animal, chosen for exhrrssiun. *I'he usc of'hrummer and ccll type comhinutions titr prutcin cxpression is generally known to those ufskill in the art t-fnuolccular hiulogy, liit-examhlr, see Sambrook el al., 1989.
The promoters emplc~yecl ntaV hc ec-ntititutive, or inducihlc, and can hr used under the appropriate conditions to dircct high level expression c-f thc introducccl 1)N/1 segment, such as is advantageous in the large-scale production ot'rccornhinant luc-tcin, cir pepti(les. Appropriate prumoter sl'stcnis contemplated tor use In hlf!h-Icvcl expression includc, hut are not limited to, thc Pichia expression vector s\=stcm (t'h,trmacia l.KIi Biotechnology).
In connection with rxrression crnhciditnents to prepare rcctinihinant proteins and peptides, it is contemplated that Iongrr 1)NA segments will must uftcn be uscd, with DNA
segmrnts encoding thr entirc peptide srducnce being rnost preferred. llowever, it will be appreciated that the use of'shurter DNA segnients to direct the cxpression ol'crystal peptides or epitopic core ret!ions, tiuch as may be used to generate anti-crytital rrotein antibodies, also falls within the scope c>Cthc ieventie.m. I)N,'1 scl;ments that encodr prptidc antigens from about 8 to about j0 amino acids in length, or niore prclcrably, from about 8 to about 3(1 amino acids in Icngth, or rvrn rnore prclerabl)=, t'ront about }t to about -10 aminc- aci(i, in length are ccmtemplated to be particularly ttsctltl. Such peptide epitopes may hc amino acid sequcnccs which ccmiprise contiguuus iunint :tci(i sequences frunt Si:O 11) NO: 10, SEQ
ID NO: 12 SF:Q II) NO:I-l, SEQ II) NO:2G,tiliQ ID NU:28, SFQ rI) NU: 30, or SFQ II) NO:34; or any peptide epitope encodcd hy the nuclcic acid sequCnces ul' til=:Q 11) NU:9, SEQ
ID NO:1 1, SEQ 11) NO:13, SEQ 11) NO:25, SF:Q 11) NO:27, SE.Q 11) N0):39, S1:(,) 11) NO:33.

Methods Icor the recombinant expression of crystal proteins and vectors useful in the expression oC DNA constt-ucts encodinf; crystal proteins are described in Intl. I'at. Appl. Publ.
/ No. WO 95/02058.

2.8 RECOMBINANT HOS"T CELLS

The recombinant host cells of the present invention which have been deposited under the terms of the Budapest Treaty are listed in "I'able 2.

STRAINS DEPOSITED WITH THE NRRL UNDER THE BUDAPEST TREATY
S7'RAIN PLASMID ACCESSION NUMBER DEPOSIT DATE
EG 11063 pEG 1068 NRRL B-21579 June 26, 1996 EG11074 pEG1077 NRRL B-21580 June 26, 1996 EG 11091 pEG 1092 NRRL B-21780 May 21, 1997 EG11092 pEG1093 NRRL B-21635 November 14, 1996 EGl 1735 pEG365 NRRL B-21581 June 26, 1996 EG11751 pEG378 NRRL B-21636 November 14, 1996 EG11768 pEG381 NRRL B-21781 May 21, 1997 2.9 DNA SEGMENTS AS HYBRIDIZATION PROBES AND PRIMERS

In addition to their use in directing the expression of crystal proteins or peptides of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA
segment of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33 will find particular utility. Also, nucleic acid segments which encode at least a 6 amino acid contiguous sequence from SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, are also preferred. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. (including all intermediate lengths and up to and including full-length sequences will also be of use in certain embodiments.

'l'he ability 01' surh ntrclric acid hrc-be, tu slVcilicatlv hvhriciize tu crystal protein-encoding sequenccs will enable thcm tt) he of usc in cletectinw the lv-esence t-f complementary sequcnccs in agiven samhlc. I lowcver, othcr uses are envisioned, including the use of thc sequcnce informatirnt liir Ihe preparation of' niutant species primers, or primers for use in preparing other benetic constructions.
Nucleic acid molectrles having secluettce regions corrsisting c-f ctmtiguous nucleotide stretches of 10-14, 15-20, 30, 50, ttr cven ot" 100-200 nuclcotiilcti or so, identical or complenicntary to I)NA seyuences of SI:QII) NO:11-, til:O 11) NO:1 1, SfiQ ID
NO:13, SEQ ID NO:25, S[;Q 1[) NC):'?7, SC:O ll) NO:29, or til;O 1t) N(>:33, are particularly contemplated as hybridiiatian probes t'or use in, e.g., tiouthcrn and Ncrrthern blotting. Smaller tcagtnents will generally find use in hybridization emhoc3iments, wherein the length of the contiguous completncntary region may hc varied, such zts hetwcen about 10- 14 and about 100 or 200 nuelcotides, but larger contiguous complement.rrity stretches may he used, according to the length completnentary sequences one wishes to detect.
Ol' course, fragments may also be obtained by other techniqucs such as, e.g., by mcchanical shearing or by restriction enzyme digestion. timall riucleic acid segments or fragments may hr readily prepared by, lor example, directly synthesizing the fragment by chenrical nieanti, us is coninionly practiced usint; an autonurtrtt ulit!onucleotide synthesizer.
Also, fragments niay be obtained by application of nucleic acid rcproeiuction technology, sueli as tlte I'CR""' technoloBY of U. S. 1'atents 4,683,195 and 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA trchniqucs generally known to those of skill in tiie art of molecular biology.
Accordingly, the nucleotide sequences ol' the invention may he used tior their ability to selectively torm duplex nwleculcs with complementary stretches of DNA
fragments.
Depending on the application envisioned, one will desire tt- etnploy varying conditions of hybridization to achieve varyinn degrees of selectivity of Pr<nce towzrds target sequence. For applications requiring liigh selectivity, one will typically desire to employ relatively stringent conditions to form tiie ltybrids, e.g., one xvill select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50 C to about 70 C. Such selective conditions tolerate little, if anv, mismatch between the prohe ancl thr temhlcUe c-r taritet str:rnd, and %vo-ulci he partictrl,u=Iv suitahle fi-r isolatittg cryslal protein-encoding 1)N;\ segments. Detrrtiun uf I)NA segtnentti via hybridization is well-known to those ul' sI:ill in the art, anJ tlre trachings uf l 1. S. 1'atcnts -1,965, 188 and 5,176,995 are exemplary of the methods of hybridization analyses. 'I'eachings such as th<-se fi-tnnd in thc tc.xts of Malt-y et u[., 1994; Segal 1976; l'rukt-p, 1991: antl I:uhv. 1994, are particularly rclcvant.

Of course, for scmie applications. lirr rxamhle, "vhcre une clcsires to preparc mutants emplovinp a mtttant printrr strand ltyhrielixed to an undrrlvinf! template or where one seeks to isolate crvstal protcin-encociing sequences Irum related spcci , functional cquivalents, or the like, less stringent hybridization conclitions will typically he nceclecl in order to allow formation of the heteruduplcz. In these circumstances, cmc may clcsire to employ conditions such as about 0.15 M to about 0.9 NI salt, at temperatures rangini-, trum about 20 C
to about 55 C.
Cross-hyhridizing species can thereby be readily identificct as positively hvhridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent hv the additicm of increasing amounts of tormamide, which serves to destabilize the hvbt-id duplex in the same nianner as increased ternperature. Thus, hybridization conditions can be readily ntaniPulated, and thus will generally be a mcthod of choice depending t-n the desired results.

In eertain embt-dintents, it will.hc advantageous tcr employ nuclcic acid sequences of thc present invention in cumbination with an apprurriatc rne.urs, such as a label, for determining hybridizatiun. A widr vat=iety c-fappropriatr indicator mcans are known in the art, including fluorescent, radioactive, cnzvnratic or other livands, such as avidin/hiotin, which are capable of giving a detcctc,hle sit;nal. In prcli=rrecl emhutiinunts, c-ne will likely desire to employ a fluorescent lahel or an enivme tag, such as urcasc, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme ta6s, colorimetric indicator substrates are known that can he employed to provide a means visible to the: hunian eye or spcctrc-phi-totnetricallv. tu identity specitic hybridization witli complementary nuclric acid-containing samples.
In gencrul, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution lrvbridization as well as in embodiments cniploying a solid phase.
In embodiments involving a solid phase, the test DNn (or RNA- is adsorbed or otherwise 3]
affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

2.10 BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. In particular embodiments of the invention, mutated crystal proteins are contemplated to be useful for increasing the insecticidal activity of the protein, and consequently increasing the insecticidal activity and/or expression of the recombinant transgene in a plant cell. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 3.

Amino Acid Codons Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU

Isoleucine Iie I AUA AUC AUU
Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological funetiunal activity, certain amino acid sequencc substitutions can be made in a protein sequetice, and, ef course, its underlying DNA codinil ticqucnce, and nevertheless obtain a protein with like propertiels. It is thus contcmplated by the invcnt(irs that various changes may be made in tlie peptide scquences of the discloseci co>nipositions, or correspondinf; DNA

sedttences wliich encode said peptides without appreciable loss of their biological utility or activity.
In niaking suclt chanpes, the hydropathic 1ndC\ l1I .Inllno acids may be considcred. 7'lie importance of the hydropathic amino acid index in conferring, interactive biologic funetion on a protein is generally understood in the art (Kyte and 1)oulittlc, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of tiie resultant protein, whic.h in turti defines the interaction of the protein with other niolecules, for exaniple, enzymcs, substrates. receptors, DNA, antibodies, antigens, and the li:.e.
Cacii amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalartittc (+2.8);
cysteine/cystine (+2.5); methioninc (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7): serine (-0.8);
tryptophan (-0.9);
tvrosinc (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutanline (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids niav be substituted by other amino acids having a siniiiar hvdropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of' amino acids whose hydropathic indices arc vithin 2 is prcfetre,: those which are within 1 are particularlv preferred, and those within 0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. IJ. S. Patent 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U. S. I'atent 4,554,101, the following hydropliilicity values have been assigned to amino acid residues: arsinine (+3.0); lysine (+3.0); aspartate (+3.0 1); glutamate (+3.0 1); serine (+0.3); asparagine (+0.2); glutaniine (+0.2); glycine (0);
threonine (-0.4);
proline (-0.5 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose liydrophilicity values are within 2 is preferred, those which are within 1 are particularly preferred, and those within 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well Icnown to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine;
glutamine and asparagine; and valine, leucine and isoleucine.

2.11 SITE-SPECIFIC 1VIUTAGENESIS

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage.
These phage are readily conunercially available and their use is generally well known to those skilled in the art.
Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest froni a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first 5 obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Kienow fragment, in order to 10 complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

15 The preparation of sequence variants of the selected peptide-encoding DNA
segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as 20 hydroxylamine, to obtain sequence variants.

2.12 CRYSTAL PROTEIN COMPOSITIONs As INSECTICIDES AND METHODS OF USE

The inventors contemplate that the chimeric crystal protein compositions disclosed herein will find particular utility as insecticides for topical and/or systemic application to field 25 crops, grasses, fruits and vegetables, and ornamental plants. In a preferred embodiment, the bioinsecticide composition comprises an oil flowable suspension of bacterial cells which expresses a novel crystal protein disclosed herein. Preferably the cells are B. thuringiensis cells, however, any such bacterial host cell expressing the novel nucleic acid segments disclosed herein and producing a crystal protein is contemplated to be useful, such as B.
30 megaterium, B. subtilis, E. coli, or Pseudomonas spp.

In another important embodiment, the bioinsecticide composition comprises a water dispersible granule. This granule comprises bacterial cells which expresses a novel crystal protein disclosed herein. Preferred bacterial cells are B. thuringiensis cells, however, bacteria such as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformed with a DNA

segment disclosed herein and expressing the crystal protein are also contemplated to be useful.
In a third important embodiment, the bioinsecticide composition comprises a wettable powder, dust, pellet, or collodial concentrate. This powder comprises bacterial cells which expresses a novel crystal protein disclosed herein. Preferred bacterial cells are B. thuringiensis cells, however, bacteria such as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformed with a DNA segment disclosed herein and expressing the crystal protein are also ' contemplated to be useful. Such dry forms of the insecticidal compositions may be formulated to dissolve immediately upon wetting, or alternatively, dissolve in a controlled-release, sustained-release, or other time-dependent manner.

In a fourth important embodiment, the bioinsecticide composition comprises an aqueous suspension of bacterial cells such as those described above which express the crystal protein. Such aqueous suspensions may be provided as a concentrated stock solution which is diluted prior to application, or alternatively, as a diluted solution ready-to-apply.

For these methods involving application of bacterial cells, the cellular host containing the crystal protein gene(s) may be grown in any convenient nutrient medium, where the DNA
construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B. thuringiensis gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
When the insecticidal compositions comprise intact B. thuringiensis cells expressing the protein of interest, such bacteria may be formulated in a variety of ways.
They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel chimeric Cry proteins may be prepared by recombinant bacterial expression systems in vitro and isolated for subsequent field application. Such protein niay be either in crude cell lysates, suspensions, colloids, etc., or alternatively may be purified, refined, buffered, and/or further processed, before formulating in an active biocidal formulation. Likewise, under certain circumstances, it may be desirable to isolate crystals and/or spores from bacterial cultures expressing the crystal protein and apply solutions, suspensions, or collodial preparations of such crystals and/or spores as the active bioinsecticidal composition.

Regardless of the method of application, the amount of the active component(s) are applied at an insecticidally-effective amount, which will vary depending on such factors as, for example, the specific coleopteran insects to be controlled, the specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating either the bacterial cell, crystal and/or spore suspension, or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, dessicated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally-acceptable carrier" covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in insecticide formulation technology; these are well known to those skilled in insecticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the insecticidal composition with suitable adjuvants using conventional formulation techniques.

The insecticidal compositions of this invention are applied to the environment of the target coleopteran insect, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. The strength and duration of insecticidal application will be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular enviroiunental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the insecticidal composition, as well as the particular formulation contemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating, spraying, aerating, misting, atomizing, and the like, are also feasible and may be required under certain circumstances such as e.g., insects that cause root or stalk infestation, or for application to delicate vegetation or ornamental plants.
These application procedures are also well-known to those of skill in the art.

The insecticidal composition of the invention may be employed in the method of the invention singly or in combination with other compounds, including and not limited to other pesticides. The method of the invention may also be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations. The insecticidal compositions of the present invention may be formulated for either systemic or topical use.

The concentration of insecticidal composition which is used for environmental, systemic, or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of biocidal activity.
Typically, the bioinsecticidal composition will be present in the applied formulation at a concentration of at least about 0.5% by weight and may be up to and including about 99% by weight. Dry formulations of the compositions may be from about 0.5% to about 99% or more by weight of the composition, while liquid formulations may generally comprise from about 0.5% to about 99% or more of the active ingredient by weight. Formulations which comprise intact bacterial cells will generally contain from about 104 to about 1012 cells/mg.

The insecticidal formulation may be administered to a particular plant or target area in one or more applications as needed, with a typical field application rate per hectare ranging on the order of from about 50 g to about 500 g of active ingredient, or of from about 500 g to about 1000 g, or of from about 1000 g to about 5000 g or more of active ingredient.

2.13 ANTIBODY COMPOSITIONS AND METHODS FOR PRODUCING

In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the crystal proteins disclosed herein.
Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1988). The methods for generating monoclonal antibodies (niAbs) generally begin along the same lines as those lor preparing polyclonal antibodies.
Briefly, a polyclonal antibody is preparcil by i:~-::1unizing an animal with an imtnttnobenic cotnposition in accordance with the present invention and collecting antisera from that immunized aninial. A wide range ul' animztl speeies can be used l'M the production of antisera.
'I'ypically the aninial used Cor production ol'anti-antisera is a rabbit, a mousc, a rat, a liamster, a guinea pig or a goat. Because of' the refatively large blood volume of rabbits, a rabbit is a prefened choice for production of' polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host itnrnune systcm, as may be achieved by coupling a peptide or polypeptide ininiunogen to a carrier. Exemplary and prefcrred carriers are keyhole limpet heniocyanin (KL.I-i) and bovine serum albuniin (E3SA). Othci albumins such as ovalbumin, mouse scruni albumin or rabbit serum albuniin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehydc, nt-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunoL!enicity ot' a particular immunogen composition can be enlianced by the use of' non-specif ic stimulators of' the immune response, known as adjuvants. Exemplary and prelcrred adjuvants include complete Freund's adjuvant (a non-specific stimulator ot' the itnmune response containing killed Mycohacteriunt luherculosis), incomplete Freund's adjttvants and aluniinurn hydroxide adjuvant.
The amount of irnrnunogen composition used in the production of' polyclonal antibodies varies upon the nature of the ininiunugen as well as the animal used for immunization. A
variety of routes can be used to administer the imniunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitotieal). The production of polyclonal antibodies may be monitored bv sarnpling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. T he process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the sertrni isolated and stored, and/or the animal can be used to generate mAbs.

d0 inAbti niay be readily prepared through use of well-known teclUnqtles, such as those cxemplificd in U. S. I'atent 4,196,265=
Typically, this tcchnidue involves immunizing a suitahle aninial with a sclccted immunol;en coniposition, e.g., a puriGed or partially puriGed crystal protein, polypeptide or peptide. The imtnunizing composition is administered in a manner eflective to stimulate antibody producing cells. Rodents such as mice and rats are preferred aninials, however, the use of rabbit, sheep frog cells is also possible. The use of rats niay provide certain advantages (Goding, 1986, pp.
60-61), but mice are preferred, with the 13ALI3/c mouse beinl; inost preferred as this is most routinely used and generally givcs a higher percentage of stable fusions.
Following inimuni=r_ation, somatic cells witll the potential for producing antibodies, specifically B lymphocytes (13 cells), are selecteci for use in the mAb generating protocol.
These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells arc preferred, thc former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will havc been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
'I'ypically, a spleen from an immunized mouse contains approximately 5 x 10' to 2 x 10 lymphocytes.
The antibody-producing 13 lymphocvtcs froni the immunized animal are then fused with cells of an immortal mycloma cell, gencrally one of the same species as the animal that was immunized. Myeloma cell lines suited for usc in hvbridoma-producing fitsion procedures preferably are non-antibody-producing, have hiph fusion et'(iciency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of ocily the desired fused cells (hybridomas).
Any one of a number of myeloma cells niay be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal is a mousc, one may use P3-X63/AgS, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag 14, FO, NSO/U, MPC-11, MPC 11-X45-GTG 1.7 and S 194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 12.3, 1R983F and 413210; and U-266. GM1500-GRG2.
LICR-LON-I-IMv2 and UC729-6 are all usef'ul in connection with hunian cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed 1-Ag4-1), which is readily available from the NIGMS I-luman Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (vol./vol.) PEG, (Gefter et al., 1977).
The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies, about I x 10-6 to 1 x 10- However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A
sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. The wild-type S-endotoxins and the relevant restriction sites that were used to construct the hybrid 6-endotoxins pertinent to the invention are diagrammed in FIG. lA. Only the DNA encoding the 8-endotoxin that is contained on the indicated plasmid (identified by the "pEG" prefix) is shown. The B. thuringiensis strains containing the indicated plasmids are identified by the "EG" prefix. The hybrid S-endotoxins described in the invention are diagrammed in FIG. 1 B and are aligned with the wild-type S-endotoxins in FIG. 1 A.

