CA2209659C - Insecticidal protein toxins from photorhabdus - Google Patents

Abstract

Proteins from the genus Photorhabdus are toxic to insects upon exposure.
Photorhabdus luminescens (formerly Xenorhabdus luminescens) have been found in mammalian clinical samples and as a bacterial symbiont of entomopathogenic nematodes of genus Heterorhabditis. These protein toxins can be applied to, or genetically engineered into, insect larvae food and plants for insect control.

Description

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2 PCT/US96/18003 INSECTICIDAL PROTEIN TOXINS FROM PHOTORHABDUS

, Field of the Invention The present invention relates to toxins isolated from bacteria and the use of said toxins as insecticides.
Background of the Invention Many insects are widely regarded as pests to homeowners, to picnickers, to gardeners, and to farmers and others whose investments in agricultural products are often destroyed or diminished as a result of insect damage to field crops.
Particularly in areas where the growing season is short, significant insect damage can mean the loss of all profits to growers and a dramatic decrease in crop yield. Scarce supply of particular agricultural products invariably results in higher costs to food processors and, then, to the ultimate consumers of food plants and products derived from those plants.
Preventing insect damage to crops and flowers and eliminating the nuisance of insect pests have typically relied on strong organic pesticides and insecticides with broad toxicities.
These synthetic products have come under attack by the general population as being too harsh on the environment and on those exposed to such agents. Similarly in non-agricultural settings, homeowners would be satisfied to have insects avoid their homes or outdoor meals without needing to kill the insects.

SUBSTITUTE SHEET (RULE 26) The extensive use of chemical insecticides has raised environmental and health concerns for farmers, companies that produce the insecticides, government agencies, public interest groups, and the public in general. The development of less intrusive pest management strategies has been spurred along both by societal concern for the environment and by the development of biological tools which exploit mechanisms of insect management.
Biological control agents present a promising alternative to chemical insecticides.
Organisms at every evolutionary development level have devised means to enhance their own success and survival.= The use of biological molecules as tools of defense and aggression is known throughout the animal and plant kingdoms. In addition, the relatively new tools of the genetic engineer allow modifications to biological insecticides to accomplish particular solutions to particular problems.
One such agent, Bacillus thuringiensis (Bt), is an effective insecticidal agent, and is widely commercially used as such. In fact, the insecticidal agent of the Bt bacterium is a protein which has such limited toxicity, it can be used on human food crops on the day of harvest. To non-targeted organisms, the Bt toxin'is a digestible non-toxic protein.
Another known class of biological insect control agents are certain genera of nematodes known to be vectors of transmission for insect-killing bacterial symbionts. Nematodes containing insecticidal bacteria invade insect larvae. The bacteria then kill the larvae. The nematodes reproduce in the larval cadaver.
The nematode progeny then eat the cadaver from within. The bacteria-containing nematode progeny thus produced can then invade additional larvae.
In the past, insecticidal nematodes in the Steinernema and Heterorhabditis genera were used as insect control agents.
Apparently, each genus of nematode hosts a particular species of bacterium. In nematodes of the Heterorhabditis genus, the symbiotic bacterium is Photorhabdus luminescens.
Although these nematodes are effective insect control agents, it is presently difficult, expensive, and inefficient to produce, maintain, and distribute nematodes for insect control.
It has been known in the art that one may isolate an insecticidal toxin from Photorhabdus luminescens that has SUBSTITUTE SHEET (RULE 26) activity only when injected into Lepidopteran and Coleopteran insect larvae. This has made it impossible to effectively exploit the insecticidal properties of the nematode or its bacterial symbiont. What would be useful would be a more practical, less labor-intensive wide-area delivery method of an insecticidal toxin which would retain its biological properties after delivery. It would be quite desirous to discover toxins with oral activity produced by the genus Photorhabdus. The isolation and use of these toxins are desirous due to efficacious reasons. Until applicants' discoveries, these toxins had not been isolated or characterized.

Summary of the Invention The native toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus, of interest are the proteins produced by the species Photorhabdus luminescens. The protein complexes, with a molecular size of approximately 1,000 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon exposure administration to a number of insects.

The present invention provides an easily administered insecticidal protein as well as the expression of toxin in a heterologous system.

The present invention also provides a method for delivering insecticidal toxins that are functional active and effective against many orders of insects.

3 In one aspect, the invention provides a polynucleotide that is operably associated with a heterologous promoter, wherein said polynucleotide encodes a protein that has toxin activity against an insect pest wherein a nucleotide molecule that codes for said protein maintains hybridization with the complement of a nucleic acid sequence after hybridization and wash wherein said hybridization is conducted at 60 C in solution containing 10% w/v PEG (polyethylene glycol, M.W. approximately 8000), 0.6X SSC, 7% w/v SDS, 10 mM
sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA, said wash is conducted in 0.25X SSC and 0.2% SDS at 60 C, and said nucleic acid sequence is selected from the group consisting of SEQ ID N0:11 and SEQ ID NO:46.

In another aspect, the invention provides a polynucleotide that encodes a protein having toxin activity against an insect pest wherein said protein comprises the amino acid sequence of SEQ ID NO:12, and SEQ ID NO:47, wherein said polynucleotide is operably associated with a heterologous promoter.

In another aspect, the invention provides a polynucleotide comprising a nucleotide sequence that encodes the protein of SEQ ID NO: 12 operably associated with a heterologous promoter.

In another aspect, the invention provides a polynucleotide comprising a nucleotide sequence that encodes the protein of SEQ ID NO:47 operably associated with a heterologous promoter.

In another aspect, the invention provides a transgenic plant cell comprising a polynucleotide as described above.

3a In another aspect, the invention provides a recombinant protein having toxin activity against an insect pest wherein a nucleotide sequence that codes for said protein maintains hybridization with a nucleic acid sequence under hybridization and wash wherein said hybridization is conducted at 60 C in solution containing 10% w/v PEG (polyethylene glycol, M.W. approximately 8000), 0.6X SSC, 7% w/v SDS, 10 mM
sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA, wherein said wash is conducted in 0.25X SSC

and 0.2% SDS at 60 C and wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO:11 and SEQ ID
NO:46.

In another aspect, the invention provides a recombinant protein having toxin activity against an insect pest wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID
NO:47.

In another aspect, the invention provides a method of controlling an insect pest wherein said method comprises feeding a protein as described above to said pest.

In another aspect, the invention provides a substantially pure culture of a Photorhabdus strain of ATCC
553597.

Objects, advantages, and features of the present invention will become apparent from the following specification.

Brief Description of the Drawings Fig. 1 is an illustration of a match of cloned DNA
isolates used as a part of sequence genes for the toxin of the present invention.

3b Fig. 2 is a map of three plasmids used in the sequencing process.

3c Fig. 3 is a map 11ustrating che inter-relationship of s-everal partial DNA fragments.
Fig. 4 is an illustration of a hcmology analysis becweeii the protein sequences of TcbAii and TcaBii proteins.
Fig. 5 is a phenogram of Photorhabdus strains. Relaticnship of Phctorhabdus Strains was defined by rep-PCR.
The upper axis of Fig. 5 measures the percentage similaricy of strains based on scoring of rep-PCR products (i.e., 0.0 (no similarity] to 1.0 (100% similarity)). At the right axis, ttie numbers and letters indicate the various strains tested; 14=W-14, Hm=Hm, H9=H9, 7=vdX-7, 1=WX-1, 2=WX-2, 88=HP88, NC-1=NC-1, 4=-WX-4, 9=WX-9, B=Wx-8, 10=Wx-10, WIR=WIR, 3=wX-3, 11=Wx-11, 5=W"/,-5, 6=WX-6, 12=WX-12, x14=14X-14, 15=WX-15, Hb=Hb, B2=B2, 48 through 52=ATCC 43948 through ATCC 43952. Vertical lines separating horizontal lines indicate the degree of relatedness (as read from the extrapolated intersection of the vertical line with the upper axis) between strains or groups of strains at the base of the horizontal lines (e.g., strain W-14 is approximately 60% si:nilar to strains H9 and Hm).
Fig. 6 is an illustration of the genomic maps of the W-14 Strain.

Detailed DescriDtion of the Invention The present inventions are directed to the discovery cF- a unique class of insecticidal protein toxins from the genus Phocorhabdus that have oral toxicity against insects. A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabdicis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbicl.
27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (Rockville, MD) ATCC #s 43948, 43949, 43950, 43951, and 43952.
It is possible that other sources could harbor.
Photorhabdus bacteria that produce insecticidal toxins. Suciz sources in the environment could be either terrestrial or aQuatlc based.

-4-The genus =hecor' abdus is taxonomically def ined as a mecrl :~L
of che Family .'ncereoaccar=aceae, although it has cercain tra=c=
acypical of this family. For example, scrains of this genus are nitrate reduction negacive, yellow and red pigmenc producing anc bioluminescenc. This latter trait is otherwise unknown within the =ncerooaccer:aceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on DPtA-DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus; or absence (Xenorhabdus) of cacalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Phocorhabdus;
Heterorhabd=cidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Phocorhabdus from Xenorhabdus.
In order to establish that the strain collection disclosed herein was comprised of Photorhabdus straiiis, the strains were characterized based on reccgnized traits which define Photorhabdus and differentiate it from other cnterobacceriaceae and Xenorhabdus species. (Farmer, 1984 Bergey's Manual of Systemic Bacteriology Vol. 1 pp.510-511; Akhurst and Boemare 1988, J. Gen. Microbiol. 134 pp.1835-1845; Boemare et al. 1993 Int. J.
Syst. Bacteriol. 43 pp.249-255.
The traits studied were the following: gram stain negative rods, organism size, colony pigmentation, inclusion bodies, presence of catalase, ability to reduce nitrace, bioluminescence, dye uptake, gelatin hydrolysis, growth on selective media, growth temperature, survival under anerobic conditions and motility. Fatty acid analysis was used to confirm that the strains herein all belong to the single genus Photorhabdus.
Currently, the bacterial genus Photorhabdus is comprised of a single defined species, Photorhabdus luminescens (ATCC T'ype strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A
variety of related strains have been described in the literacure (e.g. Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845;
Boemare et al. 1993 Izt. J. Syst. Bacteriol. 43 pp. 249-255; Pucz ec al. 1990, appl. Environ. Microbiol. , 56, 181-186) Numerous

-5-Photorhabdus strains have been characterized herein. Such strains are listed in Table 18 in the Examples. Because there is currently only one species (luminescens) defined within the genus Photorhabdus, the luminescens species traits were used to characterize the strains herein. As can be seen in Fig. 5, these ~
strains are quite diverse. It is not unforeseen that in the future there may be other Photorhabdus species that will have some of the attributes of the luminescens species as well as some different characteristics that are presently not defined as a trait of Photorhabdus luminescens. However, the scope of the invention herein is to any Photorhabdus species or strains which produce proteins that have functional activity as insect control agents, regardless of other traits and characteristics.
Furthermore, as is demonstrated herein, the bacteria of the genus Photorhabdus produce proteins that have functional activity as defined herein. Of particular interest are proteins produced by the species Photorhabdus luminescens. The inventions herein should in no way be limited to the strains which are disclosed herein. These strains illustrate for the first time that proteins produced by diverse isolates of Photorhabdus are toxic upon exposure to insects. Thus, included within the inventions described herein are the strains specified herein and any mutants thereof, as well as any strains or species of the genus Photorhabdus that have the functional activity described herein.
There are several terms that are used herein that have a particular meaning and are as follows:

By "functional activity" it is meant herein that the protein toxins function as insect control agents in that the proteins are orally active, or have a toxic effect, or are able to disrupt or deter feeding, which may or may not cause death of the insect.
when an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression, formulated protein compositions(s), sprayable protein composition(s), a bait matrix or other delivery system, the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.

-6-SUBSTITUTE SHEET (RULE 26) '29355-1 The protein toxins discussed herein are typically referred to as "insecticides". By insecticides it is meant herein that the protein toxins have a "functional activity" as further defined herein and are used as insect control agents.

By the use of the term "oligonucleotides" it is meant a macromolecule consisting of a short chain of nucleotides of either RNA or DNA. Such length could be at least one nucleotide, but typically are in the range of about 10 to about 12 nucleotides. The determination of the length of the oligonucleotide is well within the skill of an artisan and should not be a limitation herein. Therefore, oligonucleotides may be less than 10 or greater than 12.

By the use of the term "toxic" or "toxicity" as used herein it is meant that the toxins produced by Photorhabdus have "functional activity" as defined herein.

By the use of the term "genetic material" herein, it is meant to include all genes, nucleic acid, DNA and RNA.

By use of the term "Photorhabdus toxin" it is meant any protein produced by a Photorhabdus microorganism strain which has functional activity against insects, where the Photorhabdus toxin could be formulated as a sprayable composition, expressed by a transgenic plant, formulated as a bait matrix, delivered via a Baculovirus, or delivered by any other applicable host or delivery system.

Fermentation broths from selected strains reported in Table 18 were used to determine the following: breadth of insecticidal toxin production by the Photorhabdus genus, the insecticidal spectrum of these toxins, and to provide source material to purify the toxin complexes. The strains characterized herein have been shown to have oral toxicity against a variety of insect orders. Such insect orders include

7 but are not limited to Coleoptera, Homopotera, Lepidoptera, Diptera, Acarina, Hymenoptera and Dictyoptera.

As with other bacterial toxins, the rate of mutation of the bacteria in a population causes many related toxins slightly different in sequence to exist. Toxins of interest here are those which produce protein complexes toxic to a variety of insects upon exposure, as described herein.
Preferably, the toxins are active against Lepidoptera, Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina. The inventions herein are intended to capture the protein toxins homologous to protein toxins produced by the strains herein and any derivative 7a strains chereof, as we?1 as any protein toxins produced by Phocorhabdus. These homologous oroceins may differ in sequence, but do not differ in function from those toxins described her=in.
Homologous toxins are meant to include protein complexes oE
between 300 kDa to 2,000 kDa and are comprised of ac least t,=.
(2) subunits, where a subunit is a peptide which may or may n~t be the same as the ocher subunit. Various protein subunits have been identified and are taught in the Examples herein.
Typically, the protein subunits are between about 18 kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80 kDa.
As discussed above, some Photorhabdus strains can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the phylum Nemacoda, have evolved an ability to exploit insect larvae as a favored growth environment. The insect larvae provide a source of food for growing nematodes and an environment in which to reproduce. One dramatic effect that follows invasion of larvae by certain nematodes is larval death.
Larval death results from the presence of, in certain nematodes, bacteria that produce an insecticidal toxin which arrests lar-,a1 growth and inhibits feeding activity.
Interestingly, it appears that each genus of insect parasitic nematode hosts a particular species of bacterium, uniquely adapted for symbiotic growth with that nematode. In the interim since this research was initiated, the name of the bacterial genus Xenorhabdus was reclassified into the Xenorhabdus and the Photorhabdus. Bacteria of the genus Photorhabdus are characterized as being symbionts of Keterorhabditus nematodes while Xenorhabdus species are symbionts of the Steinernema species. This change in nomenclature is reflected in this specification, but in no way should a change in nomenclature alter the scope of the inventions described herein.
The peptides and genes that are disclosed herein are named according to the guidelines recently published in the Journai of Bacteriology "Instructions to Authors" p. i-xii (Jan. 1996).
The following peptides and genes were isolated from Photorhabdus strain W-14.

-8-Peptide / Gene Nomenclature Toxin complex (Tc) Peptide Gene Patent Name Name Sequence ID#
tca genomic region TcaA tcaA 12 TcaAiii tcaA 4 TcaBi tcaB 3 (19, 20) TcaBii tcaB 5 TcaC tcaC 2 tcb genomic region TcbA tcbA 16 TcbAi tcbA (pro-peptide) TcbAii tcbA 1 (21, 22, 23, 24) TcbAiii tcbA 40 tcc genomic region TccA tccA 8 TccB tccB 7 tcd genomic region TcdAi tcdA (pro-peptide) TcdAii tcdA 13, (38, 39 17, 18) TcdAiii tcdA 41, (42, 43) TcdB tcdB 14 (bracket sequence indicates internal amino acid sequence obtained by tryptic digests) The sequences listed above are grouped by genomic region.
The tcbA gene was expressed in E. coli as two protein fragments TcbA and TcbAiii as illustrated in the Examples. It may be beneficial to have proteolytic clippage of some sequences to obtain the higher activity of the toxins for commercial transgenic applications.