FIG. 2. An equal amount of each washed sporulated B. thuringiensis culture was analyzed by SDS-PAGE. Lane a: control Cry 1 Ac producing B. thuringiensis strain EG 11070, b: EG11060, c: EG11062, d: EG11063, e: EG11065, f: EG11067, g: EG11071, h:
EG 11073, i: EG 11074, j: EG 11088, k: EG 11090, and l: EG 11091.

FIG. 3. Solubilized hybrid b-endotoxins were exposed to trypsin for 0, 15, 30, 60, and 120 niinutes. The resulting material was analyzed by SDS-PAGE. The amount of active 8-endotoxin fragment remaining was quantitated by scanning densitometry using a Molecular Dynamics model 300A densitometer. The percent active toxin remaining was plotted versus time. Wild-type CrylAc b-endotoxin (open box) served as the control.

FIG. 4. Schematic diagrams of the wild-type toxins and the relevant restriction sites that were used to construct the hybrid S-endotoxin encoded by pEG381 and expressed in EG11768. Only the DNA encoding the 8-endotoxin that is contained on the indicated plasmid (identified by the "pEG" prefix) is shown.

4.0 BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
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 F.
SEQ ID NO:7 is oligonucleotide primer G.
SEQ ID NO:8 is oligonucleotide primer H.

SEQ ID NO:9 is the nucleotide and deduced amino acid sequences of the EG11063 hybrid S-endotoxin.

SEQ ID NO:10 denotes in the three-letter abbreviation form, the amino acid sequence for the hybrid 8-endotoxin specified in SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide and deduced amino acid sequences of the EG

hybrid 8-endotoxin.

SEQ ID NO:12 denotes in the three-letter abbreviation form, the amino acid sequence for the hybrid 8-endotoxin specified in SEQ ID NO: 11.

SEQ ID NO:13 is the nucleotide aiid deduced amino acid sequences of the EGl hybrid Fi-endotoxin.

SEQ ID NO:14 denotes in the three-letter abbreviation form, the amino acid sequence for the hybrid S-endotoxin specified in SEQ ID NO:13.

SEQ ID NO:15 is the 5' exchange site for pEG 1065, pEG 1070, and pEG 1074.
SEQ ID NO:16 is the 5' exchange site for pEG1067, pEG1072, and pEG1076.
SEQ ID NO:17 is the 5' exchange site for pEG 1068, pEG 1077, and pEG365.
SEQ II) NO:18 is the 5' exchange site for pEG1088 and pEG1092.

SEQ ID NO:19 is the 5' exchange site for pEG 1089 and the 3' exchange site for pEG 1070 and pEG 1072.

SEQ ID NO:20 is the 5' exchange site for pEG1091.

SEQ ID NO:21 is the 3' exchange site for pEG1065, pEG1067, pEG1068, pEG1093, pEG378, and pEG 365.

SEQ ID NO:22 is the 3' exchange site for pEG 1088.
SEQ ID NO:23 is oligonucleotide Primer I.

SEQ ID NO:24 is oligonucleotide Primer J.

SEQ ID NO:25 is the nucleic acid sequence and deduced amino acid sequence of the hybrid crystal protein-encoding gene of EG1 1092.

SEQ ID NO:26 is the three-letter abbreviation form of the amino acid sequence of the hybrid crystal protein produced by strain EG11092 encoded by SEQ ID NO:25.

SEQ ID NO:27 is the nucleic acid sequence and the deduced amino acid sequence of the hybrid crystal protein-encoding gene of EG11751.

SEQ ID NO:28 is the three-letter abbreviation form of the amino acid sequence of the hybrid crystal protein produced by strain EG 11751 encoded by SEQ ID NO:27.

SEQ ID NO:29 is the nucleic acid sequence and the deduced amino acid sequence of the hybrid crystal protein-encoding gene of EG 11091.

SEQ ID NO:30 is the three-letter abbreviation form of the amino acid sequence of the hybrid crystal protein produced by strain EG 11091 encoded by SEQ ID NO:29.

SEQ ID NO:31 is oligonucleotide primer K.

SEQ ID NO:32 is the 5' exchange site for pEG378 and pEG381.

SEQ ID NO:33 is the nucleic acid sequence and the deduced amino acid sequence of the hybrid crystal protein-encoding gene of EG 11768.

SEQ ID NO:34 denotes in the three-letter abbreviation form, the amino acid sequence of the hybrid crystal protein produced by strain EG11768 encoded by SEQ ID
NO:33.

5 SEQ ID NO:35 is the 3' exchange site for pEG 1074, pEG 1076, pEG 1077 and pEG381.
5.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

5.1 METHODS FOR CULTURING B. THURINGIENSIS TO PRODUCE CRY PROTEINS

The B. thuringiensis strains described herein may be cultured using standard known 10 media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria may be harvested by first separating the B. thuringiensis spores and crystals from the fermentation broth by means well known in the art. The recovered B.
thuringiensis spores and crystals can be formulated into a wettable powder, a liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to 15 facilitate handling and application for particular target pests. The formulation and application procedures are all well known in the art and are used with commercial strains of B.
thuringiensis (HD-1) active against Lepidoptera, e.g., caterpillars.

5.2 RECOMBINANT HOST CELLS FOR EXPRESSION OF CRY GENES

20 The nucleotide sequences of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the sites of lepidopteran insects where they will proliferate and be ingested by the insects. The results is a control of the unwanted insects.

25 Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the B. thuringiensis toxin.

Suitable host cells, where the pesticide-containing cells will be treated to prolong the 30 activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as inammals.
However, organisms which produce substances toxic to higlier organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility or toxicity to a manlmalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and Gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactobacillaceae;
Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B. thuringiensis gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.
Specific organisms include Pseudomonas aeruginosa, P. fluorescens, Saccharomyces cerevisiae, B.
thuringiensis, B. subtilis, E. coli, Streptomyces lividans and the like.

Treatment of the microbial cell, e.g., a microbe containing the B.
thuringiensis toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for stifficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehye;
anti-infectives, such as zephiran chloride and cetylpyridinium chloride;
alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol's iodine, Bouin's fixative, and Helly's fixatives, (see e.g., Humason, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to a suitable host. Examples of physical means are short wavelength radiation such as y-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. The cells employed will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.
Where the B. thuringiensis toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.
Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes eutrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. inarina, R. aurantiaca, Cryptococcus albidus, C. d ffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidiurn pollulans.

5.3 DEFINITIONS

The following words and phrases have the meanings set forth below.
Broad-Spectrum: refers to a wide range of insect species.

Broad-Spectrum Insecticidal Activity: toxicity towards a wide range of insect species.

Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

Insecticidal Activity: toxicity towards insects.

Insecticidal Specificity: the toxicity exhibited by a crystal protein towards multiple insect species.

Intraorder Specificity: the toxicity of a particular crystal protein towards insect species within an Order of insects (e.g., Order Lepidoptera).

Interorder Specificity: the toxicity of a particular crystal protein towards insect species of different Orders (e.g., Orders Lepidoptera and Diptera).

LC50: the lethal concentration of crystal protein that causes 50% mortality of the insects treated.

LC95: the lethal concentration of crystal protein that causes 95% mortality of the insects treated.

Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA
polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast or explant).

Structural Gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.
Transformed Cell: A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.
Transgene: An exogenous gene which when introduced into the genome of a host cell through a process such as transformation, electroporation, particle bombardment, and the like, is expressed by the host cell and integrated into the cells genome such that the trait or traits produced by the expression of the transgene is inherited by the progeny of the transformed cell.

Transgenic Cell: Any cell derived or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.

Transgenic Plant: A plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA
molecule not originally present in a native, non-transgenic plant of the same strain. The terms "transgenic plant" and "transformed plant" have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast as being a transgenic plant, and that usage will be followed herein.

Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.

5.4 PROBES AND PRIMERS

In another aspect, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein.
In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected crystal protein gene sequence, e.g., a sequence such as that shown in SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ lf) NO:33. The ability of such nucleic acid probes to specifically hyhridize to a cryst.il protein-cncodinc gene sequence lends them particular utilitv in zt varicty of' embodinients.
Most imPortantly, the probes niay be used in a variety of assays for detectin,, the presence of' compL-nientary sequcnces in a given xample.
5 In certain embodiments, it is advantageous to use olif!onticleoticie primers. "I'hc sequence of'such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or niutating .t defined segntcnt of a crystal protc:iii gcne fi-ont B.
tlniriitgiensis using 1'CRT"' technology. Segments ot' related crystal protein genes from othcr species may also be amplified by PCRTM using such primers.
10 To provide certain of' the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of' a crystal F:otcin-encoding sequence, such as that shown iu SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SLQ ID NO:29, or SEQ ID NO:33. A
size 15 of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequcnces over stretches greater thati 14 bases in length are generally preferrcd, though, in order to increase stability and selectivity of' the hybrid, and thereby improve the quality and degree of specific hybrid inolecules obtained. One will generally prefer to design nucleic acid 20 molecules having gene-complementary stretches of' 14 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizinb the tragnient by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patents 4,683,195, and 4,683.202, or by excising selected DNA fragments from recombinant 25 plasmids containing appropriate inserts and suitable restriction sites.
5.5 EXPRESSION VECTOR.S
The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression vector is an isolated and 30 purified DNA molecule comprising a promoter operatively linked to an coding region that encodcs a polypeptide of the present invention, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.

As used herein, the term "operatively linked" means that a promoter is connected to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.

Promoters that function in bacteria are well known in the art. Exemplary and preferred promoters for the Bacillus crystal proteins include the sigA, sigE, and sigK
gene promoters.
Alternatively, the native, mutagenized, or recombinant crystal protein-encoding gene promoters themselves can be used.

Where an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants.
Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).

A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near-constitutive, such as the CaMV 35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S, increases in polypeptide expression are found in a variety of transformed plant tissues (e.g., callus, leaf, seed and root). Alternatively, the effects of transformation can be directed to specific plant tissues by using plant integrating vectors containing a tissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The Lectin protein in soybean seeds is encoded by a single gene (Lel) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA.
The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983;
Lindstrom et al., 1990.) An expression vector containing a coding region that encodes a polypeptide of interest is engineered to he under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhii- et al., 1991).
The expression of the polypeptide is directed specifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1(Yang et al., 1990), corn alcohol dehydrogenase 1(Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP
carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP
carboxylase promoter.
The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the funetional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA
molecules.
However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et al., 1985). pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter.
In preferred embodiments, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker.

One pretcrrecl drut-, resistance marker is the gene whose expression results in kanamvcin resistance; r.e-.. the chinirric ~"ene containinW the n0paline sj-nthasr promoter. "fn.i neonivcin phosphotranslcratir Il (rT/itll) ancl n0p:rlinr ,vnthau non-translatecl refion described (Rogers el ul. , I 988).
1tNA polytnerase transcribes a cc-ciittg DNA seeluer)ce through a siic wherc polyaclenvlation occurs. '1'vpically, DNA sequence, located a few hundrecl base pairs dowtistrean) of the pulvaclcnvlation site serve to terminate transcription.
Those DNA
. sequences are referred to herein as transcription-termination regions.
'I'hose regiotis are required lor ef(icient polvadenylation of transcribecl messenger RNA (mRNA).
Means 1ior preparing expression vectors are wcll known in the art. Expression (transformation vectors) useci to transl'orni plants and mrthods o!' niaking those vectors arc described in U. S. I'atents 4.971,908, 4,940,835, 4,769,001 and 4,757,011.
Those vectors can be modified to include a coding sequence in accordance with the present invention.
A varicty of rnethods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA.
The vector and DNA segment are then foinecl by hyclrogen bondinl, between thr complementary homopolvnieric tails to li)rm recombinant DNA niolecules.
A coding region that encodes a polypeptide havin`.; the ability to confer insecticidal activity to a cell is pretcrahly a chimeric B. ,thurrirgieirsrs crystal protein-encoding gene. In preferced enlbodinients. such a polvpcptide has the an)ino acicl resiciue sequence ol' SEQ ID NO:10, Sl:(,) I1) NO:12, SEQ lD NO:14, til:O II) N0:26, SCQ ID NO:28, SEQ ID NO:30, or SE'Q II) NO:34; or a lunctlonal equivalent of one or more of thosc sequences. In accordance with sucl) embodiments, a coding region comprising the DNA
scquence of tiI:Q ID NO:9. SI:Q 11) NO: 11, SGQ 11) NO: 13, tiI:Q 11) NO:25, SrQ ID NO:27, SEQ 11) NO:29, or SFQ ID NO:33 is altio prel'errecl.

5.6 TRANSFORMEI) Olt TKANSGF:NIC 1't.ANT CELLS
A bacteriuni, a yeast cell, or a plant cell or a plant transtormed with an expression vector of the present invention is also contemplated. A transgenic bacterium.
veast cell, plant cell or plant derived from such a transformed or transgenic cell is also contemplated. Means for transforming bacteria and yeast cells are well known in the art.
Typically, means of transformation are similar to those well known means used to transform other bacteria or yeast such as E. coli or S. cerevisiae.

Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages. Thus, one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell, such as infection by A.
tumefaciens and related Agrobacterium, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like.

Technology for introduction of DNA into cells is well-known to those of skill in the art.
Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).

5.6.1 ELECTROPORATION
The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane.
DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores.

Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated 5 copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known to those of skill in the art. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more 10 susceptible to transformation, by mechanical wounding. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled 15 manner. Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.

5.6.2 MICROPROJECTILE BOM BARDMENT

20 A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being an effective 25 means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Cristou et aL, 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered 30 with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from I to 10 and average 1 to 3.
In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro-or microprojectiles.
Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

i7 'I'hc nuthocis o1'particle-me(liated transfiirmation is wcll-l:nown to those of skill in thr art. 11. S. Patent S,(11 5,59O = describes the Iransforniation of uOybeans using such a tcchniquc.
5.6.3 I(:X()13.=ICTI:R/[IAl-I~11:h1A'1'EI) IItANSF'I:It ,,1gruhactc=riunr-meeliztteci translcr is a widely applicable system f'or introciucing genes intO plant cclls bec.rusr the 1)Nn can he introducccl into whulc plant tissucs, thereby hypassint;
the neecl f(ir regeneration of un intact nlant fi=om a protoplast. 'I'he use of AfiruhacYerltcnr-mediatccl plant intel;ratint, vectors to introduce I)NA intu rlant cells is well known in thc art.

See, 1or examPle, the methods described (Fraley et crl., 1985; Rut;ers el crl., 1987). The genetic enginccring of' cotton Plants using lt,izrnhacteritnn-mediatr(1 transfer is (lcscribcd in U. S.
Patent 5,004,863, while the transformation of lettuce plants is described in U.S. Patent 5,349,124.
Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to he transfcrrcci is defined by the border sequences, and intervening DNA is usuallV lnser'le(l lnto the plant genome as clescribecl (Spielmann et al., 1986; Jorbensen et crl., 1987).
Modern Agrnhcrcterirurr transforniatiun vectors are capuhle ot' replication in G. c=uli as well as Aprnhaclc-rinm, aliowing fior convcnient manipulations ,ts deticribc(1 (Klee et al., 1985).
Moreover, recent technological aelvanccs in vectors fi-r :I,rfruhcu=rrriunr-mediatecl gcne transfer have improve(1 tiie arrangement of geties ancl restriction sites in the vectors to facilitate construction of vectot-s capable of expressing various poIvpepticle co(fing genes. The vectors describcd (Rogers et ul.. 1987), have convenient multi-link- regions flanked bv :i nromoter and a polyacienylation site for direct espressiun of insci-tcci polypeptide coclinb genes and are suitable for present purposes. In addition, : tgrnbacteriunr containing both armed and disarmed Ti genes can he used for the transformations. In th(ise plant strains where Afirobacteriturt-rnediatecl transf-)rmation is efficierit, it is the methoci of' choice because of the facile and defined nature of -he gene transfer.
Agrnhactcrirun-n)ediatecl transfnrmation of leaf ciisks and othcr tissues such as cotyledons and hypocotyls af-pears to be liniitecl to plants that :
tl,=ruhcrcteritun naturally infects.
Agrohacteriunr-tttediatr(I transforrnation is most efficient in dicotvlectonous plants. Few nionocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al., 1987).
Therefore, commercially iniportant cereal grains such as rice, corn, and wheat must usually be transformed using alternative methods. However, as mentioned above, the transformation of asparagus using Agrobacteriuin can also be achieved (see, e.g., Bytebier et al., 1987).

A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. flowever, inasmuch as use of the word "heterozygous"
usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene segregates independently during mitosis and meiosis.

More preferred is a transgenic plant that is homozygous for the added structural gene;
i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A liomozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes.
Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985;
Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (see, e.g., Fujimura et al., 1985;
Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1988). In addition, "particle gun" or high-velocity microprojectile technology can be utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.

5.6.4 GENE EXPRESSION IN PLANTS

The fact that plant codon usage more closely resembles that of humans and other higher organisms than unicellular organisms, such as bacteria, unmodified bacterial genes are often poorly expressed in transgenic plant cells. The apparent overall preference for GC content in codon position three has been described in detail by Murray et al. (1990). The 207 plant genes described in this work permitted the compilation of codon preferences for amino acids in plants. These authors describe the difference between codon usage in monocots and dicots, as well as differences between chloroplast encoded genes and those which are nuclear encoded.
Utilizing the codon frequency tables provided, those of skill in the art can engineer such a bacterial sequence for expression in plants by modifying the DNA sequences to provide a codon bias for G or C in the third position.

The work by Diehn et al. (1996) details the modification of prokaryot-derived gene sequence to permit expression in plants. lannacone et al. (1997) describe the transformation of egg plant with a genetically engineered B. thuringiensis gene encoding a cry3 class endotoxin.
Utilizing sequences which avoid polyadenylation sequences, ATTA sequences, and splicing sites a synthetic gene was constructed which permitted expression of the encoded toxin in planta. Expression of jellyfish green fluorescent protein, in transgenic tobacco has been described by Rouwendal et al. (1997). Using a synthetic gene, the third position codon bias for C+G was created to permit expression of the heterologous gene in planta.

Futterer and Hohn (1996) describe the effects of mRNA sequence, leader sequences, polycistronic messages, and internal ribosome binding site motis, on expression in plants.
Modification of such sequences by construction of synthetic genes permitted expression of viral mRNAs in transgeilic plant cells.

5 Although great progress has been made in recent years with respect to preparation of transgenic plants which express bacterial proteins such as B. thuringiensis crystal proteins, the results of expressing native bacterial genes in plants are often disappointing. Unlike microbial genetics, little was known by early plant geneticists about the factors which affected heterologous expression of foreign genes in plants. In recent years, however, several potential 10 factors have been implicated as responsible in varying degrees for the level of protein expression from a particular coding sequence. For example, scientists now know that maintaining a significant level of a particular mRNA in the cell is indeed a critical factor.
Unfortunately, the causes for low steady state levels of mRNA encoding foreign proteins are many. First, full length RNA synthesis may not occur at a high frequency. This could, for 15 example, be caused by the premature termination of RNA during transcription or due to unexpected mRNA processing during transcription. Second, full length RNA may be produced in the plant cell, but then processed (splicing, polyA addition) in the nucleus in a fashion that creates a nonfunctional mRNA. If the RNA is not properly synthesized, terminated and polyadenylated, it cannot move to the cytoplasm for translation. Similarly, in the cytoplasm, if 20 mRNAs have reduced half lives (which are determined by their primary or secondary sequence) inisufficient protein product will be produced. In addition, there is an effect, whose magnitude is uncertain, of translational efficiency on mRNA half-life. In addition, every RNA
molecule folds into a particular structure, or perhaps family of structures, which is determined by its sequence. The particular structure of any RNA might lead to greater or lesser stability in 25 the cytoplasm. Structure per se is probably also a determinant of mRNA
processing in the nucleus. Unfortunately, it is impossible to predict, and nearly impossible to determine, the structure of any RNA (except for tRNA) in vitro or in vivo. However, it is likely that dramatically changing the sequence of an RNA will have a large effect on its folded structure It is likely that structure per se or particular structural features also have a role in determining 30 RNA stability.

To overcome these limitations in foreign gene expression, researchers have identified particular sequences and signals in RNAs that have the potential for having a specific effect on RNA stability. In certain embodiments of the invention, therefore, there is a desire to optimize expression of the disclosed nucleic acid segnlents in planta. One particular method of doing so, is by alteration of the bacterial gene to remove sequences or motifs which decrease expression in a transformed plant cell. The process of engineering a coding sequence for optimal expression in planta is often referred to as "plantizing" a DNA
sequence.

Particularly problematic sequences are those which are A+T rich.
Unfortunately, since B. thuringiensis has an A+T rich genome, native crystal protein gene sequences must often be modified for optimal expression in a plant. The sequence motif ATTTA (or AUUUA
as it appears in RNA) has been implicated as a destabilizing sequence in mammalian cell mRNA
(Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich 3' untranslated regions, and these regions often have the ATTTA sequence, sometimes present in multiple copies or as multimers (e.g., ATTTATTTA...). Shaw and Kamen showed that the transfer of the 3' end of an unstable mRNA to a stable RNA (globin or VAI) decreased the stable RNA's half life dramatically. They further showed that a pentamer of ATTTA had a profound destabilizing effect on a stable message, and that this signal could exert its effect whether it was located at the 3' end or within the coding sequence. However, the number of ATTTA
sequences and/or the sequence context in which they occur also appear to be important in determining whether they function as destabilizing sequences. Shaw and Kamen showed that a trimer of ATTTA
had much less effect than a pentamer on mRNA stability and a dimer or a monomer had no effect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTA such as a pentamer automatically create an A+T rich region. This was shown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs, the ATTTA sequence may be present in only a single copy, but it is often contained in an A+T rich region. From the animal cell data collected to date, it appears that ATTTA at least in some contexts is important in stability, but it is not yet possible to predict which occurrences of ATTTA are destabiling elements or whether any of these effects are likely to be seen in plants.

Some studies on mRNA degradation in animal cells also indicate that RNA
degradation may begin in some cases with nucleolytic attack in A+T rich regions. It is not clear if these cleavages occur at ATTTA sequences. There are also examples of mRNAs that have differential stability depending on the cell type in which they are expressed or on the stage within the cell cycle at which they are expressed. For example, histone mRNAs are stable during DNA synthesis but unstable if DNA synthesis is disrupted. The 3' end of some histone mRNAs seeins to be responsible for this effect (Pandey and Marzluff, 1987). It does not appear to be niediated by ATTTA, nor is it clear what controls the differential stability of this mRNA. Another example is the differential stability of lgG mRNA in B
lymphocytes during B cell maturation (Genovese and Milcarek, 1988). A final example is the instability of a mutant (3-thallesemic globin mRNA. In bone marrow cells, where this gene is normally expressed, the mutant mRNA is unstable, while the wild-type mRNA is stable.
When the mutant gene is expressed in HeLa or L cells in vitro, the mutant mRNA shows no instability (Lim et al., 1988). These examples all provide evidence that mRNA stability can be mediated by cell type or cell cycle specific factors. Furthermore this type of instability is not yet associated with specific sequences. Given these uncertainties, it is not possible to predict which RNAs are likely to be unstable in a given cell. In addition, even the ATTTA motif may act differentially depending on the nature of the cell in which the RNA is present. Shaw and Kamen (1987) have reported that activation of protein kinase C can block degradation mediated by ATTTA.

The addition of a polyadenylate string to the 3' end is common to most eukaryotic mRNAs, both plant and animal. The currently accepted view of polyA addition is that the nascent transcript extends beyond the mature 3' terminus. Contained within this transcript are signals for polyadenylation and proper 3' end formation. This processing at the 3' end involves cleavage of the mRNA and addition of polyA to the mature 3' end. By searching for consensus sequences near the polyA tract in both plant and animal mRNAs, it has been possible to identify consensus sequences that apparently are involved in polyA addition and 3' end cleavage. The same consensus sequences seem to be important to both of these processes.
These signals are typically a variation on the sequence AATAAA. In animal cells, some variants of this sequence that are functional have been identified; in plant cells there seems to be an extended range of functional sequences (Wickens and Stephenson, 1984;
Dean et al., 1986). Because all of these consensus sequences are variations on AATAAA, they all are A+T

rich sequences. This sequence is typically found 15 to 20 bp before the polyA
tract in a mature mRNA. Studies in animal cells indicate that this sequence is involved in both polyA addition and 3' maturation. Site directed mutations in this sequence can disrupt these functions (Conway and Wickens, 1988; Wickens el a1.,.1987). However, it has also been observed that sequences up to 50 to 100 bp 3' to the putative polyA signal are also required; i.e., a gene that has a normal AATAAA but has been replaced or disrupted downstream does not get properly polyadenylated (Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984).
That is, the polyA signal itself is not sufficient for complete and proper processing. It is not yet known what specific downstream sequences are required in addition to the polyA signal, or if there is a specific sequence that has this function. Therefore, sequence analysis can only identify potential polyA signals.

In naturally occurring mRNAs that are normally polyadenylated, it has been observed that disruption of this process, either by altering the polyA signal or other sequences in the mRNA, profound effects can be obtained in the level of functional mRNA. This has been observed in several naturally occurring mRNAs, with results that are gene-specific so far.

It has been shown that in natural mRNAs proper polyadenylation is important in mRNA accumulation, and that disruption of this process can effect mRNA levels significantly.
However, insufficient knowledge exists to predict the effect of changes in a normal gene. In a heterologous gene, it is even harder to predict the consequences. However, it is possible that the putative sites identified are dysfunctional. That is, these sites may not act as proper polyA
sites, but instead function as aberrant sites that give rise to unstable mRNAs.

In animal cell systems, AATAAA is by far the most common signal identified in mRNAs upstream of the polyA, but at least four variants have also been found (Wickens and Stephenson, 1984). In plants, not nearly so much analysis has been done, but it is clear that multiple sequences similar to AATAAA can be used. The plant sites in Table 4 called major or minor refer only to the study of Dean et al. (1986) which analyzed only three types of plant gene. The designation of polyadenylation sites as major or minor refers only to the frequency of their occurrence as functional sites in naturally occurring genes that have been analyzed. In the case of plants this is a very limited database. It is hard to predict with any certainty that a site designated major or minor is more or less likely to function partially or completely when found in a heterologous gene such as those encoding the crystal proteins of the present invention.

POLYADENYLATION SITES IN PLANT GENES

PA AATAAA Major consensus site PIA AATAAT Major plant site P2A AACCAA Minor plant site P3A ATAT'AA "

P4A AATCAA "
P5A ATACTA "
P6A ATAAAA "

P l 0A ATACAT

P12A ATTAAA Minor animal site P 13A AATTAA "

P14A AATACA "
P 15A CATAAA "

The present invention provides a method for preparing synthetic plant genes which genes express their protein product at levels significantly higher than the wild-type genes which were commonly employed in plant transformation heretofore. In another aspect, the present invention also provides novel synthetic plant genes which encode non-plant proteins.

As described above, the expression of native B. thuringiensis genes in plants is often problematic. The nature of the coding sequences of B. thuringiensis genes distinguishes them from plant genes as well as many other heterologous genes expressed in plants.
In particular, B. thuringiensis genes are very rich (-62%) in adenine (A) and thymine (T) while plant genes and most other bacterial genes which have been expressed in plants are on the order of 45-55%
A+T.

Due to the degeneracy of the genetic code and the limited number of codon choices for any amino acid, most of the "excess" A+T of the structural coding sequences of some Bacillus species are found in the third position of the codons. That is, genes of sonie Bacillus species liave A or T as the tliird nucleotide in many codons. Thus A+T content in part can determine codon usage bias. In addition, it is clear that genes evolve for maximum function in the organism in which they evolve. This means that particular nucleotide sequences found in a 5 gene from one organism, where they may play no role except to code for a particular stretch of amino acids, have the potential to be recognized as gene control elements in another organism (such as transcriptional promoters or terminators, po]yA addition sites, intron splice sites, or specific mRNA degradation signals). It is perhaps surprising that such misread signals are not a more conunon feature of heterologous gene expression, but this can be explained in part by 10 the relatively homogeneous A+T content (-50%) of many organisms. This A+T
content plus the nature of the genetic code put clear constraints on the likelihood of occurrence of any particular oligonucleotide sequence. Thus, a gene from E. coli with a 50% A+T
content is much less likely to contain any particular A+T rich segment than a gene from B. thuringiensis.

Typically, to obtain high-level expression of the S-endotoxin genes in plants, existing 15 structural coding sequence ("structural gene") which codes for the S-endotoxin are modified by removal of ATTTA sequences and putative polyadenylation signals by site directed mutagenesis of the DNA comprising the structural gene. It is most preferred that substantially all the polyadenylation signals and ATTTA sequences are removed although enhanced expression levels are observed with only partial removal of either of the above identified 20 sequences. Alternately if a synthetic gene is prepared which codes for the expression of the subject protein, codons are selected to avoid the ATTTA sequence and putative polyadenylation signals. For purposes of the present invention putative polyadenylation signals include, but are not necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, 25 AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. In replacing the ATTTA
sequences and polyadenylation signals, codons are preferably utilized which avoid the codons which are rarely found in plant genomes.

The selected DNA sequence is scanned to identify regions with greater than four consecutive adenine (A) or thymine (T) nucleotides. The A+T regions are scanned for 30 potential plant polyadenylation signals. Although the absence of five or more consecutive A or T nucleotides eliminates most plant polyadenylation signals, if there are more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then the nucleotide sequence of this region is preferably altered to remove these signals while maintaining the original encoded amino acid sequence.

The second step is to consider the about 15 to about 30 or so nucleotide residues surrounding the A+T rich region identified in step one. If the A+T content of the surrounding regioii is less than 80%, the region should be examined for polyadenylation signals. Alteration of the region based on polyadenylation signals is dependent upon (1) the number of polyadenylation signals present and (2) presence of a major plant polyadenylation signal.

The extended region is examined for the presence of plant polyadenylation signals.
The polyadenylation signals are removed by site-directed mutagenesis of the DNA sequence.
The extended region is also examined for multiple copies of the ATTTA sequence which are also removed by mutagenesis.
It is also preferred that regions comprising many consecutive A+T bases or G+C
bases are disrupted since these regions are predicted to have a higher likelihood to form hairpin structure due to self-complementarity. Therefore, insertion of heterogeneous base pairs would reduce the likelihood of self-complementary secondary structure formation which are known to inhibit transcription and/or translation in some organisms. In most cases, the adverse effects may be minimized by using sequences which do not contain more than five consecutive A+T
or G+C.

5.7 PRODUCTION OF INSECT-RESISTANT TRANSGENIC PLANTS

Thus, the acnount of a gene coding for a polypeptide of interest (i.e., a bacterial crystal protein or polypeptide having insecticidal activity against one or more insect species) can be increased in plant such as corn by transforming those plants using particle bombardment methods (Maddock et al., 1991). By way of example, an expression vector containing a coding region for a B. thuringiensis crystal protein and an appropriate selectable marker is transformed into a suspension of embryonic maize (corn) cells using a particle gun to deliver the DNA coated on microprojectiles. Transgenic plants are regenerated from transformed embryonic calli that express the disclosed insecticidal crystal proteins.
Particle bombardment has been used to successfully transform wheat (Vasil et al., 1992).

1.)NA can also be introduced into plants by ciirect 1)NA transfcr into pollen as dcscribcd (Zhou et ul., 1983; lless, 1987; LuO et ul.. I988). I:xpretitiiort c-l'polypcptide ct-ding genes can he obtainecl by injection of tlie DNA into reproductive organs of a plant as (lrticribed (Pena et nl., 1987). DNA can also be injectecl directly into the cells of' imniaturc etnhryos and the rehydration of'desiccated etnbryos as described (Neuhaus cet al., 1987;
Renbrook et a1., 1986).
The dcvelopnient or regeneration of plants from either sinl;le plant protoplasts or various explants is well known in the art (Weissbach an(i Weistibach, 1998).
'T'his regeneration and growth process typically includes tlic steps of selection of tr.lnsformed cells, culturinb those individualizecl cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated.
The resulting transgenic rooted shoots are thereatter planted in an appropriate plant growth nledium sucii as soil.
'I'he development or regeneration of plants containing the toreif;n, exogenous gene that encodes a polypeptide of' interest introduced by Agrohcrcterirun from leaf' explants can be achieved by methods well laiown in the art such as described (I-lorsch et al., 1985). In this.
procedure, transformants are culturcd in the presence of'a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as clescribed (Fraley et al., 1983). In particular, U. S. Patent 5,349,124 details the creation of genetically transformed lettuce cells and plants resulting therefrom which express hybrid crystal proteins conferring insecticidal activity against Lepidopteran larvae to such plants.
This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing mediuni containing the selective agent and an antibiotic to prevent bacterial 6rowth. Shoots that rooted in the presence of the selective agent to torm plantlets are then transplanted to soil or other media to allow the production of' roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.
I'.,:fcrably, the regenerated plants are self-pollinated to provide homozygous transl;enic plants, as discussed before. Otherwise, pollen obtained froni the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines.
Conversely, pollen from plants of thosc iniportant lines is used to pollinate regenerated plants.
A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

A transgenic plant of this invention thus has an increased amount of a coding region (e.g., a cry gene) that encodes one or more of the Chimeric Cry polypeptides disclosed herein.
A preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny. A more preferred transgenic plant is homozygous for that gene, and transmits that gene to all of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for, by way of example, increased insecticidal capacity against Coleopteran insects, preferably in the field, under a range of environmental conditions. The inventors contemplate that the present invention will find particular utility in the creation of transgenic corn, soybeans, cotton, tobacco, tomato, potato, flax, rye, barley, canola, wheat, oats, rice, other grains, vegetables, fruits, fruit trees, berries, turf grass, ornamentals, shrubs and trees.

5.8 RIBOZYMES

Ribozymes are enzymatic RNA molecules which cleave particular mRNA species. In certain embodiments, the inventors contemplate the selection and utilization of ribozymes capable of cleaving the RNA segments of the present invention, and their use to reduce activity of target mRNAs in particular cell types or tissues.

Six basic varieties of naturally-occurring enzymatic RNAs are known presently.
Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid lias bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over many teclinologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis 8 virus, group I intron or RNaseP RNA (in association with an RNA
guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al.
(1992); examples of hairpin motifs are described by Hampel et al. (Eur. Pat.
EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and Cech et al. (U. S. Patent 5,631,359; an example of the hepatitis 6 virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et al. (1983); Neurospora VS
RNA
ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991;
Collins and Olive, 1993); and an example of the Group I intron is described by Cech et al.
(U.S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.

The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target.
The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA such that specitic treatnient of u disease or cnndition can be providecl with either one or several enzymatic nucleic acids. Such en=r.vmatic nucleic acid moleculcs can be delivercd exogenously to specific cells as requirecl. Alternativcly, the rihozynies can he expressed from DNA or 1tNA vectors that are deliverecl to specific cells.