The toxins described herein are quite unique in that the toxins have functional activity, which is key to developing an insect management strategy. In developing an insect management strategy, it is possible to delay or circumvent the protein degradation process by injecting a protein directly into an organism, avoiding its digestive tract. In such cases, the protein administered to the organism will retain its function until it is denatured, non-specifically degraded, or eliminated by the immune system in higher organisms. Injection into insects

-9-SUBSTITUTE SHEET (RULE 26) of an insecticidal toxin has potential application only in the laboratory, and then only on large insects which are easily injected. The observation that the insecticidal protein toxins herein described exhibits their toxic activity after oral ingestion or contact with the toxins permits the development of an insect management plan based solely on the ability to incorporate the protein toxins into the insect diet. Such a plan could result in the production of insect baits.
The Photorhabdus toxins may be administered to insects in a purified form. The toxins may also be delivered in amounts from about 1 to about 100 mg / liter of broth. This may vary upon formulation condition, conditions of the inoculum source, techniques for isolation of the toxin, and the like. The toxins may be administered as an exudate secretion or cellular protein originally expressed in a heterologous prokaryotic or eukaryotic host. Bacteria are typically the hosts in which proteins are expressed. Eukaryotic hosts could include but are not limited to plants, insects and yeast. Alternatively, the toxins may be produced in bacteria or transgenic plants in the field or in the insect by a baculovirus vector. Typically the toxins will be introduced to the insect by incorporating one or more of the toxins into the insects' feed.
Complete lethality to feeding insects is useful but is not required to achieve useful toxicity. If the insects avoid the toxin or cease feeding, that avoidance will be useful in some applications, even if the effects are sublethal. For example, if insect resistant transgenic crop plants are desired, a reluctance of insects to feed on the plants is as useful as lethal toxicity to the insects since the ultimate objective is protection of the plants rather than killing the insect.
There are many other ways in which toxins can be incorporated into an insect's diet. As an example, it is possible to adulterate the larval food source with the toxic protein by spraying the food with a protein solution, as disclosed herein. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in culture, and either applied to the food source or allowed to reside in the soil in an area in which insect eradication was desirable. Also, the protein could be genetically engineered directly into an insect food source. For

-10-SUBST(TUTE SHEET (RULE 26) 2 y-3 5 5- 1 CA 02209659 2001-01-19 instance, the major food source of many insect larvae is plant material.
By incorporacing genetic material that encodes the insecticidal propercies of the Photorhabdus toxins into che genome of a plant eaten by a particular insect pest, the adult or larvae would die after consuming the food plant. Numerous members of the mcnocotyledonous and dictyledenous genera have been transforrned. Transgenic agronmonic crops as well as fruics and vegetables are of commercial interest. Such crops include buc are not limited to maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. Several techniques exist for introducing foreign genetic material into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleracion of genetic material coated onto mic:oparticles directly into cells(U.S. Patents 4,945,050 to Cornell and 5,141,131 to DowElanco). Plants may be transformed using Agrobacceriuun technology, see U.S. Patent 5,177;010 to University of Toledo, 5,104,310 to Texas A&M, European Yatent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Patents 5,149,645, 5,469',976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, 7-uropean Patent Applications 116718, 290799, 320500 all to MaxPlanck, European Patent Applications 604662 and 627752 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Patent 5,231,019 all to Ciba Geigy, U.S. Patents 5,463,174 and 4,762,785 both to Calgene, and U.S. Patents 5,004,863 and 5,159,135 both to Agracetus. Other transformation technology includes whiskers technology, see U.S. Patents 5,302,523 and 5,464,765 both to Zeneca. Electroporation technology has also been used to transform plants, see WO
87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb, W09209696 and W09321335 both to PGS.

In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the like. Almost all plant tissues may

-11-be tr3nsfor:rle=.i dur:na jed1LL -rent_atlon using 3ppropr_-a_a c-'?,=h~.iques wichin che skill of an artisan.
Anoche= variable is che choice of a seleccable marker. The preference for a particular marker is at the discretion ot the artisan, buc any of che following seleccable markers may be used along wich any ocher gene noc listed herein which could flrnction as a selectable marker. Such seleccable markers include but are noc limited co aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiocics kanamycin, neomycin and G418, as well as those genes which code for resistance or tolerance to glyphosace; hygromycin;
methotrexate; phosphinothricin (bialophos); imidazolincnes, sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron; bromoxynil, dalapon and the like.
In addition to a seleccable marker, it may be desirous co use a reporter gene. In some instances a reporcer gene may be used without a selectable marker. Reporter genes are genes which are cypically noc presenc or expressed in the recipient organism or tissue. The reporter gene typically encodes for a protein which provides fcr some phenotypic change or enzymacic property.
Examples c'_ such genes are provided in K. Weising et a!. Ann.
Rev. Genetics, 22, 421 (1988), A preferred reporter gene is the glucuronidase (GUS) gene.
Regardless of transformation technique, the gene is preferably incorporated inco a gene transfer vector adapted to express the Phocorhabdus toxins in the planc cell by including in the vector a plant promoter. in addition to plant promoters, promoters from a variety of sources can be used efficiencly in plant cells to express foreign genes. For example. promocers of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promocer;
promoters of viral origin, such as the cauliflower mosaic =:irus (35S and 19S)and the like may be used. Plant promocers incl+lde, but are not limiced to ribulose-l,6-bisphosphate (RUEP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters. Prcmoters may also contain certain enhancer se-uence elemencs that may improve the cranscription efficiency.
?ypical enhancers include but are noc limiced to Adh-intron I and

-12-Adh-intron 6. Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP) and these promoters may also be used.
Promoters may also be are active during a certain stage of the plants' development as well as active in plant tissues and organs. Examples of such promoters include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoters and the like.

Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expression of genes in response to a specific signal, such as:
physical stimulus (heat shock genes); light (RUBP carboxylase);
hormone (Em); metabolites; and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known to the art.

In addition, it is known that to obtain high expression of bacterial genes in plants it is preferred to reengineer the bacterial genes so that they are more efficiently expressed in the cytoplasm of plants. Maize is one such plant where it is preferred to reengineer the bacterial gene(s) prior to transformation to increase the expression level of the toxin in the plant. One reason for the reengineering is the very low G+C
content of the native bacterial gene(s) (and consequent skewing towards high A+T content). This results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of the gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability.
Therefore, one goal in the design of reengineered bacterial

-13-SUBSTiTUTE SHEET (RULE 26) gene(s), more oreferably referred to as plant optimized gene!s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of plant genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) is to generate a DNA sequence that not only has a higher G+C content, but by modifying the sequence changes, should be made so as to not hinder translation.

An example of a plant that has a high G+C content is maize.
The table below illustrates how high the G+C content is in maize.
As in maize, it is thought that G+C content in other plants is also high.
Table I
Compilation of G+C contents of protein coding regions of maize genes Protein Classa Range %G+C Mean %G+C
Metabolic Enzymes (40) 44.4-75.3 59.0 (8.0) Storage Proteins Group I (23) 46.0-51.9 48.1 (1.3) Group II (13) 60.4-74.3 67.5 (3.2) Group I + II (36) 46.0-74.3 55.1 (9.6) c Structural Proteins (18) 48.6-70.5 63.6 (6.7) Regulatory Proteins (5) 57.2-68.9 62.0 (4.9) Uncharacterized Proteins (9) 41.5-70.3 64.3 (7.2) All Proteins (108) 44.4-75.3 60.8 (5.2) a Number of genes in class given in parentheses.
b Standard deviations given in parentheses.
~ Combined groups mean ignored in calculation of overall mean.

For the data in Table 1, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVectorT"" program (IBI, New Haven, CT). Intron sequences were ignored in the

-14-SUBSTITUTE SHEET (RULE 20 -alculations. Group I and II storage protein gene sequences were distinguished by their marked difference in base composition.

Due to the plasticity afforded by the redundancy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes of different organisms or classes or organisms has resulted in differential usage of redundant codons. This "codon bias" is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of redundant codons, whereas those having higher G+C contents utilize codons having G or C in the third position. It is thought that the presence of "minor" codons within a gene's mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mR1VAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by the synthesis of low levels of the encoded protein.

In order to reengineer the bacterial gene(s), the codon bias of the plant is determined. The codon bias is the statistical codon distribution that the plant uses for coding its proteins.
After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. The amino acid sequence of the protein of interest is reverse translated so that the resulting nucleic acid sequence codes for the same protein as the native bacterial gene, but the resulting nucleic acid sequence corresponds to the first preferred codons of the desired plant.
The new sequence is analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice preferred codons. other sites in the sequence which could affect the transcription or translation of the gene of interest are the exon:intron 5' or 3' junctions, poly A addition signals, or RNA polymerase termination signals. The sequence is -15-SUBSTITUTE SHEET (RULE 26) _293SS-1 f,lrt!ier anal~ zed snd modified to reduce the Ereq'1P-ncy oE T. ,r doublets. Ln addit:.on co che doublets, G or C sequence b!ocks thaC have more than about four residues thac are the same can affect transcripCion of the sequence. Therefore, these blocks are 3lso modified by replacing the codons of Eirst or second choice, etc. with the next oreferred codon of choice. It is preferred that the plant ootimized gene(s) contains abouc 630 oE
firsC choice codons, between about 22t co about 37% second choice codons, and between 15o and 01 third choice codons, wherein the lU Cocal percentage is 1001. Most preferred the plant optimized gene(s) contain about 63% of first choice codons, at le3st about 221 second choice codons, about 7.5e third choice codons, and about 7.5% fourth choice codons, wherein the total percentage i.3 100:. The method described above enables one skilled in the 3rt to modify gene(s) ttlat are foreign to a particular plant so that the genes are optimally expressed in plants. The mec'-cd is Eurcher illustraCed in International PublicaLion W097/13402.

Thus, in order to design plant optimized gene(s) the amino acid sequence of the toxins are reverse translated into a DNA
sequence, utilizing a nonredundant genetic code established E=om a codon bias table compiled for the gene DNA sequence Ear the particular plant being transformed. The resulting DNR sequence, 2:5 which is complecely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mPSIA.
It is theorized that bacterial genes may be more easily expressed in plants if the bacterial genes are expressed in the plastids. Thus, it may be possible to express bacterial genes in plants, without=optimizing the genes Eor plant expression, and obtain high express of the protein. See U.S. Patent Clos.
4,762,785; 5,451,513 and 5,545,817.

-15-~ne of the issues regarding commercial exploiting transgenic plants is resistance management. This is of particular concern with Bacillus thuringiensis toxins. There are numerous companies commerically exploiting Bacillus thuringiensis and there has been much concern about Bt toxins becoming resistant. One strataegy =
for insect resistant management would be to combine the toxins produced by Photorhabdus with toxins such as Bt, vegetative insect proteins (Ciba Geigy) or other toxins. The combinations could be formulated for a sprayable application or could be molecular combinations. Plants could be transformed with Photorhabdus genes that produce insect toxins and other insect toxin genes such as Bt as with other insect toxin genes such as Bt.

European Patent Application 0400246A1 describes transformation of 2 Bt in a plant, which could be any 2 genes.
Another way to produce a transgenic plant that contains more than one insect resistant gene would be to produce two plants, with each plant containing an insect resistant gene. These plants would be backcrossed using traditional plant breeding techniques to produce a plant containing more than one insect resistant gene.
In addition to producing a transformed plant containing plant optimized gene(s), there are other delivery systems where it may be desirable to reengineer the bacterial gene(s). Along the same lines, a genetically engineered, easily isolated protein toxin fusing together both a molecule attractive to insects as a food source and the insecticidal activity of the toxin may be engineered and expressed in bacteria or in eukaryotic cells using standard, well-known techniques. After purification in the laboratory such a toxic agent with "built-in" bait could be packaged inside standard insect trap housings.
Another delivery scheme is the incorporation of the genetic material of toxins into a baculovirus vector. Baculoviruses infect particular insect hosts, including those desirably targeted with the Photorhabdus toxins. Infectious baculovirus harboring an expression construct for the Photorhabdus toxins could be introduced into areas of insect infestation to thereby intoxicate or poison infected insects.

SUBSTITUTE SHEET (RULE 26) Transfer of the insecticidal properties requires nucleic acid sequences encoding the coding the amino acid sequences for the Photorhabdus toxins integrated into a protein expression vector appropriate to the host in which the vector will reside.
One way to obtain a nucleic acid sequence encoding a protein with insecticidal properties is to isolate the native genetic material which produces the toxins from Photorhabdus, using information deduced from the toxin's amino acid sequence, large portions of which are set forth below. As described below, methods of purifying the proteins responsible for toxin activity are also disclosed.
Using N-terminal amino acid sequence data, such as set forth below, one can construct oligonucleotides complementary to all, or a section of, the DNA bases that encode the first amino acids of the toxin. These oligonucleotides can be radiolabeled and used as molecular probes to isolate the genetic material from a genomic genetic library built from genetic material isolated from strains of Photorhabdus. The genetic library can be cloned in plasmid, cosmid, phage or phagemid vectors. The library could be transformed into Escherichia coli and screened for toxin production by the transformed cells using antibodies raised against the toxin or direct assays for insect toxicity.
This approach requires the production of a battery of oligonucleotides, since the degenerate genetic code allows an amino acid to be encoded in the DNA by any of several three-nucleotide combinations. For example, the amino acid arginine can be encoded by nucleic acid triplets CGA, CGC, CGG, CGT, AGA, and AGG. Since one cannot predict which triplet is used at those positions in the toxin gene, one must prepare oligonucleotides with each potential triplet represented. More than one DNA
molecule corresponding to a protein subunit may be necessary to construct a sufficient number of oligonucleotide probes to recover all of the protein subunits necessary to achieve oral toxicity.
From the amino acid sequence of the purified protein, genetic materials responsible for the production of toxins can readily be isolated and cloned, in whole or in part, into an expression vector using any of several techniques well-known to one skilled in the art of molecular biology. A typical expression vector is a DNA plasmid, though other transfer means SUBSTITUTE SHEET (RULE 26) including, but not limited to, cosmids, phagemids and phage are also envisioned. In addition to features required or desired for plasmid replication, such as an origin of replication and antibiotic resistance or other form of a selectable marker such as the bar gene of Streptomyces hygroscopicus or viridochromogenes, protein expression vectors normally additionally require an expression cassette which incorporates the cis-acting sequences necessary for transcription and translation of the gene of interest. The cis-acting sequences required for expression in prokaryotes differ from those required in eukaryotes and plants.
A eukaryotic expression cassette requires a transcriptional promoter upstream (5') to the gene of interest, a transcriptional termination region such as a poly-A addition site, and a ribosome binding site upstream of the gene of interest's first codon. In bacterial cells, a useful transcriptional promoter that could be included in the vector is the T7 RNA Polymerase-binding promoter.
Promoters, as previously described herein, are known to efficiently promote transcription of mRNA. Also upstream from the gene of interest the vector may include a nucleotide sequence encoding a signal sequence known to direct a covalently linked protein to a particular compartment of the host cells such as the cell surface.
Insect viruses, or baculoviruses, are known to infect and adversely affect certain insects. The affect of the viruses on insects is slow, and viruses do not stop the feeding of insects.
Thus viruses are not viewed as being useful as insect pest control agents. Combining the Photorhabdus toxins genes into a baculovirus vector could provide an efficient way of transmitting the toxins while increasing the lethality of the virus. In addition, since different baculoviruses are specific to different insects, it may be possible to use a particular toxin to selectively target particularly damaging insect pests. A
particularly useful vector for the toxins genes is the nuclear polyhedrosis virus. Transfer vectors using this virus have been described and are now the vectors of choice for transferring foreign genes into insects. The virus-toxin gene recombinant may be constructed in an orally transmissible form. Baculoviruses normally infect insect victims through the mid-gut intestinal mucosa. The toxin gene inserted behind a strong viral coat SUBSTITUTE SHEET (RULE 26) procein promocer would be expressed and should rapidly kill the infected insect.
In addition to an insect virus or baculovirus or transgenic plant delivery syscem for che protein toxins of the presenc invention, the proteins may be encapsulated using Baci:lus _huringiensis encapsulacion technology such as but not limited to U.S. Patent rJos. 4,695,455; 4,695,462; 4,861,595, Another delivery system for the protein toxins of the present invention is formulation of the protein into a bait matrix, which could then be used in above and below ground insect bait stations. Examples of such technology include but are not limited to PCT Patent Application WO
93/23998.
As is described above, it might become necessary to modify the sequence encoding the protein when expressing it in a non-native hosc, since the codon preferences of other hoscs may differ from that of Phocorhabdus. In such a case, translation may be quite inefficient in a new host unless compensating modifications to the coding sequence are made. Additionally, modifications to the amino acid sequence might be desirable to avoid inhibitory cross-reactivity with proteins of the new host, or to refine the insecticidal properties of the protein in the new host. A genetically modified toxin gene might encode a toxin exhibiting, for example, enhanced or reduced toxicity, altered insect resistance develcpment, altered stability, or modified target species specificity.
In addition to the Photorhabdus genes encoding the toxins, the scope of the present invention is intended to include relaced nucleic acid sequences which encode amino acid biopolymers homologous to the toxin proteins and which retain the toxic effect of the Photorhabdus proteins in insect species after oral ingestion.
For instance, the toxins used in the present invention seem to first inhibit larval feeding before death ensues. By manipulating the nucleic acid sequence of Photorhabdus toxins or its controlling sequences, genetic engineers placing the toxin gene into plants could modulace ics potency or its mode of action to, for example, keep the eating-inhibito ry activity while eliminating the absolute toxicity to the larvae. This change could permit the transformed planc to survive until harvest without having the unnecessarily dramatic effect on the ecosystem of wiping out all target insects. All such modifications of the gene encoding the toxin, or of the protein encoded by the gene, are envisioned to fall within the scope of the present invention.

Other envisioned modifications of the nucleic acid include the addition of targeting sequences to direct the toxin to particular parts of the insect larvae for improving its efficiency.

Strains ATCC 55397, 43948, 43949, 43950, 43951, 43952 have been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA. Amino acid and nucleotide sequence data for the W-14 native toxin (ATCC 55397)* is presented below. Isolation of the genomic DNA
for the toxins from the bacterial hosts is also exemplified herein.

Standard and molecular biology techniques were followed and taught in the specification herein. Additional information may be found in Sambrook, J., Fritsch, E.F, and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press.

*Xenorhabdus luminescens, W-14 was deposited on March 5, 1993 in the American Type Culture Collection under accession number 55397.

The following abbreviations are used throughout the Examples: Tris = tris (hydroxymethyl) amino methane; SDS =
sodium dodecyl sulfate; EDTA = ethylenediaminetetraacetic acid, IPTG = isopropylthio-B-galactoside, X-gal = 5-bromo-4-chloro-3-indoyl-B-D-galactoside, CTAB = cetyltrimethylammonium bromide;
kbp = kilobase pairs; dATP, dCTP, dGTP, dTTP, I = 2'-deoxy-nucleoside 5'-triphosphates of adenine, cytosine, guanine, thymine, and inosine, respectively; ATP = adenosine 5' triphosphate.

Example 1 Purification of toxin from P. luminescens and Demonstration of toxicity after oral delivery of purified toxin The insecticidal protein toxin of the present invention was purified from P. luminescens strain W-14, ATCC
Accession Number 55397. Stock cultures of P. luminescens were maintained on petri dishes containing 2% Proteose Peptone No. 3 (i.e., PP3, Difco Laboratories, Detroit MI) in 1.5% agar, incubated at 25 C and transferred weekly. Colonies of the primary form of the bacteria were inoculated into 200 ml of PP3 broth supplemented with 0.5%

21a polyoxyethylene sorbitan mono-stearate (Tween 60, Sigma Chemical Company=, St. Louis t4O) in a one liter flask. The broth cultures were grown for 72 hours at 30 C on a rotary shaker. The toxin proteins can be recovered from cuitures grown in the presence or absence of Tween; however, the absence of Tween can affect the form of the bacteria grow-n and the profile of proteins produced by the bacteria. In the absence of Tween;' a variant shift occurs insofar as the molecular weight of at least one identified toxin subunit shifts from about 200 kDa to about 185 kDa.
The 72 hour cultures were centri'uged at 10,000 x g for 30 minutes to remove cells and debris. The supernatant fraction that contained the insecticidal activ4-=y was decanted and brought to 50 mM K2HPO4 by adding an appropriate volume of 1.0 M KtHPO,.
The pH was adjusted to 8.6 by adding potassium hydroxide. This supernatant fraction was then mixed with DEAE-Sephacei (Pharmacia LKB Biotechnology) which had been equilibrated with 50 mM K2HPO..
The toxic activity was adsorbed to the DEAE resin. This mixture was then poured into a 2.6 x 40 cm column and washed with 50 mM
K,HPO4 at room temperature at a flow rate of 30 ml/hr until the effluent reached a steady baseline W absorbance at 280 nm. The column was then washed with 150 mM KC1 until the effluent again reached a steady 280 nm baseline. Finally the column was washed with 300 mM KC1 and fractions were collected.
Fractions containing the toxin were pooled and filter sterilized using a 0.2 micron pore membrane filter. The toxin was then concentrated and equilibrated to 100 mM KPO., pH 6.9, using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa at 4 C (Centriprep 100, Amicon Division-W.R. Grace and Company). A 3 ml sample of the toxin concentrate was applied to the top of a 2.6 x 95 cm Sephacryl S-400 HR gel filtration column (Pharmacia LKB Biotechnology) . The eluent buffer was 100 mM KPO4, pH 6.9, which was run at a flow rate of 17 ml/hr, at 4 C. The effluent was monitored at 280 nm.
Fractions were collected and tested for toxic activity.
Toxicity of chromatographic fractions was examined in a biological assay using Manduca sexta larvae. Fractions were either applied directly onto the insect diet (Gypsy moth wheat germ diet, ICN Biochemicals Division - ICN Biomedicals, Inc.) or administered by intrahemocelic injection of a 5 ul sample through the first proleg of 4th or 5th instar larva using a 30 gauge *Trade-mark needle. The weighc of each larva wichin a creatment group was recorded ac 24 hour intervals. Toxicicy was presumed if the insect ceased feeding and died within several days of consuming treated insect diet or if death occurred within 24 hours after injection of a fraction.
The toxic fractions were pooled and concentrated using the Cencriprep-100 and were then analyzed by HPLC using a 7.5 mm x 60 cm TSK-GEL * G-4000 SW gel permeacion column with 100 mM potassium phosphate, pH 6.9 eluent buffer running at 0.4 ml/min. This analysis revealed the toxin protein to be contained within a single sharp peak that eluted from the column with a retention time of approximately 33.6 minutes. This retention time corresponded to an estimated molecular weight of 1,000 kDa. Peak fractions were collected for further purification while fractions not containing this protein were discarded. The peak eluted from the HPLC absorbs W lighc at 218 and 280 nm but did not absorb at 405 nm. Absorbance at 405 nm was shown to be an attribute of xenorhabdin antibiotic compounds.
Electrophoresis of the pooled peak fractions in a non-denaturing agarose gel (Metaphor*Agarose, b'MC BioProducts) showed that two protein complexes are present in the peak. The peak material, buffered in 50 mM Tris-HC1, pH 7.0, was separated on a 1.5% agarose stacking gel buffered with 100 mM Tris-HC1 at pH 7.0 and 1.9% agarose resolving gel buffered with 200 mM Tris-borate ac pH 8.3 under standard buffer conditions (anode buffer 1M Tris-HCI, pH 8.3; cathode buffer 0.025 M Tris, 0.192 M glycine). The gels were run at 13 mA constant current at 15 C until the phenol red tracking dye reached the end of the gel. Two protein bands were visualized in the agarose gels using Coomassie brilliant blue staining.
The slower migrating band was referred to as 'protein band 1" and faster migrating band was referred to as protein band 2."
The two protein bands were present in approximately equal amounts. The Coomassie stained agarose gels were used as a guide to precisely e:ccise the two protein bands from unstained portions of the gels. The excised pieces containing the protein bands were macerated and a small amounc of sterile water was added. As a control, a portion of the gel that contained no procein was also excised and treated in the same manner as the gel pieces concaining the protein. Protein was recovered from the gel *Trade-mark pieces by electroelution into 100 mM Tris-borate pH 3.3, ar_ iOn volts (constant voltage) for two hours. Alternatively, protein was passively eluted frorn the gel pieces by adding an equal volume of 50 mM Tris-HC1, pH 7.0, to the gel pieces, then incubating at 30 C for 16 hours. This allowed the protein to diffuse from the gel into the buffer, which was then collected.
Results of insect toxicity tests using HPLC-purified toxin (33.6 min. peak) and agarose gel purified toxin demonstrated toxicity of the extracts. Injection of 1.5 ug of the HPLC
purified protein kills within 24 hours. Both protein bands 1 and 2, recovered from agarose gels by passive elution or electroelution, were lethal upon injection. The protein concentration estimated for these samples was less than 50 ng/larva. A comparison of the weight gain and the mortality between the groups of larvae injected with protein bands 1 cr 2 indicate that protein band 1 was more toxic by injection delivery.
When HPLC-purified toxin was applied to larval diet at a concentration of 7.5 jig/larva, it caused a halt in larval weight gain (24 larvae tested). The larvae begin to feed, but after consuming only a very small portion of the toxin treated diet they began to show pathological symptoms induced by the toxii: and the larvae cease feeding. The insect frass became discolorei and most larva showed signs of diarrhea. Significant insect mortality resulted when several 5 tig toxin doses were applied to the diet over a 7-10 day period.
Agarose-separated protein band 1 significantly inhibited larval weight gain at a dose of 200 ng/larva. Larvae fed similar concentrations of protein band 2 were not inhibited and gained weight at the same rate as the control larvae. Twelve larva~
were fed eluted protein and 45 larvae were fed protein-containing agarose pieces. These two sets of data indicate that proteiii band 1 was orally toxic to Manduca sexta. In this experimenT: it appeared that protein band 2 was not toxic to Manduca sexta.
Further analysis of protein bands 1 and 2 by SDS-PAGE urider denaturing conditions showed that each band was composed of several smaller protein subunits. Proteins were visualized k;y Coomassie brilliant blue staining followed by silver staining to achieve maximum sensitivity.

SUBSTITUTE SHEET (RULE 26) The protein subunits in the two bands were very similar.
Protein band i contains 8 protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical profile except that the 25.1, 60.8, and 65.6 kDa proteins were not present. The 56.2, 60.8, 65.6, and 184 kDa proteins were present in the complex of protein band 1 at approximately equal concentrations and represent 80% or more of the total protein content of that complex.
The native HPLC-purified toxin was further characterized as follows. The toxin was heat labile in that after being heated to 60 C for 15 minutes it lost its ability to kill or to inhibit weight gain when injected or fed to M. sexta larvae. Assays were designed to detect lipase, type C phospholipase, nuclease or red blood cell hemolysis activities and were performed with purified toxin. None of these activities were present. Antibiotic zone inhibition assays were also done and the purified toxin failed to inhibit growth of Gram-negative or -positive bacteria, yeast or filamentous fungi, indicating that the toxic is not a xenorhabdin antibiotic.
The native HPLC-purified toxin was tested for ability to kill insects other than Manduca sexta. Table 2 lists insects killed by the HPLC-purified P. luminescens toxin in this study.

Table 2 Insects Killed by P: luminescens Toxin Genus and Route of Common Name Order species Delivery Tobacco Lepidoptera Manduca sexta Oral and horn worm injected Mealworm Coleoptera Tenebrio molitor Oral Pharaoh ant Hymenoptera Monomorium pharoanis Oral German Dictyoptera BZattel.la gerrnanica Oral and cockroach injected Mosquito Diptera Aedes aegypti Oral SUBSTITUTE SHEET (RULE 26) Examcle 2 Insecticide Utilitv The Phccorhabdus lurninescens utility and toxicity were further characterized. Phocorhabdus Iuminescens (strain W-1,+;
culture broch was produced as follows. The production medium was 295 Bacto Proteose Peptone Number 3 (PP3, Difco Laboratories, Detroit, Michigan) in Milli-Q deionized water. Seed culture flasks consisted of 175 ml medium placed in a 500 ml tribaffied flask with a Delong neck, covered with a Kaput and autoclaved for 20 minutes, T=250 F. Producticn flasks consisted of 500 inls in a 2.8 liter 500 mi tribaffled flask with a Delong neck, covered by a Shin-etsu silicon foam closure. These were autoclaved for 45 minutes, T=250'F. The seed culture was incubated at 28"C at 150 rpm in a gyrotory shaking incubator .:ith a 2 inch throw. After 16 hours of growth, 1% of the seed cul.ture was placed in the production flask which was allowed to grow for 24 hours before harvest. Production of the toxin appears to be during log phase growth. The microbial broth was transferred to a IL centrifuge bortle and the cellular biomass was pelleted i30 minutes at 2500 RPM at 4'C, (R.C.F. =-16001 HG-4L Rotor RC3 Sorval centrifuge, Dupont, Wilmington, Delaware). The primary broth was chilled at 4*C for 8 - 15 hours and recentrifuged at least 2 hours (conditions above) to further clarify the brotti by removal of a putative mucopolysaccharide which precipitated upon standing. (An alternative processing method combined both :;teps and involved the use of a 16 hour clarification centrifugatiori, same conditions as above.) This broth was then stored at 4C
prior to bioassay or filtration.
Photorhabdus culture broth and protein toxin(s) purified from this broth showed activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects.
More specifically, the activity is seen against corn rootworni (larvae and adult), Colorado potato beetle, and turf grubs, whicb.
are members of the insect order Coleoptera. Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and weevils. Activity has also been observed against aster leafhopper, which is a member of the order, Homopcera.
Other members of the Homopcera include planthoppers, pear pysila, apple sucker, scale insects, whiteflies, and spittle bugs, as *Trade-mark -26-well as numerous hosc specific aphid species. The broth and purified fractions are also accive againsc beet armyworm, cabbage looper, black cutworm, tobacco budworm, European corn borer, corn earworm, and codling moch, which are members of the order I.epidoptera. Other typical members of this order are clothes moch, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworn, bagworm, Eastern tent caterpillar, sod webworm, and fall armyworm. Activity is also seen against fruitfly and mosquito larvae, which are members of the order Diptera. Other members of the order Dipcera are pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly, house fly, and various mosquito species. Activity is seen against carpenter ant and Argentine ant, which are members of the order that also includes fire ants, oderous house ants, and little black ants.
The broth/fraction is useful for reducing populations of insects and were used in a mechod of inhibiting an insect pepulation. The method may comprise applying to a locus of the insect an effective insecc inactivating amount of the active described. Results are reported in Table 3.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth (filter sterilized, cell-free) or purified HPLC fractions were applied directly to the surface (-1.5 cm2) of 0.25 ml of artificial diet in 30 l aliquots following dilucion in control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworin, SCR) hatched from sterilized eggs, with second instar SCR grown on artificial diet or with second instar Diabrotica virgifera virgifera (Western corn rootworm, WCR) reared on corn seedlings gros,m in Metromix . Second instar larvae were weighed prior to addition to the diet. The plates were sealed, placed in a humidified growth chamber and maintained at 27 C for the appropriate period (4 days for neonate and adult SCR, 2-5 days for WCR larvae, 7-14 days for second instar SCR). Mortality and weight determinations were scored as indicated. Generally, 16 inseccs per treatment were used in all scudies. Control mortalities were as follows: neonate larvae, <5%, adult beetles, 5s. .
*Trade-mark Activity against Colorado potato beetle was tested as follows. Photorhabdus culture broth or control medium was applied to the surface (-2.0 cm2) of 1.5 ml of standard artificial diet held in the wells of a 24-well tissue culture plate. Each well received 50 l of treatment and was allowed to air dry.
Individual second instar Colorado potato beetle (Leptinotarsa decemlineata, CPB) larvae were then placed onto the diet and mortality was scored after 4 days. Ten larvae per treatment were used in all studies. Control mortality was 3.3%.
Activity against Japanese beetle grubs and beetles was tested as follows. Turf grubs (Popillia japonica, 2-3rd instar) were collected from infested lawns and maintained in the laboratory in soil/peat mixture with carrot slices added as additional diet. Turf beetles were pheromone-trapped locally and maintained in the laboratory in plastic containers with maple leaves as food. Following application of undiluted Photorhabdus culture broth or control medium to corn rootworm artificial diet (30 .l/1.54 cm2, beetles) or carrot slices (larvae), both stages were placed singly in a diet well and observed for any mortality and feeding. In both cases there was a clear reduction in the amount of feeding (and feces production) observed.
Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well contained 200 l of aqueous solution (Photorhabdus culture broth, control medium or H20) and approximately 20, 1-day old larvae (Aedes aegypti). There were 6 wells per treatment. The results were read at 2 hours after infestation and did not change over the three day observation period. No control mortality was seen.
Activity against fruitflies was tested as follows.
Purchased Drosophila melanogaster medium was prepared using 50%
dry medium and a 50% liquid of either water, control medium or Photorhabdus culture broth. This was accomplished by placing 8.0 ml of dry medium in each of 3 rearing vials per treatment and adding 8.0 ml of the appropriate liquid. Ten late instar Drosophila melanogaster maggots were then added to each vial.
The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 3, 7 and 10 days of exposure. Incorporation of Photorhabdus culture broth into the diet media for fruitfly SUBSTITUTE SHEET (RULE 26) maggots caused a slight (17%) buc significant reduction in day-10 adult emergence as compared to wacer and control medium !30 reduction).
Activity against aster leafhopper was tested as follows.
The ingestion assay for aster leafhopper (Macrosceles severini) is designed to allow ingestion of the active without other excernal contac--. The reservoir for the active/'food solution is made by making 2 holes in the center of the bottom portion of a 35 x 10 mm Petri dish. A 2 inch ParafileM square is placed across the top of the dish and secured with an "O" ring. A I oz.
plastic cup is then infested with approximately 7 leafhoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes.
in tescs using undiluted Photorhabdus culture broth, the broth and concrol medium were dialyzed against water to reduce control mortality. Morcalicy is reported at day 2 where 26.5% concrol mortality was seen. In the tests using purified fractions (200 mg protein/ml ) a final concentration of 5% sucrose was used in all treatments to improve surrivability of the aster leafhoppers.
The assay was held in an incubator at 28 C, 70% RH with a 16/8 photoperiod. The assay was graded for mortality at 72 hours.
Control mortality was 5.5%.
Activity against Argentine ants was tested as follows. A
1.5 ml aliquot of 100% Photorhabdus culture broth, control medium or water was pipetted into 2.0 ml clear glass vials. The vials were plugged with a piece of cotton dental wick that was moistened with the appropriate treatment. Each vial was placed into a separate 60x16mm Petri dish with 8 to 12 adult Argentine ants (Linepithema humile). There were three replicates per treatment. Bioassay plates were held on a laboratory bench, at room temperature under fluorescent ceiling lights. Mortality readings were made after 5 days of exposure. Control mortality was 24%.
Activity against carpenter ant was tested as follows. Black carpenter ant workers (Camponocus pennsylvanicus) were collected from trees on DowElanco property in Indianapolis, IN. Tests with Photorhabdus culture broth were perzormed as follows. Each plastic bioassay container (7 1/8' x 3") held fifteen workers, a paper harborage and 10 mi cf broth or concrol media in a piascic shot glass. A cotton wick delivered the treatment to the ants *Trade-mark -29-through a hole in the shot glass lid. All treatments contained 5% sucrose. Bioassays were held in the dark at room temperature and graded at 19 days. Control mortality was 9%. Assays delivering purified fractions utilized artificial ant diet mixed with the treatment (purified fraction or control solution) at a rate of 0.2 ml treatment/2.0 g diet in a plastic test tube. The final protein concentration of the purified fraction was less than 10 g/g diet. Ten ants per treatment, a water source, harborage and the treated diet were placed in sealed plastic containers and maintained in the dark at 27 C in a humidified incubator. Mortality was scored at day 10. No control mortality was seen.
Activity against various lepidopteran larvae was tested as follows. Photorhabdus culture broth or purified fractions were applied directly to the surface (-1.5 cm2) of 0.25 ml of standard artificial diet in 30 l aliquots following dilution in control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively.
The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate larva. European corn borer (Ostrinia nubilalis) and corn earworm (Helicoverpa zea) eggs were supplied from commercial sources and hatched in-house, whereas beet armyworm (Spodoptera exigua), cabbage looper (Trichaplusia ni), tobacco budworm (Heliothis virescens), codling moth (Laspeyresia pomonella) and black cutworm (Agrotis ipsilon) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27 C for the appropriate period. Mortality and weight determinations were scored at days 5-7 for Photorhabdus culture broth and days 4-7 for the purified fraction. Generally, 16 insects per treatment were used in all studies. Control mortality ranged from 4-12.5%
for control medium and was less than 10% for phosphate buffer.