5 Small enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) may be used for exogenous delivery. The simple structure of these molecules increases the ability of the enzyniatic nucleic acid to invacle targeted regions of the niRNA structure.
Alternatively, catalytic RNA molectiles can be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991; hash.ani-Sabet et al., 1992; Dropulic et a1., 1991, 10 Wecrasinghc et al., 1991; Ojwang et al., 1992; Chen ct al., 1992; Sarver et al., 1990). Those skilled in the art realize that any ribozyme can he expressed in eukaryotic cells from the appropriate DNA vector. The activity of such ribozvmes can be augmented by their release from tiie primary transcript by a seconci ribozyme (Draper et al., lnt. Pat.
Appl. Publ. No. WO
93/23569, and Sullivan et a1., Int. Pat. Appl. Publ. No. WO 94/02595, 15 Ohkawa et a.1, 1992; Taira et al., 1991; Ventura et al., 1993).

Ribozymes may be added directly, or can he complexed witti cationic lipids, lipid complexes, packaged within liposomes, or otherwise ctelivered to target cells.
1'he RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo throuPh 20 injection, aerosol inhalation, infttsion pump or stent, with or without their incorporatioii in biopolymers.
Ribozymes may he designed as described in Draper et al. (Int. Pat. Appi. Publ.
No. WO
93/23569). or Sullivan et al., (Int. Pat. Appl. Publ. No. WO 94/02595) and synthesized to be tested in vitro and in vivo, as described. Such ribozvnies can also be optimized for delivery.
25 While specific examples are provided, those in the art will recognize that equivalent RNA
targets in other species can be utilized when necessary.
Flammerhead or hairpin ribozymes may bc individually analyzed by computer folding (Jaeger et al., 1989) to assess wliether the ribozvme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between 30 the binding arms and the catalytic core are eliniinated lrom consideration.
Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

Ribozymes of the hammerhead or hairpin motif inay be designed to anneal to various sites in the mRNA message, and can be cllemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al.
(1987) and in Scaringe et al. (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
Average stepwise coupling yields are typically >98%. Hairpin ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).
Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065;
Perrault et al, 1990;
Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO
93/15187; Int.
Pat. Appl. Publ. No. WO 91/03162; U.S. Patent 5,334,711; and Int. Pat. Appl.
Publ. No. WO
94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneou, or joint injectioit, aerosol inhalation, oral (tablct or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Morc detailed descriptions of ribozynic delivery and administration are provide(l in tittllivan er ul. (Int. Pat.
Aphl. Puhl. No. WC) 94/02595) and Draper et al. (Int. Pat. Aphl. Puhl. No. WO 93/23569).
Elnother means ol' accumulating high conccntrations ol' a ribozynte(s) within cells is to incorporate the ribozynic-encoding scqucnces into a DNA cxpression vector.
1'ranscription of thc ribozyme sequences are driven from a promoter tor eukaryotic RNA
polymerase I (pol I), ItNA polymerase 11 (pol 11), or RNA polvmerase 111 (pol 111). Transcripts from pol 11 or pol III

promoters will he expressed at hii;h levels in all cells; the lcvels of a given pol II promoter in a given cell type will depend on the naturc of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokarvotic RNA rolvnierase promoters may also be used, providing that the prokaryotic RNA polymerase enzynie is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al., 1993; 7.hou et al., 1990).
Ribozymes expressed from such promoters can function in mammalian cells (e.g.
Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al., 1993;
L'Huillier e1 al., 1992; Lisziewicz et al., 1993). Such transcription units can be incorporated into a variety of vectors for introduction into tnammalian cells, including httt not restricted to, plasmid DNA
vectors, viral DNA vectors (sucli as adenovirus or adeno-associated vectors), or viral RNA
vectors (such as retroviral, seniliki forest virus, sindbis virus vectors).
Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations witliin cell lines or cell tvpes. They can also be used to assess levels of thc target RNA molecule. "I'hc close relationship between ribozyme activity and the structure of the target RNA allows the detection of tnutations in any region of the molecule which alters the base-pairing and three-dimensional structure oi' the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA
structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs witlt ribozymes may be used to inhibit gene expression and deGne the role (essentially) of specified gene products in particular cells or cell types.

5.9 ISOLATING HOMOLOGOUS GENE AND GENE FRAGMENTS

The genes and 8-endotoxins according,to the subject invention include not only the full length sequences disclosed herein but also fi=agments of these sequences, or fusion proteins, which retain the characteristic insecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person skill in this art that insecticidal S-endotoxins can be identified and obtained through several means. The specific genes, or portions thereof, may be obtained from a culture depository, or constructed syntlietically, for example, by use of a gene machine. Variations of these 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 Ba13 i or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which code for active fragments may be obtained using a variety of other restriction enzymes. Proteases may be used to directly obtain active fragments of these S-endotoxins.

Equivalent 8-endotoxins and/or genes encoding these equivalent 8-endotoxins can also be isolated from Bacillus strains and/or DNA libraries using the teachings provided herein. For example, antibodies to the 8-endotoxins disclosed and claimed herein can be used to identify and isolate other 8-endotoxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the 8-endotoxins which are most constant and most distinct from other B. thuringiensis S-endotoxins. These antibodies can then be used to specifically identify equivalent 8-endotoxins with the characteristic insecticidal activity by immunoprecipitation, enzyme linked immunoassay (ELISA), or Western blotting.

A further method for identifying the 8-endotoxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a detectable label. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.
Such a probe analysis provides a rapid method for identifying formicidal 8-endotoxin genes of the subject invention.

The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.
Typical radioactive labels include 32P 1`5I, 3'S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated.
Therefore, the probes of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, by methods currently known to an ordinarily skilled artisan, and perhaps by other methods which may become known in the future.

The potential variations in the probes listed is due, in part, to the redundancy of the genetic code. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins.
Therefore different nucleotide sequences can code for a particular amino acid. Thus, the amino acid sequences of the B. thuringiensis 8-endotoxins and peptides can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein or peptide.
Accordingly, the subject invention includes such equivalent nucleotide sequences. Also, inverse or complement sequences are an aspect of the subject invention and can be readily used by a person skilled in this art. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if sucli changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a S-endotoxin encoding a gene of the invention. Such mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art.

6.0 EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

6.1 EXAMPLE 1-- CONSTRUCTION OF HYBRID B. THURINGIENSIS S-ENDOTOXINS

The B. thuringiensis shuttle vectors pEG853, pEG854, and pEG857 which are used in the present invention have been described (Baum et al., 1990). pEG857 contains the CrylAc gene cloned into pEG853 as an Sphl-BamHI DNA fragment. pEG1064 was constructed in such a way that the Kpnl site within the crylAc gene was preserved and the KpnI site in the pEG857 multiple cloning site (MCS) was eliminated. This was accomplished by sequentially subjecting pEG857 DNA to limited KpnI digestion so that only one KpnI site is cut, filling in the Kpnl 5' overhang by Klenow fragment of DNA polymerase I to create blunt DNA ends, and joining the blunt ends of DNA by T4 DNA ligase. pEG318 contains the cry1F
gene (Chambers et al., 1991) cloned into the Xhol site of pEG854 as an XhoI-SalI
DNA fragment.

pEG315 contains the cryl C gene from strain EG6346 (Chambers et al., 1991) cloned into the A'77oI-BainH1 sites of pEG854 as a Sall-13ainHl DNA fragment.

FIG. IA shows a schematic representation of the DNA encoding the complete crylAc, crylAb, cryl C, and cryl F genes contained on pEG854/pEG 1064, pEG20, pEG315, and pEG318, respectively. Unique restriction sites that were used in constructing certain hybrid genes are also shown. FIG. I B shows a schematic representation of hybrid genes pertaining to the present invention. In some cases standard PCRTM amplification with mutagenic oligonucleotide primers were used to incorporate appropriate restrictions sites into DNA
fragments used for hybrid gene construction. Certain hybrid gene constructions could not be accomplished by restriction fragment subcloning. In those instances, PCRTM
overlap extension (POE) was used to construct the desired hybrid gene (Horton Ct al., 1989). The following oligonucleotide primers (purchased from Integrated DNA Technologies Inc., Coralville, IA) were used:

Primer A: 5'-GGATAGCACTCATCAAAGGTACC-3' (SEQ ID NO:1) Primer B: 5'-GAAGATATCCAATTCGAACAGTTTCCC-3' (SEQ ID NO:2) Primer C: 5'-CATATTCTGCCTCGAGTGTTGCAGTAAC-3' (SEQ ID NO:3) Primer D: 5'-CCCGATCGGCCGCATGC-3' (SEQ ID NO:4) Primer E: 5'-CATTGGAGCTCTCCATG-3' (SEQ ID NO:5) Primer F: 5'-GCACTACGATGTATCC-3' (SEQ ID NO:6) Primer G: 5'-CATCGTAGTGCAACTCTTAC-3' (SEQ ID NO:7) Primer H: 5'-CCAAGAAAATACTAGAGCTCTTGTTAAAAAAGGTGTTCC-3' (SEQ ID NO:8) Primer I: 5'-ATTTGAGTAATACTATCC-3' (SEQ ID NO:23) Primer J: 5'-ATTACTCAA.ATACCATTGG-3' (SEQ ID NO:24) Primer K: 5'-TCGTTGCTCTGTTCCCG-3' (SEQ ID NO:31) The plasmids described in FIG. 1B containing the hybrid 8-endotoxin genes pertinent to this invention are described below. Isolation or purification of DNA
fragments generated by restriction of plasmid DNA, PCRTM amplification, or POE refers to the sequential application of agarose-TAE gel electrophoresis and use of the Geneclean Kit (Bio 101) following the manufacturer's recommendation. pEG1065 was constructed by PCRTM amplification of the cryl F DNA fragment using primer pair A and B and pEG318 as the DNA template.
The resulting PCRTM product was isolated, cut with AsuII and Kpnl, and used to replace the corresponding Asull-Kpn1 DNA fragment in pEG857. Plasmid pEG1067 was constructed using POE and DNA fragments Scrul-Kpnl oI' cry1F and AsuII-CIaI of clylAc that were isolated from pEG318 and pEG857, respectively. Tlle resulting POE product was PCRTM
arnplified with primer pair A and B, cut with Asull and Kpnl, and used to replace the corresponding Asult-KpnI fragment in pEG857.

pEG1068 was constructed by replacing the Sac1-Kpn1 DNA fragment of crylAc isolated from pEG857 with the corresponding Sacl-Kpnl DNA fragment isolated from cry1F
(pEG318). pEG1070 was constructed by replacing the Sacl-Kpnl DNA fragment isolated from pEG1065 with the corresponding Sacl-Kpnl DNA fragment isolated from crylAc (pEG857).

pEG1072 was constructed by replacing the Sacl-Kpnt DNA fragment isolated from pEG1067 with the corresponding Sac1-Kpnl DNA fragment isolated from crylAc (pEG857).
pEG1074, pEG 1076, and pEG 1077 were constructed by replacing the SphI-Xhol DNA
fragment from pEG 1064 with the PCRTM amplified Sphl-Xhot DNA fragment from pEG 1065, pEG
1067, pEG1068, respectively, using primer pairs C and D. pEG1089 was constructed by replacing the Sphl-Sacl DNA fragment of pEG1064 with the isolated and Sphl and SacI cut PCRTM
product of cryl F that was generated using primer pair D and E and the template pEG318.

pEG 1091 was constructed by replacing the Sphl-Sacl DNA fragment of pEG 1064 with the isolated and Sph1 and SacI cut PCRTM product of cryl C that was generated using primer pair D and H and the template pEG315.

pEG1088 was constructed by POE using a crylAc DNA fragment generated using primer pair B and F and a cryl C DNA fragment generated using primer pair A
and G. The Sac1-Kpnl fragment was isolated from the resulting POE product and used to replace the corresponding SacI-Kpnl fragment in pEG 1064.

pEG365 was constructed by first replacing the Sphl-Kpnl DNA fragment from pEG1065 with the corresponding crylAb DNA fragment isolated from pEG20 to give pEG364.
The Sacl-Kpnl DNA fragment from pEG364 was then replaced with the corresponding cry1F
DNA fragment isolated from pEG318.

pEG1092 was constructed by replacing the Kpnl-BamHl DNA fragment from pEG1088 with the corresponding DNA fragment isolated from pEG315. pEG1092 is distinct from the crylAb/cryl C hybrid 6-endotoxin gene disclosed in Intl. Pat. Appl. Publ. No.
WO 95/06730.

pEG1093 was constructed by replacing the S'phI-Ast.rll DNA fragment from pEG1068 with the corresponding SphI-Asull DNA fragment isolated from pEG20.

pEG378 was constructed by POE using a crylAc DNA fragment generated using primer pair B and I using pEG857 as the template and a ciyl F DNA fragment generated using primer pair A and J using pEG318 as the template. The resulting POE product was cut with AsuII and Kpnl and the resulting isolated DNA fragment used to replace the corresponding Asull-Kpnl DNA fragment in pEG 1064.

pEG381 was constructed by replacing the AsuII-XhoI DNA fragment in pEG1064 with the corresponding AsuII-Xhol DNA fragment isolated from the PCRTM
amplification of pEG378 using primer pair C and K.

6.2 EXAMPLE 2-- PRODUCTION OF THE HYBRID TOXINS IN B. THURINGIENSIS

The plasmids encoding the hybrid toxins described in Example 1 were transformed into B. thuringiensis as described (Mettus and Macaluso, 1990). The resulting B.
thuringiensis strains were grown in 50 ml of C-2 medium until the culture was fully sporulated and lysed (approximately 48 hr.). Since crystal formation is a prerequisite for efficient conunercial production of S-endotoxins in B. thuringiensis, microscopic analysis was used to identify crystals in the sporulated cultures (Table 5).

Strain Plasmid Parent 8-Endotoxins Crystal Formation EG 11060 pEG 1065 Cryl Ac + Cry1 F +
EG 11062 pEG 1067 Cryl Ac + Cryl F +
EG11063 pEG 1068 Cryl Ac + Cryl F +
EG11065 pEG1070 CrylAc + Cry1F -EG11067 pEG1072 CrylAc+Cry1F -EG 11071 pEG 1074 Cryl Ac + Cry1 F +
EG 11073 pEG 1076 Cryl Ac + Cry1 F +
EG11074 pEG1077 CrylAc + Cry1F +
EG11087 pEG1088 CrylAc + CrylC -EG11088 pEG1089 CrylF + CrylAc -EG11090 pEG1091 Cry1C + CrylAc -EG11091 pEG1092 CrylAc + CrylC +
EG11092 pEG1093 CrylAb + CrylAc + CrylF +
EG11735 pEG365 CrylAb + Cry1F + CrylAc +
EG11751 pEG378 CrylAc + CryIF +
EG11768 pEG38T- - CrylAc + CryIF +

The S-endotoxin production for some of the B. thuringiensis strains specified in Table 5 was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Baum et al., 1990. Equal volume cultures of each B. thuringiensis strain were grown in C-2 medium until fully sporulated and lysed. The cultures were centrifuged and the spore/crystal pellet was washed twice with equal volumes of distilled deionized water. The final pellet was suspended in half the culture volume of 0.005% Triton X-100 .
An equal volume of each washed culture was analyzed by SDS-PAGE as shown in FIG. 2.

The majority of hybrids involving CrylAc and Cry1F formed stable crystals in B.
thuringiensis A notable exception is EG11088 in which the active toxin fragment would be the reciprocal exchange of EG11063. Two of the three hybrids involving Cryl Ac and Cry1 C, EG11087 and EG11090, failed to produce crystal in B. thin=ingiensis even though these reciprocal hybrids mimic the activated toxin fragments of crystal-forming EG
11063 and EG 11074.

Every strain that was examined by SDS-PAGE produced some level of 6-endotoxin.
5 As expected, however, those cultures identified as crystal negative produced very little protein (e.g., lane e: EG11065, lane f: EG11067, lane j: EG11088, and lane k:
EG11090). For reference, typical yields from a crystal forming b-endotoxin is shown for Cry 1 Ac (lane a).
Several hybrid cS-endotoxins produce comparable levels of protein including EG11060 (lane b), EG11062 (lane c), EG11063 (lane d; SEQ ID NO:10), and EG11074 (lane i; SEQ ID
NO:12).

10 The data clearly show that efficient hybrid b-cndotoxin production in B.
thuringiensis is unpredictable and varies depending on the parent 8-endotoxins used to construct the hybrid.

6.3 EXAMPLE 3 -- PROTEOLYTIC PROCESSING OF THE HYBRID S-ENDOTOXINS
Proteolytic degradation of the protoxin form of the 8-endotoxin to a stable active toxin 15 occurs once b-endotoxin crystals are solubilized in the larval midgut. One measure of the potential activity of 6-endotoxins is the stability of the active S-endotoxin in a proteolytic environment. To test the proteolytic sensitivity of the hybrid b-endotoxins, solubilized toxin was subjected to trypsin digestion. The S-endotoxins were purified from sporulated B.
thuringiensis cultures and quantified as described (Chambers et al., 1991).
Exactly 250 g of 20 each hybrid S-endotoxin crystal was solubilized in 30 mM NaHCO3, 10 mM DTT
(total volume 0.5 ml). Trypsin was added to the solubilized toxin at a 1:10 ratio. At appropriate time points 50 pl aliquots were removed to 50 l Laemmli buffer, heated to 100 C for 3 min., and frozen in a dry-ice ethanol bath for subsequent analysis. The trypsin digests of the solubilized toxins were analyzed by SDS-PAGE and the amount of active 6-endotoxin at each 25 time point was quantified by densitometry. A graphic representation of the results from these studies are shown in FIG. 3.

The wild-type Cry1 Ac is rapidly processed to the active 6-endotoxin fragment that is stable for the duration of the study. The hybrid b-endotoxins from EG11063 and EG11074 are also processed to active S-endotoxin fragments which are stable for the duration of the study.

30 The processing of the EG11063 6-endotoxin occurs at a slower rate and a higher percentage of this active 6-endotoxin fragment remains at each time point. Although the hybrid b-endotoxins from EG 11060 and EG 11062 are process to active 6-endotoxin fragments, these fragments are more susceptible to further cleavage and degrade at various rates during the course of the study. The 5' exchange points between crylAc and cry1 F for the EG 11062 and 6-endotoxins result in toxins that differ by only 21 amino acid residues (see FIG. 1). However, the importance of maintaining CrylAc sequences at these positions is evident by the more rapid degradation of the EG 11062 8-endotoxin. These data demonstrate that different hybrid 8-endotoxins constructed using the same parental S-endotoxins can vary significantly in biochemical characteristics such as proteotytic stability.