SUBSTITUTE SHEET (RULE 26) Table 3 Effect of Photorhabdus luminescens (strain W-l4) Culture Broth and Purified Toxin Fraction on Mortality and Growth Inhibition of Different Insect Orders/Species Insect Order/Species Broth Purified Fraction o Mort. % G.I. % Mort. % G.I.
COLEOPTERP, =
Corn Rootworm Southern/neonate larva 100 na 100 na Southern/2"d instar na 38.5 nt nt Southern/adult 45 nt nt nt Western/2"instar na 35 nt nt Colorado Potato Beetle 93 nt nt nt 2d instar Turf Grub na a.f. nt nt 3'0 instar na a.f. nt nt adult DIPTERA
Fruit Fly (adult 17 nt nt nt emergence) 100 'na nt nt Mosquito larvae HOMOPTERA
Aster Leafhopper 96.5 na 100 na HYMENOPTERA
Argentine Ant 75 na nt na Carpenter Ant 71 na 100 na L$PIDOPT$RA
Beet Armyworm 12.5 36 18.75 41.4 Black Cutworm nt nt 0 71.2 Cabbage Looper nt nt 21.9 66.8 Codling Moth nt nt 6.25 45.9 Corn Earworm 56.3 94.2 97.9 na European Corn Borer 96.7 98.4 100 na Tobacco Budworm 13.5 52.5 19.4 85.6 Mort. = mortality, G.I. = growth inhibition, na = not applicable, nt = not tested, a.f. = anti-feedant SUBSTITUTE SHEET (RULE 26) Example 3 insectir-ide Utility Upon Soil Application Photorhabdus luminescens (strain W-14) culture broth was shown to be active against corn rootworm when applied directly to soil or a soil-mix (Metromix"). Activity against neonate SCR and WCR in Metromix was tested as follows (Table 4). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the light on moist filter paper for 6 days. After roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastic cup with 50 gm of dry Metromix . Twenty neonate SCR or WCR were then placed directly on the roots of the seedling and covered with Metromix' . Upon infestation, the seedlings were then drenched with 50 ml total volume of a diluted broth solution. After drenching, the cups were sealed and left at room temperature in the light for 7 days.
Afterwards, the seedlings were washed to remove all Metromix"' and the roots were excised and weighed. Activity was rated as the percentage of corn root remaining relative to the control plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either -, +, ++, or +++, with -representing no damage and +++ representing severe damage.
Activity against neonate SCR in soil was tested as follows (Table 5). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the light on moist filter paper for 6 days. After the roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastic cup with 150 gm of soil from a field in Lebanon, IN
planted the previous year with corn. This soil had not been previously treated with insecticides. Twenty neonate SCR were then placed directly on the roots of the seedling and covered with soil. After infestation, the seedlings were drenched with 50 ml total volume of a diluted broth solution. After drenching, the unsealed cups were incubated in a high relative humidity chamber (80%) at 78 F. Afterwards, the seedlings were washed to remove all soil and the roots were excised and weighed. Activity was rated as the percentage of corn root remaining relative to the control plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either -, +, ++, or +++, with - representing no damage and +++ representing severe damage.

SUBSTITUTE SHEET (RULE 26) Table 4 Effect of Photorhabdus luminescens (strain W-14) Culture Broth on Rootworm Larvae After Post-Infestation Drenching (Metromix ) Treatment Larvae Leaf Damage Root Weight (g) 'o Southern Corn Rootworm Water - - 0.4916 0.023 100 Medium (2.0% v/v) - - 0.4416 0.029 100 Broth (6.25%v/v) - - 0.4641 0.081 100 Water + +++ 0.1410 0.006 28.7 Media (2.0% v/v) + +++ 0.1345 0.028 30.4 Broth (1.56% v/v) + - 0.4830 0.031 104 Western Corn Rootworm Water - - 0.4446 0.019 100 Broth (2.0% v/v) - - 0.4069 0.026 100 Water + - 0.2202 0.015 49 Broth (2.0% v/v) + - 0.3879 0.013 95 Table 5 Effect of Photorhabdus luminescens (strain W-14) Culture Broth on Southern Corn Rootworm Larvae After Post-Infestation Drenching (Soil) Treatment Larvae Le- af Damage Root Weight(g) %
Water - - 0.2148 0.014 100 Broth (509. v/v) - - 0.2260 0.016 103 Water + +++ 0.0916 0.009 43 Broth (50% v/v) + - 0.2428 0.032 113 Activity of Photorhabdus luminescens (strain W-14) culture broth against second instar turf grubs in Metromix' was observed in tests conducted as follows (Table 6). Approximately 50 gm of dry Metromix" was added to a 591 ml clear plastic cup. The Metromix* was then drenched with 50 ml total volume of a 50% (v/v) diluted Photorhabdus broth solution. The dilution of crude broth was made with water, with 50% broth being prepared by adding 25 ml of crude broth to 25 ml of water for 50 ml total volume. A 1%
(w/v) solution of proteose peptone #3 (PP3), which is a 50%
dilution of the normal media concentration, was used as a broth control. After drenching, five second instar turf grubs were SUBSTITUTE SHEET (RULE 26) WO 97/17432 PCTlUS96/18003 placed on the top of the moistened Metromix . Healthy turf grub larvae burrowed rapidly into the Metromix9. Those larvae that did not burrow within lh were removed and replaced with fresh larvae.
The cups were sealed and placed in a 28 C incubator, in the dark.
After seven days, larvae were removed from the Metromix and scored for mortality. Activity was rated the percentage of mortality relative to control.

Table 6 Effect of Photorhabdus luminescens (strain W-14) Culture Broth on Turf Grub After Pre-Infestation Drenching (Metromix') Treatment Mortality* Mortality ~
Water 7/15 47 Control medium (1.0% w/v) 12/19 63 Broth (50% v/v) 17/20 85 *expressed as a ratio of dead/living larvae Example 4 Insecticide Utility Upon Leaf Application Activity of Photorhabdus broth against European corn borer was seen when the broth was applied directly to the surface of maize leaves (Table 7). In these assays Photorhabdus broth was diluted 100-fold with culture medium and applied manually to the ~
surface of excised maize leaves at a rate of -6.0 l/cm' of leaf surface. The leaves were air dried and cut into equal sized strips approximately 2 x 2 inches. The leaves were rolled, secured with paper clips and placed in 1 oz plastic shot glasses with 0.25 inch of 2% agar on the bottom surface to provide moisture. Twelve neonate European corn borers were then placed onto the rolled leaf and the cup was sealed. After incubation for 5 days at 27 C in the dark, the samples were scored for feeding damage and recovered larvae.

SUBSTITUTE SHEET (RULE 26) Table 7 Effect of Phctorhabdus luminescens (strain W-14) Culture Brot'r_ on European Corn Borer Larvae Following Pre-Infestation Application to Excised Maize Leaves Treatment Leaf Damage Larvae Recovered Weight(mg) Water Extensive 55/120 0.42 mg Control Medium Extensive 40/120 0.50 mg Broth (1.0% v/v) Trace 3/120 0.15 mg Activity of the culture broth against neonate tobacco budworm (Heliothis virescens) was demonstrated using a leaf dip methodology. Fresh cotton leaves were excised from the plant and leaf disks were cut with an 18.5 mm cork-borer. The disks were individually emersed in control medium (PP3) or Photorhabdus luminescens (strain W-14) culture broth which had been concentrated approximately 10-fold using an Amicon (Beverly, MA), Proflux M12 tangential filtration system with a 10 kDa filter.
Excess liquid was removed and a straightened paper clip was placed through the center of the disk. The paper clip was then wedged into a plastic, 1.0 oz shot glass containing approximately 2.0 ml of 1% Agar. This served to suspend the leaf disk above the agar. Following drying of the leaf disk, a single neonate tobacco budworm larva was placed on the disk and the cup was capped. The cups were then sealed in a plastic bag and placed in a darkened, 270C incubator for 5 days. At this time the remaining larvae and leaf material were weighed to establish a measure of leaf damage (Table 8).

Table 8 Effect of Photorhabdus luminescens (Strain W-14) Culture Broth on Tobacco Budworm Neonates in a Cotton-Leaf Dip Assay Final Weights (mg) Treatment Leaf Disk Larvae Control leaves 55.7 1.3 na*
Control Medium 34.0 2.9 4.3 0.91 Photorhabdus broth 54.3 1.4 0.0**
*- not applicable, ** - no live larvae found SUBSTITUTE SHEET (RULE 26) Example 5, Part A
Characterization of Toxin Peptide Components In a subsequent analysis, the toxin protein subunits of the bands isolated as in Example 1 were resolved on a 7% SDS
polyacrylamide electrophoresis gel with a ratio of 30:0.8 (acrylamide:BIS-acrylamide). This gel matrix facilitates better resolution of the larger proteins. The gel system used to estimate the Band 1 and Band 2 subunit molecular weights in Example 1 was an 18% gel with a ratio of 38:0.18 (acrylamide:BIS-acrylamide), which allowed for a broader range of size separation, but less resolution of higher molecular weight components.
In this analysis, 10, rather than 8, protein bands were resolved. Table 9 reports the calculated molecular weights of the 10 resolved bands, and directly compares the molecular weights estimated under these conditions to those of the prior example. It is not surprising that additional bands were detected under the different separation conditions used in this example. Variations between the prior and new estimates of molecular weight are also to be expected given the differences in analytical conditions. In the analysis of this example, it is thought that the higher molecular weight estimates are more accurate than in Example 1, as a result of improved resolution.
However, these are estimates based'on SDS PAGE analysis, which are typically not analytically precise and result in estimates of peptides and which may have been further altered due to post- and co-translational modifications.
Amino acid sequences were determined for the N-terminal portions of five of the 10 resolved peptides. Table 9 correlates the molecular weight of the proteins and the identified sequences. In SEQ ID NO:2, certain analyses suggest that the proline at residue 5 may be an asparagine (asn). In SEQ ID NO:3, certain analyses suggest that the amino acid residues at positions 13 and 14 are both arginine (arg). In SEQ ID NO:4, certain analyses suggest that the amino acid residue at position 6 may be either alanine (ala) or serine (ser). In SEQ ID NO:5, certain analyses suggest that the amino acid residue at position 3 may be aspartic acid (asp).

SUBSTITUTE SHEET (RULE 26) Table 9 ESTI14ATE NEW ESTIMATE* SEQ. LISTING
208 200.2 kDa SEQ ID NO:1 184 175.0 kDa SEQ ID NO:2 65.6 68.1 kDa SEQ ID NO:3 60.8 65.1 kDa SEQ ID NO:4 56.2 58.3 kDa SEQ ID NO:5 25.1 23.2 kDa SEQ ID NO:15 *New estimates are based on SDS PAGE and are not based on gene sequences. SDS PAGE is not analytically precise.
Example 5, Part B
Characterization of Toxin Peptide Components New N-terminal sequence, SEQ ID NO:15, Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr, was obtained by further N-terminal sequencing of peptides isolated from Native HPLC-purified toxin as described in Example 5, Part A, above. This peptide comes from the tcaA gene. The peptide labeled TcaAii, starts at position 254 and goes to position 491, where the TcaAiii peptide starts, SEQ ID NO:4. The estimated size of the peptide based on the gene sequence is 25,240 Da.

Example 6 Characterization of Toxin Peptide Components In yet another analysis, the toxin protein complex was re-isolated from the Photorhabdus luminescens growth medium (after culture without Tween) by performing a 10% - 80% ammonium sulfate precipitation followed by an ion exchange chromatography step (Mono Q) and two molecular sizing chromatography steps. These conditions were like those used in Example 1. During the first molecular sizing step, a second biologically active peak was found at about 100 10 kDa. Based upon protein measurements, this fraction was 20 - 50 fold less active than the larger, or primary, active peak of about 860 100 kDa (native). During this isolation experiment, a smaller active peak of about 325 50 kDa that retained a considerable portion of the starting biological activity was also resolved. It is thought that the 325 kDa peak is related to or derived from the 860 kDa peak.

SUBSTITUTE SHEET (RULE 26) A 56 kDa protein was resolved in this anaiysis. The N-terminal sequence of this protein is presented in SEQ ID N0:6.
It is noteworthy that this protein shares significant identity and conservation with SEQ ID NO:5 at the N-terminus, suggestin.:r that the two may be encoded by separate members of a gene family and that the proteins produced by each gene are sufficiently similar to both be operable in the insecticidal toxin complex.
A second, prominent 185 kDa protein was consistently present in amounts comparable to that of protein 3 from Table 9, and m,ay be the same protein or protein fragment. The N-terminal sequence of this 185 kDa protein is shown at SEQ ID NO:7.
Additional N-terminal amino acid sequence data were also obtained from isolated proteins. None of the determined N-terminal sequences appear identical to a protein identified in Table 9. Other proteins were present in isolated preparation.
One such protein has an estimated molecular weight of 108 kDa and an N-terminal sequence as shown in SEQ ID NO:8. A second such protein has an estimated molecular weight of 80 kDa and an N-terminal sequence as shown in SEQ ID NO:9.
When the protein material in the approximately 325 kDa active peak was analyzed by size, bands of approximately 51, 31, 28, and 22 kDa were observed. As in all cases in which a molecular weight was determined by analysis of electrophoretic mobility, these molecular weights were subject to error effect=
introduced by buffer ionic strength differences, electrophoresis power differences, and the like. One of ordinary skill would understand that definitive molecular weight values cannot be determined using these standard methods and that each was subject to variation. It was hypothesized that proteins of these sizes are degradation products of the larger protein species (of approximately 200 kDa size) that were observed in the larger primary toxin complex.
Finally, several preparations included a protein having ttie N-terminal sequence shown in SEQ ID NO:10. This sequence was strongly homologous to known chaperonin proteins, accessory proteins known to function in the assembly of large protein complexes. Although the applicants could not ascribe such an assembly function to the protein identified in SEQ ID NO:10, it was consistent with the existence of the described toxin protein complex that such a chaperonin protein could be involved in its SUBSTITUTE SHEET (RULE 26) 3ss?mbli . moreover, alzhough such proteins have not direcclf been suggested co have toxic accivity, this protein may be impor*_ant ro determining the overall structural nature of the protein toxin, and thus, may contribute to the toxic activity or ~urability of the complex in viro after oral delivery.
Subseq=uent analysis of the stability of the protein coxin complex co proteinase K was undertaken. It was determined that after 24 hour incubation of the complex in the presence of a 10-fold molar excess of proteinase K, activity was virtually 1() eliminated (mortality on oral application dropped to about 5e).
These data confirm the proteinaceous nature of the toxin.
The toxic activity was also retained by a dialysis membrane, again confirming the large size of the native toxin complex.

Examule 7 Isolat'_on, Characterization and Partial Amino Acid Sequencing of Phocor:iabdus Toxins Isolatien and N-Terminal Amino Acid Sequencing: In a set of experiments conducted in parallel to Examples 5 and 6, ammenium sulfate precipitation of Phocorhabdus proteins was performed by adjusting Photorhabdus broth, typically 2-3 liters, to a final concentration of either 10% or 20% 'oy the slow addition of ammonium sulfate crystals. After stirring for 1 hour at 4 C, the material was centrifuged at 12,000 x g for 30 minutes. The supernatant was adjusted to 80t ammonium sulfate, stirred ac 4 C
for 1 hour, and centrifuged at 12,000 x g for 60 minutes. The pellet was resuspended in one-tenth the volume of 10 mM Na:=POs, pH 7.0 and dialyzed against the same phosphate buffer overnight at 4 C. The dialyzed material was centrifuged at 12,000 x g for 1 hour prior to ion exchange chromatography.
A. HR 16/50 Q Sepharose"(Pharmacia) anion exchange column was equilibrated with 10 mM Nai=PO4, pH 7Ø Centrifuged, dialyzed ammonium sulfate pellet was applied to the Q Sepharose column at a rate of 1.5 ml/min and washed extensively at 3.0 ml/min with equilibration buffer until the optical density (O.D. 280) reached less than 0.100. Next, either a 60 minute NaCl gradient ranging from 0 to 0.5 M at 3 ml/min, or a series of step elutions using 0.1 M, 0.4 M and finally 1.0 NaCl for 60 minutes each was acplied to the column. Fractions were pooled and concentrated using a *Trade-mark C~encriprep 100. Alternacively, proceins could be eluted by a single 0.4 M NaC1 wash without prior elution with 0.1 M NaCl.
Two milliliter aliquocs of concer.trated Q Sepharose samples were loaded ac 0.5 ml/min onto a HR 16/50 Superose 12 (Pharmacia) gel filtratien column equilibraced wich 10 mM Nai=PO4, pH 7Ø
The column .ras washed wich the same buffer for 240 min at 0.5 mi/min and 2 min samples were collected. The void volume macerial was collected and concentrated using a Centriprep 100.
Two millilicer aliquots of concentrated Superose* 12 samples -4ere loaded at 0.5 ml/min onto a HR 16/50 Sepharose 4B-CL (Pharmacia) gel filtration column equilibrated with 10 mM Na)=PO4, pH 7Ø
The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected.
The excluded protein peak was subjected to a second =ractionacion by application to a gel filtration column that used a Sepharose CL-4B resin, which separates proteins ranging from -30 kDa to 1000 kDa. This fraction was resolved into two peaks;
a minor peak at the void volume (>1000 kDa) and a major peak which eluted ac,an apparenc molecular weight of abouc 860 kDa.
Over a one week period subsequent samples subjected to gel filtration showed the gradual appearance of a third peak (approximately 325 kDa) that seemed to arise from the major peak, perhaps by limited proteolysis. Bioassays performed on the ch:-ee peaks showed that the void peak had no activity, while the 860 kDa toxin complex fraction was highly active, and the 325 kD3 peak was less active, although quite potent. SDS PAGE analysis of Sepharose CL-4B toxin complex peaks from different fermentation productions revealed two distinct peptide patterns, denoted =P" and S. The two patterns had marked differences in che molecular weights and concentrations of peptide componencs in their fractions. The "S" pattern, produced most frequently, had 4 high molecular weight peptides (> 150 kDa) while the "P"
pattern had 3 high molecular weight peptides. In addition, che "S" peptide fraction was found to have 2-3 fold more activitr against European Corn Borer. This shift may be related to variations in protein expression due to age of inoculum and/or other factors based on growth parameters of aged cultures.
Milligram quantities of peak toxin complex fractions determined to be "P" or "S" peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine *Trade-mark -40-(Seprabuff'T" to PVDF membranes (ProBlott'''", Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides in the "S" pattern had unique N-terminal amino acid sequences compared to the sequences identified in the previous example. A 201 kDa (TcdAii) peptide set forth as SEQ ID NO:.13 below shared between 33% amino acid identity and 50% similarity with SEQ ID NO:l (TcbAii)(Table 10, in Table 10 vertical lines denote amino acid identities and colons indicate conservative amino acid substitutions). A second peptide of 197 kDa, SEQ ID NO:14 (TcdB), had 42% identity and 58%
homology with SEQ ID NO:2 (TcaC). Yet a third peptide of 205 kDa was denoted TcdAii. In addition, a limited N-terminal amino acid sequence, SEQ ID NO:16 (TcbA), of a peptide of at least 235 kDa was identical in homology with the amino acid sequence, SEQ ID
NO:12, deduced from a cloned gene (tcbA), SEQ ID NO:11, containing a deduced amino acid sequence corresponding to SEQ ID
NO:1 (TcbAii). This indicates that the larger 235+ kDa peptide was proteolytically processed to the 201 kDa peptide, (TcbAii), (SEQ ID NO:1) during fermentation, possibly resulting in activation of the molecule. In yet another sequence, the sequence originally reported as SEQ ID NO:5 (TcaBii) reported in Example 5 above, was found to contain an aspartic acid residue (Asp) at the third position rather than glycine (Gly) and two additional amino acids Gly and Asp at the eighth and ninth positions, respectively. In yet two other sequences, SEQ ID NO:2 (TcaC) and SEQ ID NO:3 (TcaBi), additional amino acid sequence was obtained. Densitometric quantitation was performed using a sample that was identical to the "S" preparation sent for N-terminal analysis. This analysis showed that the 201 kDa and 197 kDa peptides represent 7.0% and 7.2%, respectively, of the total Coomassie brillant blue stained protein in the "S" pattern and are present in amounts similar to the other abundant peptides.
It is speculated that these peptides may represent protein homologs, analogous to the situation found with other bacterial toxins, such as various CryI Et toxins. These proteins vary from 40-90% homology at their N-terminal amino acid sequence, which encompasses the toxic fragment.