6.4 EXAMPLE 4-- BIOACTIVITY OF THE HYBRID S-ENDOTOXINS

B. thuringiensis cultures expressing the desired 6-endotoxin were grown until fully sporulated and lysed and washed as described in Example 2. The 6-endotoxin levels for each culture were quantified by SDS-PAGE as described (Baum et al., 1990). In the case of bioassay screens, a single appropriate concentration of each washed S-endotoxin culture was topically applied to 32 wells containing 1.0 ml artificial diet per well (surface area of 175 mm2). A single neonate larvae was placed in each of the treated wells and the tray covered by a clear perforated mylar sheet. Larvae mortality was scored after 7 days of feeding and percent mortality expressed as the ratio of the number of dead larvae to the total number of larvae treated, 32.

In the case of LC50 determinations (S-endotoxin concentration giving 50%
mortality), S-endotoxins were purified from the B. thuringiensis cultures and quantified as described by Chambers et al. (1991). Eight concentrations of the 8-endotoxins were prepared by serial dilution in 0.005% Triton X-100 and each concentration was topically applied to wells containing 1.0 ml of artificial diet. Larvae mortality was scored after 7 days of feeding (32 larvae for each 8-endotoxin concentration). In all cases the diluent served as the control.

A comparison of the Cry 1 A/Cry 1 F hybrid toxins by bioassay screens is shown in Table 6. The hybrid 6-endotoxins from strains EGl 1063 and EGl 1074 maintain the activities of the parental CrylAc and Cry1 F 6-endotoxins. Furthermore, the hybrid 6-endotoxin from EG11735 maintains the activity of its parental CrylAb and Cry1F S-endotoxins.
The S-endotoxins produce by strains EG11061, EG11062, EG11071, and EG11073 have no insecticidal activity on the insect larvae tested despite 1) being comprised of at least one parental 6-endotoxin that is active against the indicated larvae and 2) forming stable, well-defined crystals in B. thuringiensis. These results demonstrate the unpredictable nature of hybrid toxin constructions.

For the data in Table 6. All strains were tested as washed sporulated cultures. For each insect tested, equivalent amounts of S-endotoxins were used and insecticidal activity was based on the strain showing the highest percent mortality (++++).

Strain S. frugiperda S. exigua H. virescens H. zea O. nubilalis CrylAc - - ++++ ++++ +++
Cry1 F ++++ ++ ++ ++ ++
Cryl Ab ++ + +++ ++ ++

EG11063 ++++ ++++ +++ +++ ++++

EG11074 ++++ ++++ +++ +++ ++++
EG11090 - +++ - - -EG11091 ++++ ++++ - - N.D.
EG11092 ++++ ++++ +++ +++ N.D.
EG11735 ++++ ++++ +++ +++ N.D.
EG11751 N.D.a ++++ N.D. ++++ N.D.
aN.D. = not determined.

The S-endotoxins described in FIG. 1 and that demonstrated insecticidal activity in bioassay screens were tested as purified crystals to determine their LC50 (see Table 7). The S-endotoxins purified from strains EG 11063, EG 11074, EG 11091, and EG 1173 5 all show increased arnmyworm (S: frugiperda and S. exigua) activity compared to any of the wild-type S-endotoxins tested. The EG11063 and EG11074 S-endotoxins would yield identical active toxin fragments (FIG. 1B) which is evident by their similar LC50 values on the insects examined. An unexpected result evident from these data is that a hybrid 8-endotoxin such as EG11063, EG11092, EG11074, EG11735, or EG11751 can retain the activity of their respective parental b-endotoxins, and, against certain insects such as S.
exigua, can have activity far better than either parental b-endotoxin. This broad range of insecticidal activity at doses close to or lower than the parental 6-endotoxins, along with the wild-type level of toxin production (Example 2), make these proteins particularly suitable for production in B.
thuringiensis. Although the EG11091 derived S-endotoxin has better activity against S.
frugiperda and S. exigua than its parental S-endotoxins, it has lost the H.
virescens and H. zea activity attributable to its Cry1 Ac parent. This restricted host range along with lower toxin yield observed for the EG11091 6-endotoxin (Example 2) make it less amenable to production in B. thuringiensis.

Toxin S. frugiperda S. exigua H. virescens H. zea D. nubilalis Cry 1 Ac >10000 >10000 9 100 23 CrylAb 1435 4740 118 400 17 Cry1C >10000 490 >10000 >10000 >10000 Cry1F 1027 3233 54 800 51 (Cry1Ac/1F) (Cry 1 Ac/ 1 F) EG11091 21 21 219 >10000 N.D.a (Cryl Ac/ 1 C) aN.D.=not determined.

In Table 7, the LC50 values are expressed in nanograms of purified S-endotoxin per well (175 mm) and are the composite values for 2 to 6 replications. nd = not determined.

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Table 8 describes the DNA surrounding the 5' and 3' exchange points for the hybrid S-endotoxins which are pertinent to the present invention. As evident by the SEQ ID NO, certain hybrid 6-endotoxins sliare exchange sites.

'I'o examine the effect of other small changes in the exchange site chosen for hybrid 5 endotoxin construction, the activity of EG 11751 and EG 11063 on S. exigua and H. zea were compared (Table 9). The data clearly show that hybrid 6-endotoxin improvements can be made by altering the exchange site between the two parental fi-endotoxins. In this example, the exchange site in the EG11751 6-endotoxin was moved 75 base pairs 3' compared to the EG 11063 S-endotoxin and results in improved insecticidal activity. Although no significant 10 improvement in S. exigua activity is observed between EG11063 and EG11751, a significant improvement in H. zea activity of almost 4-fold is observed for EGI 1751. It is important to note that improvements in hybrid S-endotoxin bioactivity by altering exchange sites is unpredictable. In the case of EG 11062, moving the exchange site 63 base pairs 5' of the EG 11063 exchange site abolishes insecticidal activity as shown in Table 8.

B. thuringiensis Strain LC50 Values for Washed Sporulated Cultures S. exigua H. zea To further examine the effect of changes in the exchange site for hybrid S-endotoxins, the hybrid 6-endotoxin encoded by pEG381 was compared to those encoded by pEG378 and pEG1068. In this example, the 3' exchange site for the pEG381 encoded hybrid 8-endotoxin was moved 340 base pairs 5' compared to the pEG378 hybrid 8-endotoxin. The data in Table 9 show that this change results in an increase in S. frugiperda activity compared to the pEG378 and pEG1066 encoded 8-endotoxins while maintaining the increased activity that was observed for the pEG378 encoded S-endotoxin over the pEG1068 encoded S-endotoxin (see Table 8).
This result is unexpected since the activated toxin resulting from the proteolysis of the encoded 8-endotoxins from pEG378 and pEG381 should be identical. This example further demonstrates that exchange sites within the protoxin fragment of b-endotoxins can have a profound effect on insecticidal activity.

TABI..F 10 BIOACTIVITY OF TOXINS ENCODED BY PEG378, PEG381 AND PEG1068 Plasmid LCso) Values for Purified Crystals S. frugiperda T. ni H. zea P. xylostella pEG378 464 57.7 37.5 3.02 pEG381 274 56.0 36.6 2.03 pEG 1068 476 66.7 72.7 3.83 6.5 EXAMPLE 5 -- ACTIVITY OF THE HYBRID TOXINS ON ADDITIONAL PESTS
The toxins of the present invention were also assayed against additional pests, including the southwestern corn borer and two pests active against soybean.
Toxin proteins were solubilized, added to diet and bioassayed against target pests. The hybrid toxins showed very effective control of all three pests.

LCS() AND EC5 RANGES OF H1'BRID TOXINS ON SOUTHWESTERN CORN BORERI2 EG11063 EG11074 k:G11091 EG11751 EC50 0.2-2 0.2-2 0.2-2 0.2-2 IAil values are expressed in g/ml of diet.

ZS WCB data ranges represent LC50 and EC50 ranges (as detennined by % >l st instar), respectively.

LCSp VALUES OF CHIMERIC CRYSTAL PROTEINS ON SOYBEAN PESTS~

Pest EG11063 EG11074 EG11091 EG11751 EG11768 Velvetbean caterpillar 0.9 0.6 0.3 0.1 0.06 Soybean looper 0.9 0.8 0.6 0.7 0.2 1A11 values are expressed in g/ml of diet.

2Velvetbean caterpillar (Anticarsia gemmatalis) and soybean looper (Psuedoplusi includens) are both members of the family Noctuidae.

6.6 EXAMPLE 6 -- AMINO ACID SEQUENCES OF THE NOVEL CRYSTAL PROTEINS
6.6.1 AMINO ACID SEQUENCE OF THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:10) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeu G1yGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuser GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGln TrpAspAlaPheLeuValGlnlleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis ,LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrlleAsnSerArg TyrAsnAspLeuThrArgLeulleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGlu ArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVa1 LeuAsplleValAlaLeuPheProAsnTyrAspSerArgArgTyrProlleArgThrValSerGlnLeuThr ArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu ArgSerlleArgSerProHisLeuMetAsplleLeuAsnSerlleThrlleTyrThrAspAlaHisArgGly TyrTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeu'1'yrArgArgProPheAsnlleGlylleAsnAsnGlnGlnLeuSerValLeuAsp GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis Va1SerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrpThr HisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnlleProLeuValLysAlaHis ThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAsplleLeuArgArgThrSer GlyGlyProPheAlaTyrThrlleValAsnlleAsnGlyGlnLeuProGlnArgTyrArgAlaArglleArg TyrAlaSerThrThrAsnLeuArglleTyrValThrValAlaGlyGluArgllePheAlaGlyGlnPheAsn LysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrlleAsnThrAlaPheThr PheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAsp ArgPheGluLeulleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal AsnAlaLeuPheThrSerlleAsnGlnlleGlylleLysThrAspValThrAspTyrHislleAspGlnVa1 SerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLys HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlylleAsnArgGlnLeu AspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrVa1 ThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLys AlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProlle GlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsn GluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAsp LysArgGluLysLeuGluTrpGluThrAsnlleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetlleHisAlaAlaAspLysArgVa1 HisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu LeuGluGlyArgllePheThrAlaPheSerLeuTyrAspAlaArgAsnVallleLysAsnGlyAspPheAsn AsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeu ValValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrlleLeuArg Va1ThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu LeuLysPheSerAsnCysValGluGluGlulleTyrProAsnAsnThrValThrCysAsnAspTyrThrVa1 AsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAla AspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArg G1yTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGlu Glu 6.6.2 AMINO AcID SEQUENCE OF THE EG11074 CRYS7'AL PROTEIN (SEQ ID NO:12) MetAspAsnAsnProAsnlleAsnGluCyslleProTyrAsnCysLeuSerAsnProGluValGluValLeu GlyGlyGluArgIleGluThrGlyTyrThrProlleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIlelleTrpGlyIlePheGlyProSerGln TrpAspAlaPheLeuValGlnlleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArglleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrlleAsnSerArg TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGlu ArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVa1 LeuAsplleValAlaLeuPheProAsnTyrAspSerArgArgTyrProlleArgThrValSerGlnLeuThr ArgGlulleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlylleGlu ArgSerlleArgSerProHisLeuMetAsplleLeuAsnSerlleThrlleTyrThrAspAlaHisArgGly TyrTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeuTyrArgArgProPheAsnlleGlylleAsnAsnGlnGlnLeuSerValLeuAsp G1yThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis Va1SerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrpThr HisArgSerAlaThrProThrAsnThrlleAspProGluArglleThrGlnlleProLeuValLysAlaHis ThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAsplleLeuArgArgThrSer G1yGlyProPheAlaTyrThrIleValAsnlleAsnGlyGlnLeuProGlnArgTyrArgAlaArglleArg TyrAlaSerThrThrAsnLeuArglleTyrValThrValAlaGlyGluArgllePheAlaGlyGlnPheAsn LysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrlleAsnThrAlaPheThr PheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrlleAsp ArgPheGluLeuIleProValThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaVa1 AsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsnValThrAspTyrHisIleAspGlnVa1 SerAsnLeuValThrTyrLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLys HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSerAsnPheLysAsplleAsnArgGlnPro G1uArgGlyTrpGlyGlySerThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyrVal ThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLyslleAspGluSerLysLeuLys AlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProIle G1yLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsn GluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAsp LysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetIleHisAlaAlaAspLysArgVal HisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsn 5 AsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGinArgSerValLeu Va1ValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArg Va1ThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGlulleGluAsnAsnThrAspGlu LeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrVal AsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAla 10 AspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArg GlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGlu Glu 15 6.6.3 AMINO AcID SEQUENCE OF THE EG11735 CRYSTAL PROTEIN (SEQ ID NO:14) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeu G1yGlyGluArglleGluThrGlyTyrThrProlleAsplleSerLeuSerLeuThrGlnPheLeuLeuSer GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspllelleTrpGlyllePheGlyProSerGln TrpAspAlaPheLeuValGlnIleGluGlnLeulleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla 20 IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArglleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg TyrAsnAspLeuThrArgLeulleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGlu 25 ArgValTrpGlyProAspSerArgAspTrplleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal LeuAsplleValSerLeuPheProAsnTyrAspSerArgThrTyrProIleArgThrValSerGlnLeuThr ArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu G1ySerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGly G1uTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro 30 LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeuTyrArgArgProPheAsnlleGlylleAsnAsnGlnGlnLeuSerValLeuAsp GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis Va1SerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrpThr 35 HisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGlnlleProLeuValLysAlaHis ThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAsplleLeuArgArgThrser GlyGlyProPheAlaTyrThrIleValAsnlleAsnGlyGlnLeuProGlnArgTyrArgAlaArglleArg TyrAlaSerThrThrAsnLeuArglleTyrValThrValAlaGlyGluArgllePheAlaGlyGlnPheAsn LysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrlleAsnThrAlaPheThr PheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrIleAsp ArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVa1 AsnAlaLeuPheThrSerIleAsnGlnlleGlyIleLysThrAspValThrAspTyrHisIleAspGlnVal SerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLys HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeu AspArgGlyTrpArgGlySerThrAsplleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrVa1 ThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLys AlaPheThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProlle GlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAsplleAspValGlyCysThrAspLeuAsn G1uAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAsp LysArgGluLysLeuGluTrpGluThrAsnlleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetIleHisAlaAlaAspLysArgVa1 HisSerIleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnVallleLysAsnGlyAspPheAsn AsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeu Va1ValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrlleLeuArg ValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGlulleGluAsnAsnThrAspGlu LeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrVa1 AsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAla AspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArg G1yTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGlu Glu 6.6.4 AMINO ACID SEQUENCE OF THE EG11092 CRYSTAL PROTEIN (SEQ ID NO:26) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeu GlyGlyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIleTrpGlyIlePheGlyProSerGln TrpAspAlaPheLeuValGlnIleGluGlnLeulleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrlleAsnSerArg TyrAsnAspLeuThrArgLeulleGlyAsnTyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGlu ArgValTrpGlyProAspSerArgAspTrplleArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal LeuAsplleValSerLeuPheProAsnTyrAspSerArgThrTyrProlleArgThrValSerGlnLeuThr ArgGlulleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlylleGlu ArgSerlleArgSerProHisLeuMetAsplleLeuAsnSerlleThrlleTyrThrAspAlaHisArgGly TyrTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeuTyrArgArgProPheAsnlleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp G1yThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis ValSerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrpThr HisArgSerAlaThrProThrAsnThrIleAspProGluArgIleThrGln2leProLeuValLysAlaHis ThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSer G1yGlyProPheAlaTyrThrlleValAsnlleAsnGlyGlnLeuProGlnArgTyrArgAlaArglleArg TyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsn LysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrlleAsnThrAlaPheThr PheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrlleAsp ArgPheGluLeuIleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal AsnAlaLeuPheThrSerIleAsnGlnlleGlyIleLysThrAspValThrAspTyrHisIleAspGlnVa1 SerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgG.luLeuSerGluLysValLys HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlyIleAsnArgGlnLeu AspArgGlyTrpArgGlySerThrAspIleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrVal ThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLys AlaPheThrArgTyrGlnLeuArgGlyTyrlleGluAspSerGlnAspLeuGlulleTyrLeulleArgTyr AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProlle G1yLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAsplleAspValGlyCysThrAspLeuAsn GluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAsp LysArgGluLysLeuGluTrpGluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetIleHisAlaAlaAspLysArgVa1 HisSerIleArgGluP,laTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGlu LeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsnGlyAspPheAsn AsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeu Va1ValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArg ValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGlulleGluAsnAsnThrAspGlu LeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrVa1 AsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAla AspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArg G1yTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGlu Glu 6.6.5 AMINO ACID SEQUENCE OF THE EG11751 CRYSTAL PROTEIN (SEQ ID NO:28) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeu G1yGlyGluArglleGluThrGlyTyrThrProlleAsplleSerLeuSerLeuThrGlnPheLeuLeuSer G1uPheValProGlyAlaGlyPheValLeuGlyLeuValAspllelleTrpGlyllePheGlyProSerGln TrpAspAlaPheLeuValGlnIleGluGlnLeulleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArglleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg TyrAsnAspLeuThrArgLeulleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGlu ArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVa1 LeuAsplleValAlaLeuPheProAsnTyrAspSerArgArgTyrProlleArgThrValSerGlnLeuThr ArgGluIleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlylleGlu ArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGly TyrTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeuTyrArgArgProPheAsnlleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIleArgAlaProMetPheSerTrpIle HisArgSerAlaGluPheAsnAsnllelleAlaSerAspSerIleThrGlnlleProLeuValLysAlaHis ThrLeuGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAspIleLeuArgArgThrser G1yGlyProPheAlaTyrThrIleValAsn2leAsnGlyGlnLeuProGlnArgTyrArgAlaArgIleArg TyrAlaSerThrThrAsnLeuArgIleTyrValThrValAlaGlyGluArgIlePheAlaGlyGlnPheAsn LysThrMetAspThrGlyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThr PheProMetSerGlnSerSerPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrlleAsp ArgPheGluLeulleProValThrAlaThrPheGluAlaGluTyrAspLeuGluArgAlaGlnLysAlaVal AsnAlaLeuPheThrSerIleAsnGlnlleGlyIleLysThrAspValThrAspTyrHisIleAspGlnVa1 SerAsnLeuValAspCysLeuSerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysValLys HisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheLysGlylleAsnArgGlnLeu AspArgGlyTrpArgGlySerThrAsplleThrIleGlnArgGlyAspAspValPheLysGluAsnTyrVa1 ThrLeuProGlyThrPheAspGluCysTyrProThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLys AlaPheThrArgTyrGlnLeuArgGlyTyrlleGluAspSerGlnAspLeuGlulleTyrLeulleArgTyr AsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProLeuSerAlaGlnSerProlle G1yLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCysArg AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCysThrAspLeuAsn G1uAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGlu PheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpArgAsp LysArgGluLysLeuGluTrpGluThrAsnlleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetIleHisAlaAlaAspLysArgVal HisSerlleArgGluAlaTyrLeuProGluLeuSerVallleProGlyValAsnAlaAlallePheGluGlu LeuGluGlyArgllePheThrAlaPheSerLeuTyrAspAlaArgAsnVallleLysAsnGlyAspPheAsn AsnGlyLeuSerCysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeu Va1ValProGluTrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrlleLeuArg ValThrAlaTyrLysGluGlyTyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGlu LeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsnThrValThrCysAsnAspTyrThrVal AsnGlnGluGluTyrGlyGlyAlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAla ASpTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArgArgGluAsnProCysGluPheAsnArg GlyTyrArgAspTyrThrProLeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGlu Glu 6.6.6 AMINO ACID SEQUENCE OF THE EG11091 CRYSTAL PROTEIN (SEQ ID NO:30) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeu GlyGlyGluArgIleGluThrGlyTyrThrProIleAsplleSerLeuSerLeuThrGlnPheLeuLeuSer G1uPheValProGlyAlaGlyPheValLeuGlyLeuValAspllelleTrpGlyllePheGlyProSerGln TrpAspAlaPheLeuValGlnIleGluGlnLeulleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAsp ProThrAsnProAlaLeuArgGluGluMetArglleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHis LeuSerValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAlaAlaThrlleAsnSerArg TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGlu ArgValTrpGlyProAspSerArgAspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVa1 LeuAsplleValAlaLeuPheProAsnTyrAspSerArgArgTyrProlleArgThrValSerGlnLeuThr ArgGlulleTyrThrASnProValLeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlylleGlu ArgSerlleArgSerProHisLeuMetAsp2leLeuAsnSerlleThrlleTyrThrAspAlaHisArgGly TyrTyrTyrTrpSerGlyHisGlnlleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArg ThrLeuSerSerThrLeuTyrArgArgProPheAsnlleGlylleAsnAsnGlnGlnLeuSerValLeuAsp GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyThrValAsp SerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis ValSerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrplle 5 HisArgSerAlaThrLeuThrAsnThrlleAspProGluArglleAsnGlnlleProLeuValLysGlyPhe ArgValTrpGlyGlyThrSerValIleThrGlyProGlyPheThrGlyGlyAspIleLeuArgArgAsnThr PheGlyAspPheValSerLeuGlnValAsnlleAsnSerProIleThrGlnArgTyrArgLeuArgPheArg TyrAlaSerSerArgAspAlaArgVallleValLeuThrGlyAlaAlaSerThrGlyValGlyGlyGlnVal SerValAsnMetProLeuGlnLysThrMetGlulleGlyGluAsnLeuThrSerArgThrPheArgTyrThr 10 AspPheSerAsnProPheSerPheArgAlaAsnProAspllelleGlyIleSerGluGlnProLeuPheGly AlaGlySerIleSerSerGlyGluLeuTyrlleAspLysIleGluIleIleLeuAlaAspAlaThrPheGlu AlaGluSerAspLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerSerAsnGlnlleGlyLeu LysThrAspValThrAspTyrHisIleAspGlnValSerAsnLeuValAspCysLeuSerASpGluPheCys LeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeu 15 G1nAspProAsnPheArgGlylleAsnArgGlnProAspArgGlyTrpArgGlySerThrAsplleThrlle G1nGlyGlyAspAspValPheLysGluAsnTyrValThrLeuProGlyThrValAspGluCysTyrProThr TyrLeuTyrGlnLyslleAspGluSerLysLeuLysAlaTyrThrArgTyrGluLeuArgGlyTyrlleGlu AspSerGlnAspLeuGlulleTyrLeulleArgTyrAsnAlaLysHisGlulleValAsnValProGlyThr GlySerLeuTrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsnArgCysAlaProHis 20 LeuGluTrpAsnProAspLeuAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPhe ThrLeuAspIleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLys ThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGluLysProLeuLeuGlyGluAlaLeu AlaArgValLysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGlnLeuGluThrAsnlleVal TyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGlnTyrAspArgLeuGlnValAspThr 25 AsnlleAlaMetlleHisAlaAlaAspLysArgValHisArglleArgGluAlaTyrLeuProGluLeuSer ValIleProGlyValAsnAlaAlallePheGluGluLeuGluGlyArgllePheThrAlaTyrSerLeuTyr AspAlaArgAsnValIleLysAsnGlyAspPheAsnAsnGlyLeuLeuCysTrpAsnValLysGlyHisVal AspValGluGluGlnAsnAsnHisArgSerValLeuValIleProGluTrpGluAlaGluValSerGlnGlu ValArgValCysProGlyArgGlyTyrlleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCys 30 Va1ThrlleHisGlulleGluAspASnThrAspGluLeuLysPheSerASnCysValGluGluGluValTyr ProAsnAsnThrValThrCysAsnAsnTyrThrGlyThrGlnGluGluTyrGluGlyThrTyrThrSerArg AsnGlnGlyTyrAspGluAlaTyrGlyAsnAsnProSerValProAlaAspTyrAlaSerValTyrGluGlu LysSerTyrThrAspGlyArgArgGluAsnProCysGluserAsnArgGlyTyrGlyAspTyrThrProLeu ProAlaGlyTyrValThrLysAspLeuGluTyrPheProGluThrAspLysValTrpIleGlulleGlyGlu 35 ThrGluGlyThrPheIleValAspSerValGluLeuLeuLeuMetGluGlu 6.6.7 AMINO ACID SEQUENCE OF THE EG11768 CRYS7'AL PROTEIN (SEQ ID NO:34) MetAspAsnAsnProAsnlleAsnGluCysIleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyG
lyGluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSerGluPheVa 1ProGlyAlaGlyPheValLeuGlyLeuValAspIlelleTrpGlyIlePheGlyProSerGlnTrpAspAlaPhe LeuValGlnlleGluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAlaIleSerArgLeuGluG
lyLeuSerAsnLeuTyrGlnlleTyrAlaGluSerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuAr gGluGluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsn TyrGlnValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSerVa1P
heGlyGlnArgTrpGlyPheAspAlaAlaThrlleAsnSerArgTyrAsnAspLeuThrArgLeulleGlyAsnTy rThrAspTyrAlaValArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArgAspTrpValArg TyrAsnGlnPheArgArgGluLeuThrLeuThrValLeuAsplleValAlaLeuPheProAsnTyrAspSerArgA
rgTyrProlleArgThrValSerGlnLeuThrArgGlulleTyrThrAsnProValLeuGluAsnPheAspGlySe rPheArgGlySerAlaGlnGlyIleGluArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr IleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerG
lyProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAlaGlnLeuG1 yG1nG1yValTyrArgThrLeuSerSerThrLeuTyrArgArgProPheAsnIleGlylleAsnAsnGlnGlnLeu SerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaValTyrArgLysSerGlyT
hrValAspSerLeuAspGlulleProProGlnAsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSe rHisValSerMetPheArgSerGlyPheSerAsnSerSerValSerllelleArgAlaProMetPheSerTrplle HisArgSerAlaGluPheAsnAsnllelleAlaSerAspSerlleThrGlnlleProLeuValLysAlaHisThrL
euGlnSerGlyThrThrValValArgGlyProGlyPheThrGlyGlyAsplleLeuArgArgThrSerGlyGlyPr oPheAlaTyrThrIleValAsnIleAsnGlyGlnLeuProGlnArgTyrArgAlaArglleArgTyrAlaSerThr ThrAsnLeuArglleTyrValThrValAlaGlyGluArgllePheAlaGlyGlnPheAsnLysThrMetAspThrG
lyAspProLeuThrPheGlnSerPheSerTyrAlaThrIleAsnThrAlaPheThrPheProMetSerGlnSerSe rPheThrValGlyAlaAspThrPheSerSerGlyAsnGluValTyrlleAspArgPheGluLeulleProValThr AlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAlaValAsnAlaLeuPheThrSerThrAsnGlnL
euGlyLeuLysThrAsnValThrAspTyrHislleAspGlnValSerAsnLeuValThrTyrLeuSerAspGluPh eCysLeuAspGluLysArgGluLeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsnLeuLeu G1nAspSerAsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySerThrGlyIleThrIleGlnG
lyGlyAspAspValPheLysGluAsnTyrValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTy rGlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeuArgGlyTyrlleGluAspSerGlnAsp LeuGlulleTyrLeulleArgTyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeuTrpProL
euSerAlaGlnSerProlleGlyLysCysGlyGluProAsnArgCysAlaProHisLeuGluTrpAsnProAspLe uAspCysSerCysArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAsplleAspValGlyCys ThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyA
snLeuGluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLysLysTrpAr gAspLysArgGluLysLeuGluTrpGluThrAsnlleValTyrLysGluAlaLysGluSerValAspAlaLeuPhe ValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnlleAlaMetlleHisAlaAlaAspLysArgValHisS

erlleArgGluAlaTyrLeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluG1 yArgIlePheThrAlaPheSerLeuTyrAspAlaArgAsnVallleLysAsnGlyAspPheAsnAsnGlyLeuSer CysTrpAsnValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerValLeuValValProGluTrpG
luAlaGluValSerGlnGluValArgValCysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluG1 yTyrGlyGluGlyCysValThrlleHisGluIleGluAsnAsnThrAspGluLeuLysPheSerAsnCysValGlu G1uGlulleTyrProAsnAsnThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGlyAlaTyrT
hrSerArgAsnArgGlyTyrAsnGluAlaProSerValProAlaAspTyrAlaSerValTyrGluGluLysSerTy rThrAspGlyArgArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrProLeuProValGlyTyr Va1ThrLysGluLeuGluTyrPheProGluThrAspLysValTrplleGluIleGlyGluThrGluGlyThrPheI
leValAspSerValGluLeuLeuLeuMetGluGlu 6.7 EXAMPLE 7 -- DNA SEQUENCES ENCODING THE NOVEL CRYSTAL PROTEINS
6.7.1 DNA SEQUENCE ENCODING THE EG11063 CRYSTAL PROTEIN (SEQ ID NO:9) 6.7.2 DNA SEQUENCE ENCODING THE EG11074 CRYSTAL PROTEIN (SEQ ID NO:11) 6.7.3 DNA SEQUENCE ENCODING THE EG11735 CRYSTAL PROTEIN (SEQ ID NO:13) GAT GAA TTT TGT CTG GAT GAA AAG CGA GAA TTG TCC GAG AAA. GTC AAA 2016 6.7.4 DNA SEQUENCE ENCODING THE EGII092 CRYSTAL PROTEIN (SEQ ID NO:25) 6.7.5 DNA SEQUENCE ENCODING THE EG11751 CRYSTAL PROTEIN (SEQ ID NO:27) $ GTT GTT CCG GAA TGG GAA GCA GAA GTG TCA CAA GAA GTT CGT GTC TGT 3072 6.7.6 DNA SEQUENCE ENCODING THE EG11091 CRYSTAL PROTEIN (SEQ ID NO:29) CAP. AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC 2448 6.7.7 DNA SEQUENCE ENCODING THE EG11768 CRYSTAL PROTEIN (SEQ ID NO:33) WO 98/22595 Ill PCT/US97/21587 TAC ACT TCT CGT AAT CGA GGA TAT AAC GA.P. GCT CCT TCC GTA CCA GCT 3312 6.8 EXAMPLE 8-- ISOLATION OF TRANSGENIC PLANTS RESISTANT

TO CRY* VARIANTS
6.8.1 PLANT GENE CONSTRUCTION

The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA
polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus.

This processing involves a 3' non-translated region which adds polyadenylate nucleotides to the 3' end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA
usually referred to as the "promoter". The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA
using one of the DNA strands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have been described in the literature. Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of 11~' ribulose 1,5-bisphosphatc carhoxylatie (ssRUL3lSCO, a very ahtinciant plant polypeptide), and the I~igwort Mosaic Virtts WMV) 35S promoter. All ol'these promoters have been used to create various types of I)NA constructs which have hcen expressed in plants (see e.g., U. S.
Patent No. 5,463,175).

The pa-:ict:lar promoter selected should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective aniount of protein.
One set of preferred promoters are constitutive. promoters such as the CaMV35S
or FMV35S
promoters that yield high levels ot' expression in most plant organs (U. S.
Patent No.
5,378,619). Another set of preferred promoters are root enhanced or specific promoters such as the CaMV derived 4 as-I
promoter or the wheat POXI promoter (U. S. Patent No.5,023,179, specifically incorporated herein by reference; Herti}; el al., 1991). The root enhaneed or specific promoters would be particulariv prelerred for the control of corn rootworm (Dinhrolirrrs spp.) in transgenic corn plants.
The pronioters used in the DNA constructs (i.e. chimeric plant genes) of the present invention niav be modified, if desired, to affect their control characteristics. For examplc, the CaMV35S promotcr may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of' light, to create a promoter which is active in leaves but not in roots. 1'he resulting chinieric promoter may he used as described herein. For purposes of this description, the phrase "CaMV35S" promoter thus includes variations of' CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the protnoters may be altered to contain multiple "enhancer sequences" to assist in elevating gcne espression.
The RNA produced by a DNA construct of the present invention also contains a 5' non-translated leader sequence. This seqttence can bc: derived from the promoter selected to express the gene, and can be specifically niodiGed so as to increase translation of the mRNA.
1'he 5' non-translated regions can also be obtained froni viral RNA's, from suitable eucaryotic genes, or from a synthetic gene sequence. Tlie present invention is not limited to constructs wherein the non-translated region is derived froni the 5' non-translated sequence that accompanies the promoter sequence.
For optimized expression in monocotyledenotts plants such as niaize, an intron should also be included in the DNA expression constnict. "I'his intron would typically be ---- -- ----- --It3 rlacctl near thc 5' cntl of thc mI:NA in untranslated scclucnce. 1'his intron could be obtainccl fiutn, but not limitccl tu, a set (i(' introus c(-rtsistinb of the niaizr h.c()7(1 intrc-n (U. S. l'atent Nu.
5,424,412) or'the rice Actl intron (McElroy et ul., 1990). As shown below, (hc maiic {-s/)70 intron is useful in the Prrscnt invention.
As noted above, ilic 3' non-tr;enslated region of the chlltltrlc hlant genes of thc present invention contains a polyadenylation signal which fttnctions in plants to cause the addition of adenvlate nucleotidcs to the 3' end of' tlie ItNA. t:xamples of preferrcd 3' regions are (1) the 3' transcribed, non-translated regions cemtaining ilic holyadenylate signal of Agrnhuctrrita tunior-inducing ("I'i) plasmid l;enes, such as thc nopalinc;
synthase (NOS) gene 7n(i (2) plant genes such as the pea ssRt)131SCY) C9 gene Wischhol'f et al., 1987).

6.8.2 Pt.AN'1' TRANtiF'OIZNIATION ANI) I:\1'Itl:SS10N
A chimeric trans,~enc containing a structural ruclin4, scclttcnce of tlie present invention can he inserteci into thr f;enonie of a plant by any suitable inetttod such as those detailed herein. Suitable plant transllorni;ition vectors incluLle those clcrivcd lrom a Ti plasmid of A~,~rohucteritint ttnn~Jitcicns, ati well as those discloscd, e.g., by Iferrera-Fstrella (1983), Bevan (1983), Klee (1985) and l-lur. Pat. Appl. I'ubl. No. L:I'0120516. In addition to plant transformation vectors derived from ilic Ti or root-inducint; WZi) hl;ismicls of Agrohacteritint, altcrnative niethods can be used to insert ilic DNA construct, uf this invention into plant cells.
Suclt niethods mav involve. fior example, thc usr of liposomes, elcctroporation, chenticals that increase free [)N11 uptakr, fire 1)NA dclivcry via microprojrctile bombardnient, and transfonn:ttion usinf; viruscs tir pollcn (Fromn- et ul., 1986; Armstrong c-t a/., 1990; Fromni et al., 1990).

6.8.3 CONSTIZIICI'ION 01~ I'LANT F.XPIt1:SSION Vb:C'1'Oltti FOR CR}'*
TItANS(:ENES
For efficient expression of' the crr* variants disclosed herrin in tr;tnsgenic plants, the gene encoding the variants must h:tvc a suitablc sequcnc:c cumposition (Dirltn et al., 1996).
To place a cr),* gene in a vector sttitable fior exprcssi(+n in nionocotyledonous plants (i_e. under control of ilic enh;tnec(l Cauliflower Mosaic Virus i5S promoter and link to the hsp70 intron followed by a nopaline synthase polyadenvlation sitc as in lJ. S.
I'atent No.
5,424,412), the vector is digested with II=1 appropriate enzvmes such as A4=nl anel 1:'c=~~RI. The larL;er vector band of approximately 4.6 kb is then clcctropltoresrcl, puriiir(l, ancl lir:ucLl %vith 'I'-} I)NA ligase tc) the appropriate restriction fragment containimg, the plantized c=--Y* gene. The ligation mix is thrn transforlnecl into L'. coli, carbenlctllltl resistant ciilonies rccoverc(l and plasmid C)Nn t'ecovcrecl by DNA miniprep procedures. 7'he DNA may then be ,ubjectecl to restriction endonuclease analysis with enzymes such as Nc=ul and EcoRl (together), Noil, and I'.,=11 to identify clones containing the crl'* gene coding sequence fused to the It.-=p,O intron ttnder control of tlte enhanced CaMV35S
pronioter).