SUBSTITUTE SHEET (RULE 26) Internal Amino Acid Sequencing: To facilitate cloning of toxin peptide genes, internal amino acid sequences of selected peptides were obtained as followed. Milligram quantities of peak 2A fractions determined to be "P" or "S" peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (SeprabuffT" to PVDF membranes (ProBlottT'4, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides, referred to as TcbAii (containing SEQ ID NO:1), TcdAii, and TcaBi (containing SEQ ID NO:3) were subjected to trypsin digestion by Harvard MicroChem followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal peptides were sequenced for the peptide TcaBi (205 kDa peptide) referred to as TcaBi-PT111 (SEQ ID NO:17) and TcaBz-PT79 (SEQ ID
NO:18). Two internal peptides were sequenced for the peptide TcaB; (68 kDa peptide) referred to as TcaB!-PT158 (SEQ ID NO:19) and TcaBi-PT108 (SEQ ID NO:20). Four internal peptides were sequenced for the peptide TcbAii (201 kDa peptide) referred to as TCBAII-PT103 (SEQ ID NO:21), TcbAii-PT56 (SEQ ID NO:22), TcbAii-PT81(a) (SEQ ID NO:23), and TcbAii-PT81(b) (SEQ ID NO:24).

Table 10 N-Terminal Amino Acid Sequences -201 kDa (33% identity & 50% similarity to SEQ ID NO.1) L I G Y N N Q F S LIGYNNQFSGA A SEQ ID NO:13 . i I I . I
F I Q G Y S D L F G N - A SEQ ID NO:1 197 kDa (42% identity & 58% similarity SEQ ID NO.2) M Q N S Q T F S V G E L SEQ ID NO.14 I I. I i.. I
M Q D S P E V S I T T L SEQ ID NO.2 Example 8 Construction of a cosmid library of Photorhabdus lurninescens W-14 genomic DNA and its screening to isolate genes encoding peptides comprising the toxic protein preparation As a prerequisite for the production of Photorhabdus insect toxic proteins in heterologous hosts, and for other uses, it is - 45 necessary to isolate and characterize the genes that encode those SUBSTITUTE SHEET (RULE 26) peptides. 'rni.s oD7ective was pursueci in parallel. One approach, described later, was based on the use of monoclonal and polyclonal antibodies raised against the purified toxin which were then used to isolate clones from an expression library. The other approach, described in this example, is based on the use of the N-terminal and internal amino acid sequence data to design degenerate oligonucleotides for use in PCR amplication. Either method can be used to identify DNA clones that contain the peptide-encoding genes so as to permit the isolation of the respective genes, and the determination of their DNA base sequence.

GENOMIC DNA ISOLATION: Photorhabdus luminescens strain W-14 (ATCC accession number 55397) was grown on 2% proteose peptone #3 agar (Difco Laboratories, Detroit, MI) and insecticidal toxin competence was maintained by repeated bioassay after passage, using the method described in Example 1 above. A 50 ml shake culture was produced in a 175 ml baffled flask in 2% proteose peptone #3 medium, grown at 28 C and 150 rpm for approximately 24 hours. 15 ml of this culture was pelleted and frozen in its medium at -20 C until it was thawed for DNA isolation. The thawed culture was centrifuged, (700 x g, 30 min) and the floating orange mucopolysaccharide material was removed. The remaining cell material was centrifuged (25,000 x g, 15 min) to pellet the bacterial cells, and the medium was removed and discarded.
Genomic DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Current Protocols in Molecular Biology (Ausubel et al. eds, John Wiley & Sons, 1994) (modified to include a salt shock and with all volumes increased 10-fold].
The pelleted bacterial cells were resuspended in TE buffer (10 mM
Tris-HC1, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12 ml of 5 M NaCl was added; this mixture was centrifuged 20 min at 15,000 x g. The pellet was resuspended in 5.7 ml TE and 300 ml of 10% SDS and 60 ml of 20 mg/ml proteinase K (Gibco BRL
Products, Grand Island, NY; in sterile distilled water) were added to the suspension. This mixture was incubated at 37 C for I hr; then approximately 10 mg lysozyme (Worthington Biochemical =
Corp., Freehold, NJ) was added. After an additional 45 min, I ml of 5 M NaCl and 800 ml of CTAB/NaCl solution (10% w/v CTAB, 0.7 M

SUBST(ME SHEET (RULE 20 L y.5 5 5- 1 CA 02209659 2001-01-19 NaCl) were aaded. 'rnis preparation was incuoaced 10 min at 63c'_'.
then gently agitated and zurther incubated and agitaced for approximately 20 min to assist clearing of the cellular material.
An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently and centrifuged. After two extractions with an equal volume of PCI
(phenol/chloro=o r=n/isoamyl alcohol; 50:49:1, v/v/v; equilibrated with I M Tris-HC1, pH 8.0; Intermountain Scientific Corporation, Kaysville, UT), the DNA was precipitated with 0.6 volume of isopropanol. The DNA precipitate was gently removed with a glass rod, washed twice with 70% ethanol, dried, and dissolved in 2 cnl STE (10 mM Tris-HC1 pH 8.0, 10 mM NaCl, 1 mM EDTA). This preparation contained 2.5 mg/ml DNA, as determined by optical density at 260 nm (i.e., OD26a) .
The molecular size range of the isolated genomic DNA was evaluated for suitability for library construction. CHEF gel analysis was performed in 1.5% agarose (Seakeni LE, FMC
BioProducts, Rockland, ME) gels with 0.5 X TBE buffer (44.5 mti Tris-HCI pH 8.0, 44.5 mM H)BOI, 1 mM EDTA) on a BioRad CHEF-DR IZ
apparatus with a Pulsewave 760 Switcher (Bio-Rad Laborator;.es.
Inc., Richmond, CA). The running parameters were: initial A
time,"3 sec: final A time, 12 sec; 200 volts; running temperature, 4-18 C; r.in time, 16.5 hr. Ethidium bromide staining and examination of the gel under ultraviolet light indicated the DNA ranged from 30-250 kbp in size.

CONSTRUCTiON OF LIBRARY: A partial Sau3A I digest was i::.;d=
of this Phoccrhabdus genomic DNA preparation. The method was based on section 3.1.3 of Ausubel (supra.). Adaptions included running smaller scale reactions under various conditions until nearly optimal results were achieved. Several scaled-up large reactions with varied conditions were run, the results analyzed on CHEF gels, and only the best large scale preparation was carried forward. In the optimal case, 200 g of Phocorhabdus genomic DNA was incubated with 1.5 units of Sau3A 1(New England Biolabs, "NEB", Beverly, MA) for 15 min at 37 C in 2 ml total volume of 1X NEB 4 buffer (supplied as lOX by the manufacturer).
The reaction was stopped by adding 2 ml of PCI and centrifuging at 8000 x g for 10 min. To the supernatant were added 200 l ci 5 M NaCl plus 6 ml of ice-cold ethanol. This preparation was *Trade-mark -44-chilled for 30 min at -20 C, then centrifuged at 12,000 :-: g for 15 min. The supernatant was removed and the precipitate was dried in a vacuum oven at 40 C, then resuspended in 400 l STE.
Spectrophotometric assay indicated about 40% recovery of the input DNA. The digested DNA was size fractionated on a sucrose gradient according to section 5.3.2 of CPMB (op. cit.). A 10%
to 40% (w/v) linear sucrose gradient was prepared with a gradient maker in Ultra-C1earT"' tubes (Beckman Instruments, Inc., Palo Alto, CA) and the DNA sample was layered on top. After centrifugation, (26,000 rpm, 17 hr, Beckman SW41 rotor, 20 C!, fractions (about 750 l) were drawn from the top of the gradient and analyzed by CHEF gel electrophoresis (as described earlier).
Fractions containing Sau3A 1 fragments in the size range 20-40 kbp were selected and DNA was precipitated by a modification (amounts of all solutions increased approximately 6.3-fold) -,f the method in section 5.3.3 of Ausubel (supra.). After overnight precipitation, the DNA was collected by centrifugation (17,O(j0 x g, 15 min), dried, redissolved in TE, pooled into a final volume of 80 .l, and reprecipitated with the addition of 8 l 3 M s~.-dium acetate and 220 l ethanol. The pellet collected by centrifugation as above was resuspended in 12 l TE.
Concentration of the DNA was determined by Hoechst 33258 dye (Polysciences, Inc., Warrington, PA) fluorometry in a Hoefer TKO100 fluorimeter (Hoefer Scientific Instruments, San Francisco, CA). Approximately 2.5 g of the size-fractionated DNA was recovered.

Thirty g of cosmid pWE15 DNA (Stratagene, La Jolla, CA) was digested to completion with 100 units of restriction enzyme b3mH
1(NEB) in the manufacturer's buffer (final volume of 200 l, 37 C, 1 hr). The reaction was extracted with 100 l of PCI and DNA was precipitated from the aqueous phase by addition of 20 l 3M sodium acetate and 550 l -20 C absolute ethanol. After :0 min at -70 C, the DNA was collected by centrifugation (17,000 x g, 15 min), dried under vacuum, and dissolved in 180 l of 10 mM

Tris-HC1, pH B.O. To this were added 20 N.1 of lOX CIP buffe!_ (100 mM Tris-HC1, pH 8.3; 10 mM ZnC12; 10 mM MgC12), and 1 l (0.25 units) of 1:4 diluted calf intestinal alkaline phosphatase SUBSTIME SHEET (RULE 26) (9oehringer Mannheim Corporation. Indianapolis, IN). After 30 min at 37 C, the following additions were made: 2 l 0.5 M EDTA, pH 8.0; 10 l 10% SDS; 0.5 l of 20 mgiml proteinase K (as above), followed by incubation at 55 C for 30 min. Following sequential extractions with 100 l of PCI and 100 l phenol (Incermountain Scientific Corporation, equilibrated with I M
Tris-HCI, pH 8.0), the dephosphorylated DNA was precipitated by addition of 72 l of 7.5 M ammonium acetate and 550 l -20 C
ethanol, incubation on ice for 30 min, and centrifugation as lU above. The pelleted DNA was washed once with 500 l -20 C 70e ethanol, dried under vacuum, and dissolved in 20 l of TE buffer.
Ligation of the size-fractionated Sau3A 1 fragments to the BamH 1-digesced and phosphatased pWE15 vector was accomplished using T4 ligase (NEB) by a modification (i.e., use of premixed lOX ligation buffer supplied by the manufacturer) of the protoci1 in section 3.33 of Ausubel. Ligation was carried ouc overnight in a total volume of 20 l at 15 C, followed by storage at -C.
Four l of the cosmid DNA ligation reaction, containing 20 about 1 g of DNA, was packaged into bacteriophage lambda usinq a commercial packaging extract (Gigapac)~ III Gold Packaging Extract, Stratagene), following the manufacturer's directions.
The packaged preparation was stored at 4 C until use. The packaged cosmid preparation was used to infect Escherichia coli XL1 Blue MR cells (Stratagene) according to the Gigapack' III ~.=Ad protocols ("Titering the Cosmid Library"), as follows. :CL1 Hlue MR cells were grown in LB medium (g/L: Bacto-tryptone, 10; Bacto-yeast extract, 5; Bacto-agar, 15; NaCl, 5; (Difco Laboratories, Detroit, MIl) containing 0.2% (w/v) maltose plus 10 mM MgSO4, at 37 C. After 5 hr growth, cells were pelleted at 700 x g (15 min) and resuspended in 6 ml of 10 mM MgSO.. The culture density was adjusted with 10 mM MgSO. to OD,soo= 0.5. The packaged cosmid library was diluted 1:10 or 1:20 with sterile SM medium (0.1 M
NaCl, 10 mM MgS04,50 mM Tris-HCI pH 7.5, 0.01% w/v gelatin), an-3 25 l of the diluted preparation was mixed with 25 l of the diluted XLI Blue MR cells. The mixture was incubated at 25 i_ E.-)r 30 min (without shaking), then 200 l of LB broth was added, aild incubation was continued for approximately 1 hr with occasional *Trade-mark -46-gefltle shaking. ?liquots (20-40 l) of this culture were spre_=i on LB agar plates containing 100 mg/1 ampicillin (i.e. , LB-An:n;..,) and incubated overnight at 37 C. To store the library withouc amplification, single colonies were picked and inoculated into individual wells of sterile 96-well microwell plates; each weil containing 75 l of Terrific Broth (TB media: 12 g/1 Bacto-tryptone, 24 g/1 Bacto-yeast extract, 0.4% v/v glycerol. 17 mM
KH2PO4, 72 mM K2HP04) plus 100 mg/1 ampicillin (i.e., TB-Amp;) and incubated (without shaking) overnight ac 37 C. After replicaci.ig lU the 96-well plate into a copy plate, 75 l/well of filter-sterilized TB:glycerol (1:1, v/v; with, or without, 100 mg/i ampicillin) was added to the plate, it was shaken briefly at 100 rpm, 37 C, and then closed with Parafilm' (American National I:aii, Greenwich, CT) and placed in a-70 C freezer for storage. Cupy plates were grown and processed identically to the master plac=s.
A total of 40 such master plates (and their copies) were prepared.

SCREENING OF THE LI3RARY WITH rZADIOL?.BELED DNA PROBES : T::
prepare colony filters for probing with radioactively labeled probes, ten 96-well plates of the library were thawed at 25 C
(bench top at room temperature). A replica plating tool wit!i 2i prongs was used to inoculate a fresh 96-well copy plate containing 75 )tl/well of TB-Amploo. The copy plate was grown overnight (stationary) at 37 C, then shaken about 30 min at 100 rpm at 37 C. A total of 800 colonies was represented in these copy plates, due to nongrowth of some isolates. The replica tool was used to inoculate duplicate impressions of the 96-well aLrays onto Magna tIT (MSI, Westboro, MA) nylon membranes (0.45 micr=n.
220 x 250 mm) which had been placed on solid LB-Amptoo (100 mi/dish) in Bio-assay plastic dishes (Nunc. 243 x 243 x 18 mm;
Curtin Mathison Scientific, Inc., Wood Dale, IL). The colonies were grown on the membranes at 37 C for about 3 hr.
A positive control colony (a bacterial clone containing a GZ4 sequence insert, see below) was grown on a separate Magn:t t1T
membrane (Nunc, 0.45 micron, 82 mm circle) on LB medium supplemented with 35 mg/1 chloramphenicol (i.e., LB-Cam,;), az:d processed alongside the library colony membranes. Bacterial colonies on the membranes were lysed, and the DNA was denatured *Trade-mark and neutralized according to a protocol taken from the Genius'M
System User's Guide version 2.0 (Boehringer Mannheim, Indianapolis, IN). Membranes were placed colony side up on filter paper soaked with 0.5 N NaOH plus 1.5 M NaCl for 15 min to denature, and neutralized on filter paper soaked with I M Tris-HC1 pH 8.0, 1.5 M NaCl for 15 min. After UV-crosslinking using a Stratagene W Stratalinker set on auto crosslink, the membraries were stored dry at 25 C until use. Membranes were trimmed irito strips containing the duplicate impressions of a single 96-well plate, then washed extensively by the method of section 6.4.1 in CPMB (op. cit.): 3 hr at 25 C in 3X SSC, 0.1% (w/v) SDS, followed by 1 hr at 65 C in the same solution, then rinsed in 2X SSC in preparation for the hybridization step (20X SSC = 3 M NaCl, 0.3 M
sodium citrate, pH 7.0).
Amplification of a specific genomic fragment of a tcaC -rene.
Based on the N-terminal amino acid sequence determined for ttie purified TcaC peptide fraction [disclosed herein as SEQ ID NO:2], a pool of degenerate oligonucleotides (pool S4Psh) was synthesized by standard 0-cyanoethyl chemistry on an Applied BioSystem AB1394 DNA/RNA Synthesizer (Perkin Elmer, Foster City, CA). The oligonucleotides were deprotected 8 hours at 55 C, dissolved in water, quantitated by spectrophotometric measurement, and diluted for use. This pool corresponds to the determined N-terminal amino acid sequence of the TcaC peptide.
The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents inosine:
Amino Met Gln Asp Ser Pro Glu Val Acid S4Psh 5' ATG CA(A/G) GA(T/C) (T/A)(C/G)(T/A) CCI GA(A/G) GT 3' Another set of degenerate oligonucleotides was synthesi-ed (pool P2.3.5R), representing the complement of the coding strand for the determined amino acid sequence of the SEQ ID NO:17:
Amino Acid Ala Phe Asn Ile Asp Asp Val Codons 5' GCN TT(T/C) AA(T/C) AT(A/T/C) GA(T/C) GA(T/C) GT 3' P2.3.5R 3'CG(A/C/G/T) AA(A/G) TT(A/G) TA(T/A/G) CT(A/G) CT(A/G) CA 5' These oligonucleotides were used as primers in Polymerase Chain Reactions (PCe, Roche Molecular Systems, Branchburg, NJ) to SUBSTITUTE SHEET (RULE 26) amplify a specitic UNA tragment from genomic DNA prepared from Photorhabdus strain W-14 (see above). A typical reaction (50 1) contained 125 pmol of each primer pool P2Psh and P2.3.5R, 253 ng of genomic template DNA, 10 nmol each of dATP, dCTP, dGTP, and dTTP, 1X GeneAmp") PCR buffer, and 2.5 units of AmpliTaq'9 DNA
polymerase (both from Roche Molecular Systems; lOX GeneAmpo buffer is 100 mM Tris-HC1 pH 8.3, 500 mM KC1, 0.01% w/v gelatin).
Amplifications were performed in a Perkin Elmer Cetus DNA Thermal Cycler (Perkin Elmer, Foster City, CA) using 35 cycles of 94 C
(1.0 min), 55 C (2.0 min), 72 C (3.0 min), followed by an extension period of 7.0 min at 72 C. Amplification products were analyzed by electrophoresis through 2% w/v NuSieve 3:1 agarose (FMC BioProducts) in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0). A specific product of estimated size 250 bp was observed amongst numerous other amplification products by ethidium bromide (0.5 g/ml) staining of the gel and examination under ultraviolet light.
The region of the gel containing an approximately 250 bp product was excised, and a small plug (0.5 mm dia.) was removed and used to supply template for PCR amplification (40 cycles).
The reaction (50 l) contained the same components as above, minus genomic template DNA. Following amplification, the ends of the fragments were made blunt and were phosphorylated by incubation at 25 C for 20 min with 1 unit of T4 DNA polymerase (NEB), 1 nmol ATP, and 2.15 units of T4 kinase (Pharmacia Biotech Inc., Piscataway, NJ).
DNA fragments were separated from residual primers by electrophoresis through 1% w/v GTe agarose (FMC) in TEA. A gel slice containing fragments of apparent size 250 bp was excisecl, and the DNA was extracted using a Qiaex kit (Qiagen Inc., Chatsworth, CA).
The extracted DNA fragments were ligated to plasmid vector pBC KS(+) (Stratagene) that had been digested to completion with restriction enzyme Sma 1 and extracted in a manner similar to that described for pWE15 DNA above. A typical ligation reaction (16.3 l) contained 100 ng of digested pBC KS(+) DNA, 70 ng oF
250 bp fragment DNA, 1 nmol 1Co(NH3)61C1,, and 3.9 Weiss units of T4 DNA ligase (Collaborative Biomedical Products, Bedford, MA), in IX ligation buffer (50 mM Tris-HC1, pH 7.4; 10 mM MgClz; 10 mM