I, o place the gene in a vector stlitable lbr recovery of stably transformed and insect resistant plants, thc restriction fragment from pMON33708 containing the lysinc oxidase coding sequence fused to the h.cn'D intron under control of the erihanced CaMV35S promoter may be isolated by gel electrophoresis and purification. 'fhis fragment can then be ligated with a vector such as pMON30460 treated with iVutl and calf ;ntcstinal alkaline phosphatase (pMON30460 contains the neomycin phosphotransferase coding sequence un(ler control of the CaMV35S promoter). Kanamycin resistant colonies niay then be obtaincd by transformation of this ligation mix into li. cnli and colonies containing the resulting plastnid can be identified by restriction endonuclease digestion of plasniid miniprep DNAs. Restriction enzynies such as rVnll, LcoRV, llindlll, Ncol, EcoRl, and 13zlII can be used to identify the appropriate clones containing the restriction fragment properlv inserted in the corresponding site of pMON30460, in the orientation suclt that both genes are in tandeni (i.e. the 3' end of thc c=rv* gene expression cassette is linked to the 5' end of the )1p111 c.xpression cassette).
Expression of the Cry* protein by the resulting vector is then confirmed in plant protoplasts by electroporation ol' the vector into protoplasts followed by protein blot and ELISA analysis. This vector can be introduced into the genomic DNA of' plant embryos such as niaizr bv particle gun bombardment followed by = paromomycin selection to obtain corn plants expressing the crY* gene essentially as described in U.S. Patent No. 5,424,412. In this example, the vector was introduced. via cobombttrdnient with a hygromvcin resistance conferring plasniid into immature embryo scutella (ies) of maize, followed bv hygronivcin selection, and regeneration.
transgenic coni lines expressing the cry* protein are then identified by elisa analysis. progeny seed from these evcnts are then subsequently tested for protection from susceptible insect feeding.

7.0 REFERENCES

The following references provide exemplary procedural or other details supplementary to those set forth herein.

U. S. Patent 4,196,265, issued April 1, 1980.
U. S. Patent 4,554,101, issued Nov. 19, 1985.
U. S. Patent 4,683,195, issued Jul. 28, 1987.
U. S. Patent 4,683,202, issued Jul. 28, 1987.
U. S. Patent 4,702,914, issucd Oct. 27, 1987.
U. S. Patent 4,757,011 , issued Jul. 12, 1988.
U. S. Patent 4,769,061, issued Sept. 6, 1988.
U. S. Patcnt 4,940,835, issued Jul 10, 1990.
U. S. Patent 4,965,188, issued Oct. 23, 1990.
U. S. Patent 4,971,908, issued Nov. 20, 1990.
U. S. Patent 4,987,071. issued January 22, 1991.
[J. S. Patent 5,004,863, issued Apr. 2, 1991.
U. S. Patent 5,015,580, issucd May 14, 1991.
U. S. Patent 5,023,179, issued June 11, 1991.
U. S. Patcnt 5,055;294. issued Oct. ', 1991.
U. S. Patent 5,128,130, issued Jul. 7, 1992.
U. S. Patent 5,176,995, issued Jan. 5, 1993.
U. S. Patent 5,349,124, issucd Sept. 20, 1994.
U. S. Patent 5,378,619, issued January 3, 1995.
U. S. Patcnt 5,380,831, issued Jan. 10, 1995.
U. S. Patent 5,384,253, issued Jan. 24, 1995.
U. S. Patent 5,416,102, issued May 16, 1995.
U. S. Patent 5,424,412, issued June 13, 1995.

U. S. Patent 5,441,884, issued Aug. 15, 1995.

U. S. I'atent 5,449,681, issued Sept. 12, 1995.
U. S. Patent 5,463,175, issued October 31, 1995.
U. S. Patent 5,500,365, issued Mar. 19, 1996.

U. S. Patent 5,631,359, issued May 20, 1997.
U. S. Patent 5,659,123, issued Aug. 19, 1997.
Eur. Pat. EP 0120516, issued October 3, 1984.
Eur. Pat. EP 0360257, issued March 28, 1990.

Intl. Pat. Appl. Publ. No. WO 91/10725, published Jul. 25, 1991.
Intl. Pat. Appl. Publ. No. WO 93/07278, published Apr. 15, 1993.
Intl. Pat. Appl. Publ. No. WO 95/02058, published Jan. 19, 1995.
Intl. Pat. Appl. Publ. No. WO 95/06730, published Mar. 9, 1995.
Intl. Pat. Appl. Publ. No. WO 95/30752, published Nov. 16, 1995.
Intl. Pat. Appl. Publ. No. WO 95/30753, published Nov. 16, 1995.
Intl. Pat. WO 92/07065, issued April 30, 1992, Intl. Pat. WO 93/15187, issued August 5, 1993.
Intl. Pat. WO 93/23569, issued November 25, 1993.
Intl. Pat. WO 94/02595, issued February 3, 1994.
Intl. Pat. WO 94/13688, issued June 23, 1994.

Intl. Pat.WO 91/03162, issued March 21, 1991.
Abdullah et al., Biotechnology, 4:1087, 1986.

Adami and Nevins, In: RNA Processing, Cold Spring Harbor Laboratory, p. 26, 1988.
Adang et al., In: Molecular Strategies for Crop Protection, Alan R. Liss, Inc., pp. 345-353, 1987.

Adelman et al., DNA, 2/3:183-193, 1983.

Allen and Choun, "Large unilamellar liposomes with low uptake into the reticuloendothelial system," FEBS Lett., 223:42-46, 1987.

Altschul, et al., "Basic local alignment search tool," J. Mol. Biol., 215:403-410, 1990.
Arvidson et al., Mol. Biol., 3:1533-1534, 1989.
Barton et al., Plant Physiol., 85:1103-1109, 1987.
Baum et al., Appl. Environ. Microbiol., 56:3420-3428, 1990.

Benbrook et al., In: Proceedings Bio Expo 1986, Butterworth, Stoneham, MA, pp.
27-54, 1986.

Bevan et al., Nature, 304:184, 1983.
Bolivar el al., Gene, 2:95, 1977.

Bosch et al., " Recombinant Bacillus thuringiensis Crystal Proteins with New Properties:
Possibilities for Resistance Management," Bio/Technology, 12:915-918, 1994.

Brady and Wold, In: RNA Processing, Cold Spring Harbor Laboratory, p. 224, 1988.
Brown, Nucl. Acids Res., 14(24):9549, 1986.

Bytebier et al., Proc. Natl. Acad. Sci. USA, 84:5345, 1987.
Callis et al., Genes Develop. 1:1183, 1987.

Callis, Fromm, Walbot, Genes and Develop., 1:1183-1200, 1987.

Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burden and Von Knippenberg, Eds. pp. 75-83, Elsevier, Amsterdam, 1984.

Capecchi, "High efficiency transformation by direct microinjection of DNA into cultured mammalian cells," Cell, 22(2):479-488, 1980.

Cashmore el al., Gen. Eng. ofPlants, Plenum Press, New York, 29-38, 1983.
Chambers et al., J. Bacteriol., 173:3966-3976, 1991.
Chang et al., Nature, 375:615, 1978.

Chau et al., Science, 244:174-181, 1989.

Chen et al., Nucl. Acids Res., 20:4581-9, 1992.

Clapp, "Somatic gene theraphy into hematopoietic cells. Current status and future implications," Clin. Perinatol. 20(1):155-168, 1993.
Collins and Olive, Biochem., 32:2795-2799, 1993.

Conway and Wickens, In: RNA Processing, Cold Spring Harbor Laboratory, p. 40, 1988.
Cornellssen et al., EMBO J., 5(1):37-40, 1986.

Couvreur et al., "Nanocapsules, a new lysosomotropic carrier," FEBS Lett., 84:323-326, 1977.
Couvreur, "Polyalkyleyanoacrylates as colloidal drug carriers," Crit. Rev.
Ther. Drug Carrier Syst., 5:1-20, 1988.

Crickmore et al., Abstr. 28th Annu. Meet. Soc. Invert. Pathol., Cornell University, Ithaca, NY, 1995.

Cristou et al., Plant Phvsiol, 87:671-674, 1988.

Curie], Agarwal, Wagner, Cotten, "Adenovirus enhancement of transferrin-polylysine-mediated gcne delivery," Proc. Natl. Acad Sci. USA, 88(19):8850-8854, 1991.

Curiel, Wagner, Cotten, Birnstiel, Agarwal, Li, Loechel, Hu, "High-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes," Hum. Gen. Ther., 3(2):147-154, 1992.

Daar et al., In: RNA Pi-ocessing, Cold Spring Harbor Laboratory, p. 45, 1988.
Dean et al., Nucl. Acids Res., 14(5):2229, 1986.

Dedrick ct al., J. Biol. Chem., 262(19):9098-1106, 1987.
Dhir et al., Plant Cell Reports, 10:97, 1991.

Diehn, De Rocher, Green., "Problems that can limit the expression of foreign genes in plants:
Lessons to be learned from B.t. toxin genes," In: Genetic Engineering, Vol.
18, Edited by J.K. Setlow, Plenum Press, N.Y., 1996.

Donovan et al., J. Biol. Chem., 263(1):561-567, 1988.
Doyle et al., J. Biol. Chem., 261(20):9228-9236, 1986.
Dropulic et al., J. Virol., 66:1432-41, 1992.

Eglitis and Anderson, "Retroviral vectors for introduction of genes into mammalian cells,"
Biotechniques, 6(7):608-614, 1988.

Eglitis, Kantoff, Kohn, Karson, Moen, Lothrop, Blaese, Anderson, "Retroviral-mediated gene transfer into hemopoietic cells," Adv. Exp. Med. Biol., 241:19-27, 1988.

Eichenlaub, J. Bacteriol., 138(2):559-566, 1979.
Elionor et al., Mol. Gen. Genet., 218:78-86, 1989.

Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA, 87:6743-7, 1990.
Fiers et al., Nature, 273:113, 1978.

Fischhoff et al., Bio/Technology, 5:807, 1987.

Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803, 1983.
Fraley et al., Bio/Technology, 3:629-635, 1985.

Fromm, Taylor, Walbot, Nature, 319:791-793, 1986.

Fromm, Taylor, Walbot, "Expression of genes transferred into monocot and dicot plant cells by electroporation," Proc. Natl. Acad. Sci. USA, 82(17):5824-5828, 1985.

Fromm et al., "Inheritance and expression of chimeric genes in the progeny of transgenic maize plants," Biotechnolof,ry (NY), 8(9):833-839, 1990.
Fujimura et al., 1'lant Tissue Culture Letters, 2:74, 1985.

Futterer and Hohn, "Translation in plants - rules and exceptions." Plant Mol.
Biol., 32:159-189, 1996.

Fynan, Webster, Fuller, Haynes, Santoro, Robinson, "DNA vaccines: protective immunizations by parenteral, mucosal, and gene gun inoculations," Proc. Natl. Acad. Sci.
USA, 90(24):11478-11482, 1993.

Gallego and Nadal-Ginard, ln: RNA Processing, Cold Spring Harbor Laboratory, p. 61, 1988.
Gao and Huang, Nucl. Acids Res., 21:2867-72, 1993.

Gawron-Burke and Baum, Genet. Engineer., 13:237-263, 1991.
Gefter et al., Somat. Cell Genet., 3:231-236, 1977.

Genovese and Milcarek, ln: RNA Processing, Cold Spring Harbor Laboratory, p.
62, 1988.
Gil and Proudfoot, Nature, 312:473, 1984.
Gill et al., J. Biol. Chem., 270:27277-27282, 1995.

Goding, "Monoclonal Antibodies: Principles and Practice," pp. 60-74. 2nd Edition, Academic Press, Orlando, FL, 1986.
Goeddel et al., Nature, 281:544, 1979.
Goeddel et al., Nucl. Acids Res., 8:4057, 1980.

Goodall et al., ln: RNA Processing, Cold Spring Harbor Laboratory, p. 63, 1988.

Graham and van der Eb, "Transformation of rat cells by DNA of human adenovirus 5,"
Virology, 54(2):536-539, 1973.

Green, Nucl. Acids Res. 16(1):369. 1988.
Grochulski et al., J. Mol. Biol., 254:447-464, 1995.

Gross et al., ln: RNA Processing, Cold Spring Harbor Laboratory, p. 128, 1988.
Guerrier-Takada et al., Cell, 35:849, 1983.

Hampel and Tritz, Biochem., 28:4929, 1989.
Hampel et al., Nucl. Acids Res., 18:299, 1990.

Hampson and Rottman, In: RNA Processing, Cold Spring Harbor Laboratory, p. 68, 1988.
Hanley and Schuler, Nucl. Acids Res., 16(14):7159, 1988.

Harlow and Lane, In: Antibodies: A Laborcttory Manzial, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.

Helfinan and Ricci, In: RNA Processing, Cold Spring Harbor Laboratory, p. 219, 1988.
Henry-Michelland et al., "Attachment of antibiotics to nanoparticles;
Preparation, drug-release and antimicrobial activity in vitro, Int. J. Pharm., 35:121-127, 1987.

Herrera-Estrella et al., Nature, 303:209, 1983.
Hess et al., J. Adv. Enzyme Reg., 7:149, 1968.

Hertig et al., "Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)," Plant Mol. Biol., 16(1):171-174, 1991.

Hilber, Bodmer, Smith, Koller, "Biolistic transformation of conidia of Botryotinia.fuckeliana,"
Curr. Genet., 25(2):124-127, 1994.

Hitzeman et al., J. Biol. Chem., 255:2073, 1980.

Hoekema et al., Molecular and Cellular Biology, 7:2914-2924, 1987.
H6fte and Whiteley, Microbiol. Rev., 53:242-255, 1989.
Holland et al., Biochemistry, 17:4900, 1978.
Honee et al., Mol. Microbiol., 5:2799-2806, 1991.
Honee et al., Nucl. Acids Res., 16(13), 1988.

Hoover et al., (Eds.), In: Remington's Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., Easton, PA, 1975.

Horsch et al., Science, 227:1229-1231, 1985.
Horsch et al., Science, 227:1229, 1985.
Horton et al., Gene, 77:61-68, 1989.

Humason, "Animal Tissue Techniques," W. H. Freeman & Company, New York, 1967.
lannacone, Pasquale, Grieco, Francesco, Cellini, "Specific sequence modifications of a cry3B
endotoxin gene results in high levels of expression and insect resistance,"
Plant, Mol.
Biol., 34:485-496, 1997.

Itakura et al., Science, 198:1056, 1977.

Jaeger et al., Proc. Natl. Acad. Sci. USA, 86: 7706-7710, 1989.

Jameson and Wolf, "The Antigenic Index: A Novel Algorithm for Predicting Antigenic Determinants," Compu. Appl. Biosci., 4(1):181-6, 1988.
Jarret et al., In Vitro, 17:825, 1981.

Jarret et al., Physiol. Plant, 49:177, 1980.

Johnston and Tang, "Gene gun transfection of animal cells and genetic immunization,"
Methods Cell. Biol., 43(A):353-365, 1994.
Jones, Genetics, 85:12 1977.

Jorgensen et al., Mol. Gen. Genet., 207:471, 1987.

Kaiser et al., "Amphiphilic secondary structure: design of peptide hormones,"
Science, 223(4633):249-255, 1984.

Kashani-Saber et al., Antisense Res. Dev., 2:3-15, 1992.
Kay et al., Science, 236:1299-1302, 1987.

Keller et al., EMBO J., 8:1309-14, 1989.

Kessler et al., In: RNA Processing, Cold Spring Harbor Laboratory, p. 85, 1988.
Kingsman et al., Gene, 7:141, 1979.

Klee et al., Bio/Technology, 3:637, 1985.
Klee et al., Bio/Technology, 3:637-642, 1985.
Klein et al., Nature, 327:70, 1987.

Klein et al., Proc. Natl. Acad. Sci. USA, 85:8502-8505, 1988.
Knight et al., J. Biol. Chem., 270:17765-17770, 1995.
Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.
Kohler and Milstein, Nature, 256:495-497, 1975.
Kozak, Nature, 308:241-246, 1984.

Krebbers et al., Plant Molecular Biology, 11:745-759, 1988.

Kuby, Immunology 2nd Edition, W. H. Freeman & Company, New York, 1994.

Kunkel, "Rapid and efficient site-specific mutagenesis without phenotypic selection," Proc.
Nail. Acad. Sci. US.A., 82(2):488-492, 1985.

Kunkel et al., "Rapid and efficient site-specific mutagenesis without phenotypic selection,"
Methods Enzymol, 154:367-382, 1987.

Kyte and Doolittle, "A simple method for displaying the hydropathic character of a protein," J.
Mol. Biol., 157(1):105-132, 1982.

Langridge et al., Proc. Natl. Acad. Sci. USA, 86:3219-3223, 1989.
Lee et al., Biochem. Biophys. Res. Comm., 216:306-312, 1995.
L'Huillier et al., EMBO J., 11:4411-8, 1992.

Lieber et al., Methods Enzymol., 217:47-66, 1993.

Lisziewicz et al., Proc. Natl. Acacl. Sci. U.S.A., 90:8000-4, 1993.

Lim et al., "Hunlan bcta-globin mRNAs that llarbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II
that have a cap-like structure at the 5' terniini," EMBO J., 11(9):3271-3278, 1992.
Lindstrom et al., Develop. Genet., 11:160, 1990.
Lorz et al., Mol. Gen. Genct., 199:178, 1985.

Lu, Xiao, Clapp, Li, Broxmeyer, "High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic sten-dprogenitor cells from human umbilical cord blood," J. Exp. Med. 178(6):2089-2096, 1993.
Luo et al., Plant Mol. Biol. Report, 6:165, 1988.

Maddock et al., Third Intl. Congr. Plant Mol. Biol., Abstr. No. 372, 1991.

Maloy et al., In: Microbial Genetics, 2nd Ed., Jones & Bartlett Publishers, Boston, MA, 1994.
Maniatis et al., In: Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.

Marcotte et al., Nature, 335:454, 1988.

Marrone et al., J. Econ. Entomol., 78-290-293, 1985.

Marzluff and Pandey, In: RNA Processing, Cold Spring Harbor Laboratory, p.
244, 1988.
Masson et al., J. Biol. Chem., 270:20309-20315, 1995.
McCabe et al., Biotechnology, 6:923, 1988.
McCormick et al., Plant Cell Reports, 5:81-84, 1986.
McDevitt et al., Cell, 37:993-999, 1984.