SUBSTITUTE SHEET (RULE 26) dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin). Following overnight incubation at 14 C, the ligate%.l = products were transformed into frozen, competent Escherichia .=oii DH5a cells (Gibco BRL) according to the suppliers' recommendations, and plated on LB-Cam3splates, containing IPT13 (119 g/ml) and X-gal (50 g/ml). Independent white colonies were picked, and plasmid DNA was prepared by a modified alkalinF-lysis/PEG precipitation method (PRISMT'" Ready Reaction DyeDeor.YTM
Terminator Cycle Sequencing Kit Protocols; ABI/Perkin Elmer).
The nucleotide sequence of both strands of the insert DNA was determined, using T7 primers [pBC KS(+) bases 601-623:
TAAAACGACGGCCAGTGAGCGCG) and LacZ primers [pBC KS(+) bases 792-816: ATGACCATGATTACGCCAAGCGCGC) and protocols supplied with the PRISMTM sequencing kit (ABI/Perkin Elmer). Nonincorporated dye-terminator dideoxyribonucleotides were removed by passage through Centri-Sep 100 columns (Princeton Separations, Inc., Adelphia, NJ) according to the manufacturer's instructions. The DNA
sequence was obtained by analysis of the samples on an ABI Model 373A DNA,Sequencer (ABI/Perkin Elmer). The DNA sequences of two isolates, GZ4 and HB14, were found to be as illustrated in Figure 1.
This sequence illustrates the following features: 1) bases 1-20 represent one of the 64 possible sequences of the S4Psh degenerate oligonucleotides, ii) the sequence of amino acids 1-3 and 6-12 correspond exactly to that determined for the N-terminus of TcaC (disclosed as SEQ ID NO:2), iii) the fourth amino acid encoded is a cysteine residue rather than serine. This difference is encoded within the degeneracy for the serine codons (see above), iv) the fifth amino acid encoded is proline, corresponding to the TcaC N-terminal sequence given as SEQ ID
NO:2, v) bases 257-276 encode one of the 192 possible sequences designed into the degenerate pool, vi) the TGA termination codon introduced at bases 268-270 is the result of complementarity to the degeneracy built into the oligonucleotide pool at the corresponding position, and does not indicate a shortened reading frame for the corresponding gene.

Labeling of a TcaC peptide gene-specific probe. DNA
fragments corresponding to the above 276 bases were amplified (35 SUBSTITUTE SHEET (RULE 26) =.:cies) by PCR' in a 100 l reaction volume, using 100 pmcl each of'P2Psh and P2.3.5R primers, 10 ng of piasmids GZ4 or HB14 as cemplaces, 20 nmol each of dAT?, dCT?, dGTP, and dTTP, 5 units of AmpliTAq' DNA polymerase, and IX concencracion of GeneAmp" buf'er, under the same temperature regimes as described above. The amplification pr-~ducts were extracted from a 190- GTG' agarose gel by Qiae *kit and quantitated by fluoromecry.
The extracted amplification products from plasmid HB14 template (approximately 400 ng) were split into five aliquots and labeled with ''P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) according to the manufacturer's instructions. Nonincorporated radioisotope was removed by passage through NucTrap' Probe Purification Columns (Stracagene), according to the supplier's instructions. The specific accivity of the labeled DNA product was determined by scintillation counting to be 3.11 x l0jdpm/ g. This labeled DNA was used to probe membranes prepared frcm 800 members of the gencmic Library.

Screening with a TcaC-neptide gene specific Drobe. The radiolabeled HB14 probe was boiled approximately 10 min, then added to "minimal hyb" solution. (Note: The "minimal hyb" method is taken from a CERES protocol; "Restriction Fragment Length Polymorphism Laboratory Manual version 4.0", sections 4-40 and 4-4'; CERES/NPI, Salt Lake City, UT. NPI is now defunct, with its successors operating as Linkage Genetics) . "Minimal hyb"
solution contains 10% w/v PEG (polyethylene glycol, M.W. approx.
8000), 7% w/v SDS; 0.6X SSC, 10 mM sodium phosphate buffer (from a 1M stock containing 95 g/l NaH2PO4-1H2O and 84.5 g/l Na,HPO4=7H2O), 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA. Membranes were blotted dry briefly then, without prehybridization, 5 strips of membrane were placed in each of 2 plastic boxes containing 75 ml of =minimal hyb" and 2.6 ng/ml ol.
radiolabeled HB14 probe. These were incubated overnight with slow shaking (50 rpm) at 60 C. The filters were washed three times for approximately 10 min each at 25 C in "minimal hyb wash solution" (0.25X SSC, 0.2% SDS), followed by two 30-min washes with slow shaking at 60 C in the same solution. The filters were placed on paper covered with Saran wrap' (Dow Brands, Indianapolis, IN) in a light-tight autoradiographic cassette and exposed to X-Omat*X-ray film (Kodak, Rochescer, NY) with two *Trade-mark -51-DuPont CrCn :: L13hCZ=nQ-?Lus~
-1 enhancers (Sigma C iemlcal Cc., Sc, Louis, MO), for 4 hr 3C -70 C._ Upon developmenc (scandarci phocographic procedures) , significant signals were evident in boch replicates amongst a high background of weaker, more irregular signals. The filters were again washed for about 4 hr ac 69 C in "minimal hyb wash solucicn" and then placed again in che casseccas and film was exposed overnight at -70 C. Tuelve possible posi:ives were identified due to strong signals cn boch of che duplicate 96-well colony impressions. No signal was seen wich negaci=re concrol membranes (colonies of XL1 Blue MP, cells concaining pWE15), and a very strong signal was seen wizh positive concrol membranes (DH5a cells containing the GZ4 isolace of the PCR producc) that had been processed concurrently with the experimental samples.
The twelve pucative hybridization-positive colonies were retrieved from the frozen 96-well library plates and grc,rn overnighc at 37 C on solid LB-Amp,oo medium. They were then patched (3/plate, plus three negative controls: XL1 31ue MR cells containing the pWE15 vector) onto solid LB-Amptoo= Two sets of membranes (Magna NT nylon, 0.45 micron) were prepared for hybridization. The first sec was prepared by placing a filter direccly onto the colonies on a patch plate. then removing it with adherent bacterial cells, and processing as below. ?il--ers of che secor.d set -were placed on plates containing L3-,ampt0q medium, then inoculated by transferring cells from the patch plates onto the filters. Afcer overnight growth at 37 C, che filters were removed from the plates and processed.
3acterial cells on the filters were lysed and DNA denatured by placing each filter colony-side-up on a pool (1.0 ml) o: 0.5 tt ttaOH in a plastic plate for 3 min. The filters were blocced dr-I
on a paper towel, then the process was repeaced with fresh 0.5 ~1 NaOH. After blotting dry, the filters were neutralized by placing each on a 1.0 ml pool of 1 M Tris-HC1, pH 7.5 for 3 min, blotted dry, and reneutralised with fresh buffer. Th'-s was followed by two similar soakings (5 min each) on pools of 0.5 t4 Tris-HC1 pH 7.5 plus 1.5 M NaC1. After blotting dry, the DNA was UV crosslinked to the filter (as above), and the filzers -.:ere washed (25 C, 100 rpm) in about 100 ml of 3X SSC plus 0.1a;=~~v) SDS (4 times, 30 min each with fresh solution for each wash).
They were then placed in a minimal volume of prehybridlz3tion *Trade-mark soi':ticn (6X SSC plus 1l w.'v each of Fico11* 400 (Pharmacia;
polyvinylpyrroiidone (av. 4I.W. 360,000; Sigma ) and bovine ser,lm albumin Fraction V; (Sigma)J for 2 hr ac 65 C, 50 rpm. The prehybridization solution was removed, and replaced with the HB14 ''P-labeled probe that had been saved frcm the previous hybridization of the library membranes and which had been denatured at 95 C for 5 min. Hybridization was performed at 60 C
for 16 hr with shaking at 50 rpm.
Following removal of the labeled probe solution, the membranes were washed 3 times at 25 C (50 rpm, 15 min) in 3X SSC
(about 150 ml each wash). They were then washed for 3 hr ac 63 C
(50 rpm) in 0.25X SSC plus 0.2% SDS (minimal hyb wash solution), and exposed to X-ray film as described above for 1.5 hr at 25 C
(no enhancer screens). This exposure revealed very strong hybridization signals to cosmid isolates 22G12, 25A10, 26A5, and 26310, and a very weak signal with cosmid isolate 8310. No signal was seen wi:h the negative control (pWE15) colonies, and a very strong signal was seen with positive control membranes (DH5(X
cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.
AmDlification of a sDecific gencmic fragment of a c=a? gene.
Based on the N-terminal amino acid sequence determined for the purified TcaB, peptide fraction (disclosed here as SEQ ID 170:3) a pool of degenerate oligonucleotides (pool P8F) was synthesized as described for peptide TcaC. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents inosine:
>mino Acid Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg P8F 5' TTT ACI CA(A/G) ACI (C/T)TI AAA GAA CCI (A/C)C 3' (C/T)TI

Another set of degenerate oligonucleotides was synthesized (pool P8.108.3R), representing the complement of the coding strand for the determined amino acid seqsence of the TcaBi-PT108 internal peptide (disclosed herein as SEQ ID NO:20):
Amino Acid Met 'I'yr T'yr Ile Gln Ala GLn Gin *Trade-mark -53-Codons ATG TA(T/C) TA(T/C) AT(T/C/A) CA(?./G) GC(A/C/G!T) CA(A/G Ca(;;iG) c'8.1 3F. 3' AT(A/G) AT(A/G) TA(A/G/T) GT(T/C) CGI GT(T,'C) GT 5' TAC
These oligonucleotides were used as primers for PCR using HotStart 50 TubesTM' (Molecular Bio-Products, Inc., San Diego, CA) to amplify a specific DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above). A typical reaction (50 l) contained (bottom layer) 25 pmol of each primer pool PBF and P8.108.3R, with 2 nmol each of dATP, dCTP, dGTP, and dTTP, in 1X
GeneAmp'9 PCR buffer, and (top layer) 230 ng of genomic template DNA, 8 nmol each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of AmpliTaq'0 DNA polymerase, in 1X GeneAmpo PCR buffer.
Amplifications were performed by 35 cycles as described for the TcaC peptide. Amplification products were analyzed by electrophoresis through 0.7% w/v SeaKemA LE agarose (FMC) in TEA
buffer. A specific product of estimated size 1600 bp was observed.
Four such reactions were pooled, and the amplified DNA was extracted from a 1.0% SeaKee LE gel by Qiaex kit as described for the TcaC peptide. The extracted DNA was used directly as the template for sequence determination (PRISMTM Sequencing Kit) using, the P8F and P8.108.3R primer pools. Each reaction contained about 100 ng template DNA and 25 pmol of one primer pool, and was processed according to standard protocols as described for the TcaC peptide. An analysis of the sequence derived from extension of the P8F primers revealed the short DNA sequence (and encoded amino acid sequence):
GAT GCA TTG NTT GCT
Asp Ala Leu (Val) Ala which corresponds to a portion of the N-terminal peptide sequence disclosed as SEQ ID NO:3 (TcaBi).

Labeling of a TcaBi-peptide gene-specific probe.
Approximately 50 ng of gel-purified TcaBi DNA fragment was labeled with "-P-dCTP as described above, and nonincorporated radioisotopes were removed by passage through a NICK Column (Pharmacia). The specific activity of the labelled DNA was determined to be 6 x 10" dpm/ g. This labeled DNA was used to SUBSTITUTE SHEET (RULE 26) probe colony membranes prepared from members of the genomic library that had hybridized to the TcaC-peptide specific probe.
The membranes containing the 12 colonies identified in the TcaC-probe library screen (see above) were stripped of radioactive TcaC-specific label by boiling twice for approximately 30 min each time in 1 liter of 0.1X SSC plus 0.1 %
SDS. Removal of radiolabel was checked with a 6 hr film exposure. The stripped membranes were then incubated with the TcaBi peptide-specific probe prepared above. The labeled DNA was denatured by boiling for 10 min, and then added to the filters that had been incubated for 1 hr in 100 ml of "minimal hyb"
solution at 60 C. After overnight hybridization at this temperature, the probe solution was removed, and the filters were washed as follows (all in 0.3X SSC plus 0.1% SDS): once for 5 min at 25 C, once for 1 hr at 60 C in fresh solution, and once for 1 hr at 63 C in fresh solution. After 1.5 hr exposure to X-ray film by standard procedures, 4 strongly-hybridizing colonies were observed. These were, as with the TcaC-specific probe, isolates 22G12, 25A10, 26A5, and 26B10.
The same TcaBiprobe solution was diluted with an equal volume (about 100 ml) of "minimal hyb" solution, and then used to screen the membranes containing the 800 members of the genomic library. After hybridization, washing, and exposure to X-ray film as described above, only the four cosmid clones 22G12, 25A10, 26A5, and 26B10, were found to hybridize strongly to this probe.

ISOLATION OF SUBCLONES CONTAINING GENES ENCODING TcaC PlID
TcaBi PEPTIDES, AND DETERMINATION OF DNA BASE SEQUENCE THEP.EOF:
Three hybridization-positive cosmids in strain XL1 Blue MR were grown with shaking overnight (200 rpm) at 30 C in 100 ml TB-Ampt,,. After harvesting the cells by centrifugation, cosmid DNA
was prepared using a commercially available kit (BIGprepTM', 5 Prime 3 Prime, Inc., Boulder, CO), following the manufacturer's protocols. Only one cosmid, 26A5, was successfully isolated by this procedure. When digested with restriction enzyme EcoR 1 (NEB) and analyzed by gel electrophoresis, fragments of approximate sizes 14, 10, 8 (vector), 5, 3.3, 2.9, and 1.5 kbp were detected. A second attempt to isolate cosmid DNA from the 40 same three strains (8 ml cultures; TB-Ampluu, 30 C) utilized a -55-SUBSTITUTE SHEET (RULE 26) boiling miniprep method (Evans G. and G. Wahl., 1987, "Cosmid vectors for genomic walking and rapid restriction mapping." in Guide to Molecular Cloning Techniques. Meth. Enzymology, vol.
152, S. Berger and A. Kimmel, eds., pgs. 604-610). Only one cosmid, 25A10, was successfully isolated by this method. When digested with restriction enzyme EcoR 1(NEB) and analyzed by gel electrophoresis, this cosmid showed a fragmentation pattern identical to that previously seen with cosmid 26A5.
A 0.15 .g sample of 26A5 cosmid DNA was used to transform 50 ml of E. co1.i DH5a cells (Gibco BRL), by the supplier's protocols. A single colony isolate of that strain was inoculated into 4 ml of TB-Ampl:.,u, and grown for 8 hr at 37 C.
Chloramphenicol was added to a final concentration of 225 g/ml, incubation was continued for another 24 hr, then cells were harvested by centrifugation and frozen at -200C. Isolation of the 26A5 cosmid DNA was by a standard alkaline lysis miniprep (Maniatis et al., op. cit., p. 382), modified by increasing all volumes by 50% and with stirring or gentle mixing, rather than vortexing, at every step. After washing the DNA pellet in 70%
ethanol, it was dissolved in TE containing 25 g/ml ribonuclease A (Boehringer Mannheim).