McElroy et al., "Characterization of the rice (Oryza sativa) actin gene family," Plant Mol Biol., 15(2):257-268, 1990.

Mettus and Macaluso, Appl. Environ. Microbiol., 56:1128-1134, 1990.
Murashige and Skoog, Physiol. Plant, 15:473, 1962.

Murray, Lotzer, Eberle, "Codon usage in plant genes," Nucleic Acids Research, 17:(2)477-498, 1989.

Neuhaus et al., Theor. Appl. Genet., 75:30, 1987.
Odell et al., Nature, 313:810, 1985.
Odell et al., Nature, 313:810, 1985.

Ojwang et al., Proc. Natl. Acad. Sci. USA, 89:10802-6, 1992.

Ohkawa et al., Nucl. Acids Svmp. Ser., 27:15-6, 1992.
Omirulleh et al., I'lant Mol. Biol., 21:415-428, 1993.

Pandey and Marzluff, ln: RNA Processing, Cold Spring Harbor Laboratory, p.
133, 1987.
Pieken et al., Science, 253:314, 1991.

Pena et al., Nature, 325:274, 1987.
Perrault et al, Nature, 344:565, 1990.
Poszkowski et al., EMBO J., 3:2719, 1989.
Potrykus et al., Mol. Gen. Genet., 199:183, 1985.

Poulsen et al., Mol. Gen. Genet., 205:193-200, 1986.

Prokop and Bajpai, "Recombinant DNA Technology I" Ann. N. Y. Acad. Sci., Vol.
646, 1991.
Proudfoot et al., ln: RNA Processing, Cold Spring Harbor Laboratory, p. 17, 1987.
Reines et al., J. Mol. Biol., 196:299-312, 1987.

Rogers et al., In: Methods For Plant Molecular Biology, A. Weissbach and H.
Weissbach, eds., Academic Press Inc., San Diego, CA 1988.

Rogers et al., Methods Enzymol., 153:253-277, 1987.
Rossi et al., Aid.s Res. Hum. Retrovir., 8:183, 1992.

Rouwendal, Odette Mendes, Wolbert Douwe de Boer, "Enhanced expression in tobaccoo of the gene encoding green fluorescent protein by modification of its codon usage," Plant Mol. Biol., 33:989-999, 1997.

Ruud et al., "Different Domains of Bacillus thuringiensis 8-Endotoxins Can Bind to Insect Midgut Membrane Proteins on Ligand Blots," Appl. Environ. Microbiol., 62(8):2753-2757, 1996.

Ruud et al., "Domain III Substitution in Bacillus thuringiensis Delta-Endotoxin CryIA(b) Results in Superior Toxicity for Spodoptera exigua and Altered Membrane Protein Recognition," Appl. Environ. Microbiol., 62(5):1537-1543, 1996.

Sadofsky and Alwine, Molecular and Cellular Biology, 4(8):1460-1468, 1984.

Sambrook et al., In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
Sanders et al., Nucl. Acids Res., 15(4):1543, 1987.
Saville and Collins, Cell, 61:685-696, 1990.

Saville and Collins, Proc. Natl. Acad. Sci. USA, 88:8826-8830, 1991.

Scanlon et cil., Proc. Natl. Acad. Sci. USA, 88:10591-5, 1991.
Scaringe et al., Nucl. Acids Res., 18:5433-5441, 1990.
Schnepf el al., J. Biol. Cherrt., 265:20923-20930, 1990.

Schuler et al., Nucl. Acids Res., 10(24):8225-8244, 1982.

Segal, ln: Biochemical Ccilculations, 2nd Ed., John Wiley & Sons, New York, 1976.
Shaw and Kamen, Cell, 46:659-667, 1986.

Shaw and Kamen, ln: RNA Processing, Cold Spring Harbor Laboratory, p. 220, 1987.
Simpson, Science, 233:34, 1986.

Spielmann et al., Mol. Gen. Genet., 205:34, 1986.
Spoerel, Methods Enzymol., 152:588-597, 1987.

Stemmer, "DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution," Proc. Natl. Acad. Sci. U. S. A., 91(22):10747-10751, 1994.
Stinchcomb et al., Nature, 282:39, 1979.

Taira et al., Nucl. Acids Res., 19:5125-30, 1991.
Thompson et al., Genet. Engineer., 17:99-117, 1995.
Toriyama et al., Theor. Appl. Genet., 73:16, 1986.
Trolinder and Goodin, Plant Cell Reports, 6:231-234, 1987.
Tschemper et al., Gene, 10:157, 1980.

Tsurushita and Korn, In: RNA Processing, Cold Spring Harbor Laboratory, p.
215, 1987.
Tumer et al., Nucl. Acids Reg., 14:8, 3325, 1986.

Uchimiya et al., Mol. Gen. Genet., 204:204, 1986.

Usman and Cedergren, Trends in Biochem. Sci., 17:334, 1992.
Vaeck et al., Nature, 328:33, 1987.

Van Tunen et al., EMBO J., 7:1257, 1988.

Vasil et al., "Herbicide-resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus," Biotechnology, 10:667-674, 1992.
Vasil, Biotechnology, 6:397, 1988.

Velten and Schell, Nucl. Acids Res., 13:6981-6998, 1985.
Velten et al., EMBO J., 3:2723-2730, 1984.

Ventura et al., Nucl. Acids Res., 21:3249-55, 1993.

Visser et al., Mol. Gen. Genet., 212:219-224, 1988.
Vodkin et al., Cell, 34:1023, 1983.

Vogel et al., J. Cell Biocheni., (Suppl.) 13D:312, 1989.

Wagner, Zatloukal, Cotten, Kirlappos, Mechtler. Curiel, 13irnstiel, "Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor- mediated gene delivery and expression of transfected genes," Proc. Natl. Acad. Sci., USA
89(l 3):6099-6103, 1992.
Webb et al., Plant Sci. Letters, 30:1, 1983.
Weerasinghe et al., J. Virol., 65:5531-4, 1991.

Weissbach and Weissbach, Methods for Plant Molecular Biology, (eds.). Academic Press, Inc., San Diego, CA, 1988.

Wenzler et al., Plant Mol. Biol., 12:41-50, 1989.
Wickens and Stephenson, Science, 226:1045, 1984.

Wickens et al., ln: RNA Processing, Cold Spring Harbor Laboratory, p. 9, 1987.
Wiebauer et al., Molecular and Cellular Biology, 8(5):2042-2051. 1988.

Woolf et al., Proc. Natl. Acad. Sci. USA, 89:7305-7309, 1992.

Wolf et al., "An Integrated Family of Amino Acid Sequence Analysis Programs,"
Compu.
App1. Biosci., 4(1):187-91, 1988.

Wong and Neumann, "Electric field mediated gene transfer." Biochini. Biophys.
Res.
Commun., 107(2):584-587, 1982.
Yamada et al., Plant Cell Rep., 4:85, 1986.

Yamamoto and lizuka, Archives of Biochemistry and Biophysics, 227(1):233-24I, 1983.
Yang et al., Proc. Natl. Acad. Sci., USA, 87:4144-48, 1990.

Zhou et al., Methods Enzymol., 101:433, 1983.
Zhou et al., Mol. Cell Biol., 10:4529-37, 1990.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chenlically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All sucll siniilar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ECOGEN, INC.
(B) STREET: 205 Cabot Boulevard West (C) CITY: Langhorne (D) STATE: Pennsylvania (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 19047-3023 (ii) TITLE OF INVENTION: BROAD-SPECTRUM delta-ENDOTOXINS
(iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: McFadden Fincham (B) STREET: 606-225 Metcalfe Street (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: K2P 1P9 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,272,843 (B) FILING DATE: 20-NOV-1997 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US97/21587 (B) FILING DATE: 20-NOV-1997 (C) CLASSIFICATION: C12N 15/32 (vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McFadden Fincham (B) REGISTRATION NUMBER: 3083 (C) REFERENCE/DOCKET NUMBER: 1987-132 (viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 234-1907 (B) TELEFAX: (613) 234-5233 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CCAAGAAAAT ACTAGAGCTC TTGTTAAAAP. AGGTGTTCC 39 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3531 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..3531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gin Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Vai Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Giu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr.

Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gin Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gin Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3531 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..3531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gin Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gin Leu Gly Leu Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys CAT GCG AAG.CGA CTC AGT GAT GAA CGC AAT TTA CTC CAA GAT TCA AAT 2064 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 12:

( i ) SEQUENCE CFiARP.CTERI STICS :
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu Cys.Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gin Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg G1y Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3531 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..3531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile G1y Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val , 1565 1570 1575 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Va1 Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Giy Tyr Ile Leu Arg'Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Q
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Giu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gin Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gin Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gin Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Vai Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..3531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gin Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gin Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Giy Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Vai Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Giy His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..3531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp,Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gin Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gin Ile Glu Gln Leu Ile Asn G1n Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg G1y Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3579 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

GCAGGTTCTA TTAGTAGCGG TGAACTTTAT ATAGATAAAP. TTGAAATTAT TCTAGCAGAT 1860 GCAACATTTG AAGCAGAATC TGATTTAGAA AGAGCACAAP. AGGCGGTGAA TGCCCTGTTT 1920 -- -- -------- ---(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1193 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GAACAGTTAA TTAACCAAAG AATAGAAGAA TTCGCTAGGA ACCAAGCCP.T TTCTAGATTA 300 AACAGTATAA CCATCTATAC GGATGCTCAT AGGGGTTATT ATTATTGGTC AGGGC,ATCAA 960 ATGGGAAATG CAGCTCCACA ACAACGTATT GTTGCTC,AAC TAGGTCAGGG CGTGTATAGA 1080 CGACGAAC,AA GTGGAGGACC ATTTGCTTAT ACTATTGTTA ATATAAATGG GCAATTACCC 1560 CTGGATGAAP. AGCGAGAATT GTCCGAGAAA GTCAAACATG CGAAGCGACT CAGTGATGAA 2040 TCAAAATTAA AAGCCTTTAC CCGTTATCAA TTAAGAGGGT ATATCGAAGA TAGTC,AAGAC 2280 (2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1177 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Vai Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Vai His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val Va1 Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Giu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu (2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Claims (50)

CLAIMS:

1. An isolated nucleic acid segment comprising a gene which encodes a polypeptide having the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34

2. The nucleic acid segment of claim 1, wherein said gene encodes a polypeptide having insecticidal activity against Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, Helicoverpa zea or Ostrinia nubilalis.

3. The nucleic acid segment of claim 1 or 2, wherein said nucleic acid segment is isolatable from Bacillus thuringiensis NRRL B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, or NRRL B-21781.

4. The nucleic acid segment of any one of claims 1 to 3, wherein said nucleic acid segment specifically hybridizes to a nucleic acid sequence of a complement of SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:33.

5. The nucleic acid segment of any one of claims 1 to 4, wherein said nucleic acid segment comprises the nucleic acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:33.

6. The nucleic acid segment of any one of claims 1 to 5, further defined as a DNA segment.

7. The nucleic acid segment of any one of claims 1 to 6, wherein said gene is operably linked to a promoter.

8. The nucleic acid segment of any one of claims 1 to 7, comprised within a recombinant vector.

9. The nucleic acid segment of any one of claims 1 to 8, comprised within a plasmid, cosmid, phage, phagemid, viral, baculovirus, bacterial artificial chromosome, or yeast artificial chromosome recombinant vector.

10. The nucleic acid segment of any one of claims 1 to 9, comprised within the plasmid vector pEG1068, pEG1077, pEG1093, pEG365, pEG378, or pEG381.

11. A nucleic acid segment in accordance with any one of claims 1 to 10 for use in a recombinant expression method to prepare a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:10, 12, 14, 26, 28 or 34.

12. A nucleic acid segment in accordance with any one of claims 1 to 10 for use in the preparation of an insect-resistant transgenic plant.

13. A method of using a nucleic acid segment in accordance with any one of claims 1 to 10, comprising expressing said gene in a host cell and collecting the expressed polypeptide.

14. Use of a nucleic acid segment in accordance with any one of claims 1 to 10 in the preparation of a recombinant polypeptide composition comprising the amino acid sequence of SEQ ID NO:10, 12, 14, 26, 28 or 34.

15. Use of a nucleic acid segment in accordance with any one of claims 1 to 10 in the generation of a vector for use in producing an insect-resistant transgenic plant.

16. Use of a nucleic acid segment in accordance with any one of claims 1 to 10 in the generation of an insect-resistant transgenic plant.

17. A host cell comprising a nucleic acid segment in accordance with any one of claims 1 to 10.

18. The host cell of claim 17, wherein said host cell is a bacterial cell.

19. The host cell of claim 17 or 18, wherein said cell is an E. coli, B.
thuringiensis, B. subtilis, B. megaterium, or a Pseudomonas spp. cell.

20. The host cell of any one of claims 17 to 19, wherein said cell is B.
thuringiensis EG11063, EG11074, EG11092, EG11735, EG11751, or EG11768, deposited under NRRL B-21579, NRRL B-21580, NRRL B-21635, NRRL B-21581, NRRL B-21636, or NRRL B-21781, respectively.

21. The host cell of claim 17, wherein said cell is a eukaryotic cell.

22. The host cell of claim 21, wherein said host cell is a plant cell.

23. The host cell of claim 21 or 22, wherein said cell is a grain, tree, vegetable, fruit, berry, nut, grass, cactus, succulent, or ornamental plant cell.

24. The host cell of any one of claims 21 to 23, wherein said cell is a corn, rice, tobacco, potato, tomato, flax, canola, sunflower, cotton, wheat, oat, barley, soybean or rye cell.

25. The host cell of any one of claims 22 to 24, wherein said cell is comprised within a transgenic plant.

26. The host cell of any one of claims 21 to 25, wherein said cell produces a polypeptide having insecticidal activity against Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, Helicoverpa zea or Ostrinia nubilalis, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:10, 12, 14, 26, 28 or 34.

27. A host cell in accordance with any one of claims 17 to 26 for use in the expression of a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:10, 12, 14, 26, 28 or 34.

28. A host cell in accordance with any one of claims 22 to 26 for use in the preparation of a transgenic plant.

29. Use of a host cell in accordance with any one of claims 22 to 26 in transforming plant cells.

30. Use of a host cell in accordance with any one of claims 17 to 26, in the preparation of an insecticidal polypeptide formulation comprising the amino acid sequence of SEQ
ID
NO:10, 12, 14, 26, 28 or 34.

31. A composition comprising an isolated polypeptide that comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34, and an agriculturally-acceptable carrier.

32. The composition of claim 31, wherein said polypeptide is insecticidally active against Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, Helicoverpa zea, or Ostrinia nubilalis.

33. The composition of claim 31 or 32, wherein said polypeptide is isolatable from Bacillus thuringiensis EG11063, EG11074, EG11092, EG11735, EG11751, or EG11768, deposited under NRRL B-21579, NRRL B-21580, NRRL B-21635, NRRL B-21581, NRRL B-21636, or NRRL B-21781, respectively.

34. The composition of any one of claims 31 to 33, wherein said polypeptide comprises from about 0.5% to about 99% by weight of said composition.

35. The composition of any one of claims 31 to 34, wherein said polypeptide comprises from about 50% to about 99% by weight of said composition.

36. A composition comprising a polypeptide and an agriculturally-acceptable carrier, wherein said polypeptide is preparable by a process comprising the steps of:

(a) culturing a B. thuringiensis EG11063, EG11074, EG11092, EG11735, EG11751 or EG11768, deposited under NRRL B-21579, NRRL B-21580, NRRL B-21635, NRRL B-21581, NRRL B-21636, or NRRL B-21781, respectively, cell under conditions effective to produce a composition comprising a B. thuringiensis polypeptide of SEQ ID
NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34; and (b) obtaining said polypeptide from said cell.

37. A composition in accordance with any one of claims 31 to 36 for use in killing an insect cell.

38. Use of a composition in accordance with any one of claims 31 to 36 in the preparation of an insecticidal formulation.

39. Use of a composition in accordance with any one of claims 31 to 36 in the preparation of a plant protective spray formulation.

40. A method of preparing a B. thuringiensis crystal protein comprising:
(a) culturing a B. thuringiensis EG11063, EG11074, EG11092, EG11735, EG11751 or EG11768, deposited under NRRL B-21579, NRRL B-21580, NRRL B-21635, NRRL B-21581, NRRL B-21636, or NRRL B-21781, respectively, cell under conditions effective to produce a B. thuringiensis crystal protein of SEQ ID NO:10, SEQ ID NO:12, SEQ
ID
NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34; and (b) obtaining said B. thuringiensis crystal protein from said cell.

41. A method of killing an insect cell, comprising providing to an insect cell an insecticidally-effective amount of a composition in accordance with any one of claims 31 to 36.

42. The method of claim 41, wherein said insect cell is comprised within an insect.

43. The method of claim 42, wherein said insect ingests said composition by ingesting a plant coated with said composition.

44. The method of claim 42 or 43, wherein said insect ingests said composition by ingesting a transgenic plant which expresses said composition.

45. A purified antibody generated by using a polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34 as an immunogen.

46. The antibody of claim 45, operatively attached to a detectable label.

47. An immunodetection kit comprising, in suitable container means, an antibody according to claim 45, and an immunodetection reagent.

48. A method for detecting an insecticidal polypeptide in a biological sample comprising contacting a biological sample suspected of containing said insecticidal polypeptide with an antibody in accordance with claim 45, under conditions effective to allow the formation of immunecomplexes, and detecting the immunecomplexes so formed.

49. A method for producing an insect resistant transgenic plant, comprising the steps of:
(a) transforming a plant cell with a nucleic acid segment encoding an insecticidal polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34, wherein the nucleic acid segment is operably linked to a promoter; and (b) generating from the plant cell a transgenic plant that comprises the nucleic acid segment.

50. A method for producing an insect resistant plant, comprising the steps of:
(a) crossing an insect resistant plant, comprising a nucleic acid sequence encoding an insecticidal polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:34, with another plant;
(b) obtaining at least one progeny plant derived from the cross of (a); and (c) selecting progeny that is insect resistant and comprises the nucleic acid sequence encoding an insecticidal polypeptide comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, or SEQ
ID NO:34.