Identification of EcoR 1 fragments hybridizing to GZ4-derived and TcaBi- probes. Approximately 0.4 g of cosmid 25A10 (from XL1 Blue MR cells) and about 0.5 g of cosmid 26A5 (from chloramphenicol-amplified DH5(x cells) were each digested with about 15 units of EcoR 1 (NEB) for 85 min, frozen overnight, then heated at 65 C for five min, and electrophoresed in a 0.7%
agarose gel (Seakem" LE, 1X TEA, 80 volts, 90 min). The DNA was stained with ethidium bromide as described above, and photographed under ultraviolet light. The EcoR 1 digest of cosmid 25A10 was a complete digestion, but the sample of cosmid 26A5 was only partially digested under these conditions. The agarose gel containing the DNA fragments was subjected to depurination, denaturation and neutralization, followed by Southern blotting onto a Magna NT nylon membrane, using a high salt (20X SSC) protocol, all as described in section 2.9 of Ausubel et al. (CPMB, op. cit.). The transferred DNA was then UV-crosslinked to the nylon membrane as before.

SUBSTITUTE SHEET (RULE 26) An TcaC-peptide specific DtIA fragment corresponding to the insert of plasmid isolate GZ4 was amplified by PCR in a 100 ml reaction volume as described previously above. The amplification products from three such reactions were pooled and were extracted from a 1% GTG' agarose gel by Qiaex kit, as described above, and quantitated by fluorometry. The gel-purified DNA (100 ng) was labeled with "P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) as described above, to a specific activity of 6.34 x 108 dpm/ g.
The 32P-labeled GZ4 probe was boiled 10 min, then added to "minimal hyb" buffer (at 1 ng/ml), and the Southern blot membranA
containing the digested cosmid DNA fragments was added, and incubated for 4 hr at 60 C with gentle shaking at 50 rpm. The membrane was then washed 3 times at 25 C for about 5 miri each (minimal hyb wash solution), followed by two washes for :0 min each at 60 C. The blot was exposed to film !with enhancer screens) for about 30 min at -70 C. The GZ4 probe hybridized strongly to the 5.0 kbp (apparent size) EcoR 1 fragmeiit ~.>f both these two cosmids, 26A5 and 25A10.
The membrane was stripped of radioactivity by boiling for about 30 min in 0.1X SSC plus 0.1 % SDS, and absence of radiolabel was checked by exposure to film. It was then hybridized at 60 C for 3.5 hours with the (denatured) TcaBi probe in "minimal hyb" buffer previously used for screening the colony membranes (above), washed as described previously, and exposed to film for 40 min at -70 C with two enhancer screens. With both cosmids, the TcaBi probe hybridized lightly with the about 5.0 kbp EcoR 1 fragment, and strongly with a fragment of approximately 2.9 kbp.
The sample of cosmid 26A5 DNA previously described, (from DH5a cells) was used as the source of D14A from which to subclone the bands of interest. This DNA (2.5 ug) was digested with about 3 units of EcoR 1 (NEB) in a total volume of 30 l for 1.5 hr, to give a partial digest, as confirmed by gel electrophoresis. Ten g of pBC KS (+) DNA (Stratagene) were digested for 1.5 hr with 20 units of EcoR 1 in a total volume of 20 l, leading to total digestion as confirmed by electrophoresis. Both EcoR 1-cut DNA
preparations were diluted to 50 l with water, to each an equal volume of PCI was added, the suspension was gently mixed, spun in SUBSTITUTE SHEET (RULE 26) a microcentrifuge and the aqueous supernatant was collected. Ltin was precipitated by 150 41 ethanol, and the mixture was placed ac -20 C overnight. Following centrifugation and drying, the EcoR
1-digested pBC KS (+) was dissolved in 100 41 TE; the partially digested 26A5 was dissolved in 20 l TE. DNA recovery was checked by fluorometry.
In separate reactions, approximately 60 ng of EcoR 1-digested pBC KS(+) DNA was ligated with approximately 180 ng or 270 ng of partially digested cosmid 26A5 DNA. Ligations were carried out in a volume of 20 l at 15 C for 5 hr, using T4 ligase and buffer from New England BioLabs. The ligation mixture, diluted to 100 l with sterile TE, was used to transform frozen, competent DH5a cells (Gibco BRL) according to the supplier's instructions. Varying amounts (25-200 l) of the transformed cells were plated on freshly prepared solid LB-Cam;S
medium with 1 mM IPTG and 50 mg/l X-gal. Plates were incubated at 37 C about 20 hr, then chilled in the dark for approximately 3 hr to intensify color for insert selection. White colonies were picked onto patch plates of the same composition and incubated overnight at 37 C.
Two colony lifts of each of the selected patch plates were prepared as follows. After picking white colonies to fresh plates, round Magna NT nylon membranes were pressed onto the patch plates, the membrane was lifted off, and subjected to denaturation, neutralization and W crosslinking as described above for the library colony membranes. The crosslinked colony lifts were vigorously washed, including gently wiping off the excess cell debris with a tissue. One set was hybridized with the GZ4(TcaC) probe solution described earlier, and the other set was hybridized with the TcaBi probe solution described earlier, according to the 'minimal hyb' protocol, followed by washing and film exposure as described for the library colony membranes.
Colonies showing hybridization signals either only with the GZ4 probe, with both GZ4 and TcaBi probes, or only with the TcaBi probe, were selected for further work and cells were streaked for single colony isolation onto LB-Cam35 media with IPTG and X-gal as before. Approximately 35 single colonies, from 16 different isolates, were picked into liquid LB-Cam35 media and grown SUBSTITUTE SHEET (RULE 26) overnight at 37 C; the cells were collected by centrifugation and plasmid DNA was isolated by a standard alkaline lysis miniprep according to Maniatis et al. (op. cit. p. 368). DNA pellets were dissolved in TE + 25 g/ml ribonuclease A and DNA concentration was determined by fluorometry. The EcoR 1 digestion pattern was analyzed by gel electrophoresis. The following isolates were picked as useful. Isolate A17.2 contains religated pBC F:S(+) only and was used for a (negative) control. Isolates D38.3 and C44.1 each contain only the 2.9 kbp, TcaBi -hybridizing EcoR 1 fragment inserted into pBC KS(+). These plasmids, named pDAB2000 and pDAB2001, respectively, are illustrated in Fig. 2.
Isolate A35.3 contains only the approximately 5 kbp, GZ4)-hybridizing EcoR 1 fragment, inserted into pBC KS(+). This plasmid was named pDAB2002 (also Fig. 2). These isolates provided templates for DNA sequencing.
Plasmids pDAB2000 and pDAB2001 were prepared using the BIGprepr" kit as before. Cultures (30 ml) were grown overnight in TB-Cam35 to an ODsoo of 2, then plasmid was isolated according to the manufacturer's directions. DNA pellets were redissolved in 100 ul TE each, and sample integrity was checked by EcoR 1 digestion and gel electrophoretic analysis.
Sequencing reactions were run in duplicate, with one replicate using as template pDAB2000 DNA, and the other replicate using as template pDAB2001 DNA. The reactions were carried out using the dideoxy dye terminator cycle sequencing method, as described above for the sequencing of the GZ4/HB14 DNAs. Initial sequencing runs utilized as primers the LacZ and T7 primers described above, plus primers based on the determined sequence of the TcaB1 PCR amplification product (TH1 =
aTTGCAGACTGCCAATCGCTTCGG, TH12 = GAGAGTATCCAGACCGCGGATGATCTG).
After alignment and editing of each sequencing output, each was truncated to between 250 to 350 bases, depending on the integrity of the chromatographic data as interpreted by the Perkin Elmer Applied Biosystems Division SeqEd 675 software.
Subsequent sequencing "steps" were made by selecting appropriate sequence for new primers. With a few exceptions, primers (synthesized as described above) were 24 bases in length with a 50% G+C
composition. Sequencing by this method was carried out on both strands of the approximately 2.9 kbp EcoR 1 fragment.

SUBSTITUTE SHEET (RULE 26) To further sert:e as template for DNA sequencing, plasmid DtrA
from isolate pDAB2002 was prepared by BIGprep"kit. Sequencing reactions were performed and analyzed as described above.
Initially, a T3 primer (pBS SK (+) bases 774-796:
CGCGCAATTAACCCTCACTAAAG) and a T7 primer (pBS KS (+) bases 621-643: GCGCGTAATACGACTCACTATAG) were used to prime the sequencing reactions from the flanking vector sequences, reading into the insert DNA. Another set of primers, (GZ4F:
GTATCGATTACAACGCTGTCACTTCCC; TH13: GGGAAGTGACAGCGTTGTAATCGATAC;
TH14: ATGTTGGGT(3CGTCGGCTAATGGACATAAC; and LW1-204:
GGGAAGTGACAGCGTTGTAATCGATAC) was made to prime from internal sequences, which were determined previously by degenerate oligonucleotide-mediated sequencing of subcloned TcaC-peptide PCR
products. From the data generated during the initial rounds of sequencing, new sets of primers were designed and used to walk the entire length of the -5 kbp fragment. A total of 55 oligo primers was used, enabling the identification of 4832 total bp of contiguous sequence.
When the DNA sequence of the EcoR 1 fragment insert of pDAB2002 is combined with part of the determined sequence of the pDaB2000/pDAB2001 isolates, a total contiguous sequence of 6005 bp was generated (disclosed herein as SEQ ID NO:25). When long open reading frames were translated into the corresponding amino acids, the sequence clearly shows the TcaBi N-terminal peptide (disclosed as SEQ ID NO:3), encoded by bases 19-75, immediately following a methionine residue (start of translation). Upstream lies a potential ribosome binding site (bases 1-9), and downstream, at bases 166-228 is encoded the TcaBi-PT158 internal peptide (disclosed herein as SEQ ID NO:19). Further downstream, in the same reading frame, at bases 1738-1773, exists a secr.ience encoding the TcaBi-PT108 internal peptide (disclosed herein as SEQ ID NO:20). Also in the same reading frame, at bases 1897-1923, is encoded the TcaBii N-terminal peptide (disclosed herein as SEQ ID NO:5), and the reading frame continues uninterrupted t~:) a translation termination codon at nucleotides 3586-3588.
The lack of an in-frame stop codon between the end of the sequence encoding TcaBi-PT108 and the start of the TcaBii encoding region, and the lack of a discernible ribosome binding site immediately upstream of the TcaBii coding region, indicate that SUBSTfTUTR SHEET (RULE 26) peptides TcaBii and TcaBi are encoded by a single open readir.-a frame of 3567 bp beginning at base pair 16 in SEQ ID NO:25), and are most likely derived from a single primary gene product of 1189 amino acids (131,586 Daltons; disclosed herein as SEQ ID
NO:26) by post-translational cleavage. If the amino acid immediately preceding the TcaBii N-terminal peptide represents the C-terminal amino acid of peptide TcaBi, then the predicted mass of TcaBii (627 amino acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28), somewhat higher than the size observed by SDS-PAGE (68 kDa). This peptide would be encoded by a contiguous stretch of 1881 base pairs (disclosed herein as SEQ ID
N0:27). It is thought that the native C-terminus of TcaBi lies somewhat closer to the C-terminus of TcaBi-PT108. The molecular mass of PT108 [3.438 kDa; determined during N-terminal amino acid sequence analysis of this peptidej predicts a size of 30 amino acids. Using the size of this peptide to designate the C-terminus of the TcaBi coding region [Glu at position 604 of SEQ
ID NO:281, the derived size of TcaBi is determined to be 604 amino acids or 68,463 Daltons, more in agreement with experimental observations.
Translation of the TcaBii peptide coding region of 1686 base pairs (disclosed herein as SEQ ID NO:29) yields a protein of 562 amino acids (disclosed herein as SEQ ID NO:30) with predicted mass of 60,789 Daltons, which corresponds well with the cbserved 61 kDa.
A potential ribosome binding site (bases 3633-3638) is found 48 bp downstream of the stop codon for the tcaB open reading frame. At bases 3645-3677 is found a sequence encoding the N-terminus of peptide TcaC, (disclosed as SEQ ID NO.2). The open reading frame initiated by this N-terminal peptide continues uninterrupted to base 6005 (2361 base pairs, disclosed herein as the first 2361 base pairs of SEQ ID NO.31). A gene (tcaC) encoding the entire TcaC peptide, (apparent size -165 kDa; -1500 amino acids), would comprise about 4500 bp.
Another isolate containing cloned EcoR 1 fragments of cosmid 26A5, E20.6, was also identified by its homology to the previously mentioned GZ4 and TcaBiprobes. Agarose gel analysis of EcoR 1 digests of the DNA of the plasmid harbored by this strain (pDAB2004, Fig. 2), revealed insert fragments of estimated SUBSTITUTE SHEET (RULE 26) sizes 2.9, 5, and 3.3 kbp. DNA sequence analysis initiated from primers designed from the sequence of plasmid pDAB2002 revealed that the 3.3 kbp EcoR 1 fragment of pDAB2004 lies adjacent to the kbp EcoR 1 fragment represented in pDAB2002. The 2361 base 5 pair open reading frame discovered in pDAB2002 continues uninterrupted for another 2094 bases in pDAB2004 [disclosed herPin as base pairs 2362 to 4458 of SEQ ID NO:31]. DNA sequence analysis using the parent cosmid 26A5 DNA as template confirmed the continuity of the open reading frame. Altogether, the open reading frame (TcaC SEQ ID NO:31) comprises 4455 base pairs, and encodes a protein (TcaC) of 1485 amino acids [disclosed herein as SEQ ID NO:32]. The calculated molecular size of 166,214 Daltons is consistent with the estimated size of the TcaC peptide (165 kDa), and the derived amino acid sequence matches exactly that disclosed for the TcaC N-terminal sequence [SEQ ID NO:2].
The lack of an amino acid sequence corresponding to SEQ ID
NO:17; used to design the degenerate oligonucleotide primer pool in the discovered sequence indicates that the generation of the PCR products found in isolates GZ4 and HB14, which were used as probes in the initial library screen, were fortuitously generated by reverse-strand priming by one of the primers in the degenerate pool. Further, the derived protein sequence does not include the internal fragment disclosed herein as SEQ ID NO:18. These sequences reveal that plasmid pDAB2004 contains the complete coding region for the TcaC peptide.

Example 9 Screening of the Photorhabdus genomic library for genes encoding the TcbAii peptide This example describes a method used to identify DNA clones that contain the TcbAii peptide-encoding genes, the isolation of the gene, and the determination of its partial DNA base sequence.
Primers and PCR reactions The TcbAii polypeptide of the insect active preparation is -206 kDa. The amino acid sequence of the N-terminus of this peptide is disclosed as SEQ ID NO:l. Four pools of degenerate oligonucleotide primers ("Forward primers": TH-4, TH-5, TH-6, and SUBSTITUTE SHEET (RULE 26) TH-7) were synthesized to encode a portion of this amino acid sequence, as described in Example 8, and are shown below.

Table 11 Amino Acid Phe Ile Gln Gly Tyr Ser Asp Leu Phe TH-4 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) CTI TT-3' TH-5 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) AG(T/C) GA(T/C) CTI TT-3' }
TH-6 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) TT(A/G) TT-3' TH-7 5'-7P(T/C) ATI CA(A/G) GGI TA(T/C) AG(T/C) GA(T/C) TT(A/G) TT-3' In addition, a primary ("a") and a secondary ("b") sequence of an internal peptide preparation (TcbAii-PT81) have been determined and are disclosed herein as SEQ ID No:23 and SEQ ID
No:24, respectively. Four pools of degenerate oligonucleotides ("Reverse Primers": TH-8, TH-9, TH-10 and TH-11) were similarly designed and synthesized to encode the reverse complement of sequences that encode a portion of the peptide of SEQ ID NO:23, as shown below.

SUBSTITUTE SHEET (RULE 26) u, in u~ in 1 1 , a [~ [~
F F

!d H H H H
~ U U U U
DD C.J U U U

U U U U
E4 Ei E+ E-4 q '.

U U U U
E-4 E-4 Ey E-+
U U U U
v U
a c~ t~ t~ t~
F in a a a a a ~a a~ a~ ~a a Q

f1 H H ~ ~
4) 0 C.D H V
E-+

.C7 C7 CH7 CH7 E E H E+ Eq ~-~l ta7 Ca7 C7 t~ C7 C~
~ a' RC Q
Q E-, Q
H H H H
C7 C7 C.~ C7 E E+ E E-4 [-1 M M M M
O O
a 'Ll W 01 -i .-~
=rl =~ 1 1 . ~ a F F F F

SUBSTITUTE SHEET (RULE 26) Sets of these primers were used in PCR reactions to ampii_y TcbAii- encoding gene fragments from the genomic Photorhabcius luminescens W-14 DNA prepared in Example 6. All PCR10 reactions were run with the "Hot Start" technique using AmpliWaxT'" gems and other Perkin Elmer reagents and protocols. Typically, a mixture (total volume 11 l) of MgCl, dNTP's, 10X GeneAmp"0 PCR Buffer II, and the primers were added to tubes containing a single wax beaa.
[lOX GeneAmp9 PCR Buffer II is composed of 100 mM Tris-HCI, pH
8.3; and 500 mM KC1.] The tubes were heated to 80 C for 2 minutes and allowed to cool. To the top of the wax seals, a solution containing lOX GeneAmp'0 PCR Buffer II, DNA template, and AmpliTaql DNA polymerase were added. Following melting of the wax seal and mixing of components by thermal cycling, finai reactioii conditions (volume of 50 l) were: 10 mM Tris-HC1, pH 8.3; 50 iiit-1 KC1; 2.5 mM MgC12; 200 M each in dATP, dCTP, dGTP, dTTP; 1.25 mM
in a single Forward primer pool; 1.25 M in a single Reverse primer pool, 1.25 units of AmpliTaq' DNA polymerase, and 170 ng of template DNA.
The reactions were placed in a thermocycler (as in Example 8) and run with the following program:

Table 13 Temperature Time Cycle Repetition 94 C 2 minutes 1X
94 C 15 seconds 55-65 C 30 seconds 30X

72 C 1 minute 72 C 7 minutes ix 15 C Constant SUBSTITUTE SHEET (RULE 26) A series of amplifications was run at three different annealing temperatures (55 , 600, 65 C) using the degenerate primer pools. Reactions with annealing at 65 C had no amplification products visible following agarose gel electrophoresis. Reactions having a 60 C annealing regime and containing primers TH-5+TH-10 produced an amplification product that had a mobility corresponding to 2.9 kbp. A lesser amount of the 2.9 kbp product was produced under these conditioris with primers TH-7+TH-10. When reactions were annealed at 55 C, these primer pairs produced more of the 2.9 kbp product, and this product was also produced by primer pairs TH-5+TH-8 and TH-5+TH-11. Additional very faint 2.9 kbp bands were seen in lanes containing amplification products from primer pairs TH-7 plus TH-8, TH-9, TH-10, or TH-11.
To obtain sufficient PCR amplification product for cloning and DNA sequence determination, 10 separate PCR reactions were set up using the primers TH-5+TH-i0, and were run using the above conditions with a 55 C annealing temperature. All reactions were pooled and the 2.9 kbp product was purified by Qiaex extraction from an agarose gel as described above.
Additional sequences determined for TcbAii internal peptides are disclosed herein as SEQ ID NO:21 and SEQ ID NO:22. As before, degenerate oligonucleotides (Reverse primers TH-17 and TH-18) were made corresponding to the reverse complement of sequences that encode a portion of the amino acid sequence of these peptides.

Table 14 From SEQ ID NO:21 Amino Acid Met Glu Thr Gln Asn Ile Gln Glu Pro TH-17 3'-TAC CTT/C TGI GTT/C TTA/G TAI GTT/C GTT/C GG-5' Table 15 From SEQ ID NO:22 = Amino Acid Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp TH-18 3'-TT(A/G) GGI TAI TT(A/G) TAI TT(A?G) TGI CCI TAI CT(A/G)-5' SUBSTITUTE SHEET (RULE 26) Deoenerate oliaonucleotides TH-18 and TH-17 were used in an amplification experiment with Photorhabcius luminescens W-14 DN.y as template and primers TH-4, TH-5, TH-6, or TH-7 as the 51-(Forward) primers. These reactions amplified products of approximately 4 kbp and 4.5 kbp, respectively. These DNAs were transferred from agarose gels to nylon membranes and hybridized sx:ith a''P-labeled probe (as described above) prepared from the 2.9 kbp product amplified by the TH-5+TH10 primer pair. Both the 4 kbp and the 4.5 kbp amplification products hybridized strongly to the 2.9 kbp probe. These results were used to construct a map ordering the TcbAii internal peptide sequences as shown in Fig. 3. Approximate distances between the primers are shown in nucleotides in Fig. 3.

DNA Sequence of the 2.9 kbp TcbAii-encoding fragment Approximately 200 ng of the purified 2.9 kbp fragment (prepared above) was precipitated with ethanol and dissolved in 17 ml of water. One-half of this was used as sequencing template with 25 pmol of the TH-5 pool as primers, the other half was used as template for TH-10 priming. Sequencing reactions were as given in Example B. No reliable sequence was produced using the TH-10 primer pool; however, reactions with TH-5 primer pool produced the sequence disclosed below:.

61 TATTNGAGGG At1TNGTCCCG TGAGGCCAAA AANTGGAATG AAAGAAGTTC AATTTNTTAC
121 CTAGATAAAC GTCGCCCGGtI TTTAGAAAGN TTANTGNTCA GCCAGAAAAT TTTGGTTGAG
181 GAAATTCCAC CGNTGGTTCT CTCTATTGAT TNGGGCCTGG CCGGGTTCGA ANNP.AAACNA

301 CCTTNTCANC ACGNTTNTGA AACTGTCCGG GAAATCGTCC ATGANCGTGA fICCAGGNTTPI

Based on this sequence, a sequencing primer (TH-21, 5'-CCGGGCGACGTTTATCTAGG-3') was designed to reverse complement bases 120-139, and initiate polymerization towards the 5' end (i.e., TH-5 end) of the gel-purified 2.9 kbp TcbAii-encoding PCR
fraament. The determined sequence is shown below, and is compared to the biochemically determined N-terminal peptide sequence of TcbAii SEQ ID NO:1.

SUBSTITUTE SHEET (RULE 26) T,::b,4.ii 2.9 kbD PCR fragment Sequence Confirmation [L'nderlined amino acids = encoded by degenerate oligonucieotides;
SEQ ID NO:l F I Q G Y S D L F G - - A

2.9 kbp seq GC ATG CAG GGG TAT AGT GAC CTG TTT GGT AAT CGT GCT
M Q G Y S D L F G N R A:From the homology of the derived amino acid sequence to the = 10 biochemically determined one, it is clear that the 2.9 kbp PCR
fragment represents the TcbA coding region. This 2.9 kbp fragment was then used as a hybridization probe to screen the Photorhabdus W-14 genomic library prepared in Example 8 for cosmids containing the TcbAii-encoding gene.
Screening the Photorhabdus cosmid library The 2.9 kb gel-purified PCR fragment was labeled with ''P
using the Boehringer Mannheim High Prime labeling kit as described in Example 8. Filters containing remnants of approximately 800 colonies from the cosmid library were screened as described previously (Example 8), and positive clones were streaked for isolated colonies and rescreened. Three clones (8A11, 25G8, and 26D1) gave positive results through several screening and characterization steps. No hybridization of the TcbAii-specific probe was ever observed with any of the four cosmids identified in Example 8, and which contain the tcaB and tcaC genes. DNA from cosmids 8A11, 25G8, and 26D1 was digested with restriction enzymes Bgl 2, EcoR 1 or Hind 3 (either alone or in combination with one another), and the fragments were separated on an agarose gel and transferred to a nylon membrane as described in Example 8. The membrane was hybridized with 'P-labeled probe prepared from the 4.5 kbp fragment (generated by amplification of Photorhabdus genomic DNA with primers TH-5+TH-17). The patterns generated from cosmid DNAs 8Al1 and 26D1 were identical to those generated with similarly-cut genomic DNA on the same membrane. It is concluded that cosmids 8A11 and 26D1 are accurate representations of the genomic TcbAii encoding locus. However, cosmid 25G8 has a single Bgl 2 fragment which is slightly larger than the genomic DNA. This may result from positioning of the insert within the vector.

SUBSTITUTE SHEET (RULE 26) DNA sequence of the tcbA-encoding gene The membrane hybridization analysis of cosmid 26D1 revealed that the 4.5 kbp probe hybridized to a single large EcoR 1 fragment (greater than 9 kbp). This fragment was gel purified and ligated into the EcoR 1 site of pBC KS (+) as described in Example 8, to generate plasmid pBC-S1/R1. The partial DNA
sequence of the insert DNA of this plasmid was determined by "primer walking" from the flanking vector sequence, using procedures described in Example 8. Further sequence was generated by extension from new oligonucleotides designed from the previously determined sequence. When compared to the determined DNA sequence for the tcbA gene identified by other methods (disclosed herein as SEQ ID NO:ll as described in Example 12 below), complete homology was found to nucleotides 1-272, 319-826, 2578-3036, and 3068-3540 (total bases = 1712). It was concluded that both approaches can be used to identify DNA
fragments encoding the TcbAii peptide.

Analysis of the derived amino acid sequence of the tcbA gene.
The sequence of the DNA fragment identified as SEQ ID NO:ll encodes a protein whose derived amino acid sequence is disclosed herein as SEQ ID NO:12. Several features verify the identity of the gene as that encoding the TcbAii protein. The TcbAii N-terminal peptide (SEQ
ID NO:1; Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is encoded as amino acids 88-100. The TcbAii internal peptide TcbAii-PT81(a) (SEQ ID NO:23) is encoded as amino acids 1065-1077, and TcbAii-PT81(b) (SEQ ID NO:24) is encoded as amino acids 1571-1592. Further, the internal peptide TcbAii-PT56 (SEQ ID NO:22) is encoded as amino acids 1474-1488, and the internal peptide TcbAii-PT103 (SEQ ID NO:24) is encoded as amino acids 1614-1639. It is obvious that this gene is an authentic clone encoding the TcbAii peptide as isolated from insecticidal protein preparations of Photorhabdus luminescens strain w-14.
The protein isolated as peptide TcbAii is derived from cleavage of a longer peptide. Evidence for this is provided by the fact that the nucleotides encoding the TcbAii N-terminal peptide SEQ ID NO:1 are preceded by 261 bases (encoding 87 N-terminal-proximal amino acids) of a longer open reading frame (SEQ ID NO:11). This reading frame begins with nucleotides that encode the amino acid sequence Met Gln Asn Ser -69- =
SUBSTITUTE SHEET (RULE 26) Leu, which corresponds to the N-terminal sequence of the large peptide TcbA, and is disclosed herein as SEQ ID NO:16. It is thought that TcbA is the precursor protein for TcbAii' Relationship of tcbA, tcaB and tcaC genes.

The tcaB and tcaC genes are closely linked and may be transcribed as a single mRNA (Example 8). The tcbA gene is borne on cosmids that apparently do not overlap the ones harboring the tcaB and tcaC cluster, since the respective genomic library screens identified different cosmids.

However, comparison of the amino sequences encoded by the tcaB
and tcaC genes with the tcbA gene reveals a substantial degree of homology. The amino acid conservation (Protein Alignment Mode of MacVectorTM Sequence Analysis Software, scoring matrix pam250, hash value = 2; Kodak Scientific Imaging Systems, Rochester, NY) is shown in Fig. 4A and Fig 4B. On the score line of each panel in Fig. 4A and Fig. 4B, up carats (~) indicate homology or conservative amino acid changes, and down carats (v) indicate nonhomology.

This analysis shows that the amino acid sequence of the TcbA peptide from residues 1739 to 1894 is highly hornologous to amino acids 441 to 603 of the TcaBi peptide (162 of the total 627 amino acids of P8; SEQ ID NO:28). In addition, the sequence of TcbA amino acids 1932 to 2459 is highly homologous to amino acids 12 to 531 of peptide TcaBii (520 of the total 562 amino acids; SEQ ID NO:30). Considering that the TcbA peptide (SEQ ID NO:12) comprises 2505 amino acids, a total of 684 amino acids (27%) at the C-proximal end of it is homologous to the TcaBi or TcaBii peptides, and the homologies are arranged colinear to the arrangement of the putative TcaB preprotein (SEQ ID NO:26). A sizeable gap in the TcbA homology coincides with the Junction between the TcaBi and TcaBii portions of the TcaB preprotein. Clearly the TcbA and TcaB gene products are evolutionarily related, and it is proposed that they share some common function(s) in Photorhabdus.

- 70a -Example 10 Characterization of zinc-metalloproteases in Photorhabdus Broth:
Protease Inhibition, Classification, and Purification Protease Inhibition and Classification Assays: Protease assays were performed using FITC-casein dissolved in water as substrate (0.08% final assay concentration). Proteolysis reactions were performed at 25 C for 1 h in the appropriate buffer with 25 ul of Photorhabdus broth (150 til total reaction volume). Samples were also assayed in the presence and absence of dithiothreitol. After incubation, an equal volume of 12%
trichloroacetic acid was added to precipitate undigested protein.
Following precipitation for 0.5 h and subsequent centrifugation, 100 ul of the supernatant was placed into a 96-well microtiter plate and the pH of the solution was adjusted by addition of an equal volume of 4N NaOH. Proteolysis was then quantitated using a Fluoroskan II fluorometric plate reader at excitation and emission wavelengths of 485 and 538 nm, respectively. Protease activity was tested over a range from pH 5.0-10.0 in 0.5 units increments. The following buffers were used at 50 mM final concentration: sodium acetate (pH 5.0 - 6.5); Tris-HCL (pH 7.0 -8.0); and bis-Tris propane (pH 8.5-10.0). To identify the class of protease(s) observed, crude broth was treated with a variety of protease inhibitors (0.5 ug/ul final concentration) and then examined for protease activity at pH 8.0 using the substrate described above. The protease inhibitors used included E-64 (L-trans-expoxysaccinylleucylamido[4-,-guanidino]-butane), 3,4 dichloroisocoumarin, Leupeptin, pepstatin, amastatin, ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline.
Protease assays performed over a pH range revealed that indeed protease(s) were present which exhibited maximal activity at - pH 8.0 (Table 16). Addition of DTT did not have any effect on protease activity. Crude broth was then treated with a variety of protease inhibitors (Table 17). Treatment of crude broth with the inhibitors described above revealed that 1,10 phenanthroline caused complete inhibition of all protease activity when added at a final concentration of 50 pg, with the IC50 = 5 ug in 100 ul of a 2 mg/ml crude broth solution. These =
data indicate that the most abundant protease(s) found in the SUBSTITUTE SHEET (RULE 26) Photoriiabdus broth are from the zinc-metalloprotease class cf enzymes.

Table 16 Effect of pH on the protease activity found in a Day 1 production of Photorhabdus luminescens (strain W-14).

pH Flu. Unitsa Percent Activityb 5.0 3013 78 17 5.5 7994 448 45 6.0 12965 483 74 6.5 14390 1291 82 7.0 14386 1287 82 7.5 14135 198 80 8.0 17582 831 100 8.5 16183 953 92 9.0 16795 760 96 9.5 16279 1022 93 10.0 15225 210 87 a Flu. Units = Fluorescence Units (Maximum =--28,000;
background = - 2200).
b. Percent activity relative to the maximum at pH 8.0 SUBSTITUTE SHEET (RULE 26) Table 17 Effect of different protease inhibitors on the protease activitl at pH 8 found in a Day 1 production of Photorhabdus lurninescens (strain W-14).
Inhibitor Corrected Flu. Unitsa Percent Inhibitionb Control 13053 0 1,10 Phenanthrolinec 15 99 =
3,4 Dichloroisocoumarind 7956 39 Leupeptin 13074 0 Pepstatinc 13441 0 Amastatin 12474 4 DMSO Control 12005 8 Methanol Control 12125 7 a. Corrected Flu. Units = Fluorescence Units -background(2200 flu. units).
b. Percent Inhibition relative to protease activity at pH
8Ø
c. Inhibitors were dissolved in methanol.
d. Inhibitors were dissolved in DMSO.

The isolation of a zinc-metalloprotease was performed by applying dialyzed 10-80% ammonium sulfate pellet to a Q Sepharose column equilibrated at 50 mM Na2PO4, pH 7.0 as described in Example 5 for Photorhabdus toxin. After extensive washing, a 0 to 0.5 M NaCl gradient was used to elute toxin protein. The majority of biological activity and protein was eluted from 0.15 - 0.45 M NaCl. However, it was observed that the majority of proteolytic activity was present in the 0.25-0.35 M NaCl traction with some activity in the 0.15-0.25 M NaCl fraction. SDS PAGE
analysis of the 0.25-0.35 M NaCl fraction showed a major peptide band of approximately 60 kDa. The 0.15-0.25 M NaCl fraction contained a similar 60 kDa band but at lower relative protein concentration. Subsequent gel filtration of this fraction using a Superose 12 HR 16/50 column resulted in a major peak migratirig at 57.5 kDa that contained a predominant (> 90% of total stained protein) 58.5 kDa band by SDS PAGE analysis. Additional analysis of this fraction using various protease inhibitors as described above determined that the protease was a zinc-metalloprotease.
Nearly all of the protease activity present in Photorhabdus broth at day 1 of fermentation corresponded to the -58 kDa zinc-metalloprotease.
In yet a second isolation of zinc-metalloprotease(s), W-14 Photorhabdus broth grown for three days was taken and protease SUBSTITUTE SHEET (RULE 26) activity was visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) laced with gelatin as described in Schmidt, T.M., Bleakley, B. and Nealson, K.M.
1988. SDS running gels (5.5 x 8 cm) were made with 12.5 %
polyacrylamide (40% stock solution of acrylamide/bis-acr-ylamide;
Sigma Chemical Co., St. Louis, MO) into which 0.1% gelatin final concentration (Biorad EIA grade reagent; Richmond CA) was incorporated upon dissolving in water. SDS-stacking gels (1.0 x 8 cm) were made with 5% polyacrylamide, also laced with 0.1%
gelatin. Typically, 2.5 g of protein to be tested was diluted in 0.03 ml of SDS-PAGE loading buffer without dithiothreitol (DTT) and loaded onto the gel. Proteins were electrophoresed in SDS running buffer (Laemmli, U.K. 1970. Nature 227, 680) at 00 C
and at 8 mA. After electrophoresis was complete, the gel was washed for 2 h in 2.5% (v/v) Triton X-100. Gels were then incubated for 1 h at 37 C in 0.1 M glycine (pH 8.0). After incubation, gels were fixed and stained overnight with 0.1% amido black in methanol-acetic acid- water (30:10:60, vol./vol./vol.;
Sigma Chemical Co.). Protease activity was visualized as light areas against a dark, amido black stained background due to proteolysis and subsequent diffusion of incorporated gelatin. At least three distinct bands produced by proteolytic activity at 58-, 41-, and 38 kDa were observed.
Activity assays of thedifferent proteases in W-14 day three culture broth were performed using FITC-casein dissolved in water as substrate (0.02% final assay concentration). Proteolysis experiments were performed at 37. C for 0-0.5 h in 0.1M Tris-HC1 (pH 8.0) with different protein fractions in a total volume of 0.15 ml. Reactions were terminated by addition of an equal volume of 12% trichloroacetic acid (TCA) dissolved in water.
After incubation at room temperature for 0.25 h, samples were centrifuged at 10,000 x g for 0.25 h and 0.10 ml aliquots were removed and placed into 96-well microtiter plates. The solution was then neutralized by the addition of an equal volume of 2 21 sodium hydroxide, followed by quantitation using a Fluoroskan II
fluorometric plate reader with excitation and emission wavelengths of 485 and 538 nm, respectively. Activity measurements were performed using FITC-Casein with different protease concentrations at 37 C for 0-10 min. A unit of SUBSTITUTE SHEET (RULE 26) activity was arbitrarily defined as the amount of enzyme needed to produce 1000 fluorescent units/min and specific activity was defined as units/mg of protease.
Inhibition studies were performed using two zinc-metalloprotease inhibitors; 1,10 phenanthroline and N-(a-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp(phosphoramidon) with stock solutions of the inhibitors dissolved in 100% ethanol and water, respectively. Stock concentrations were typically 10 mg/ml and 5 mg/mi for 1,10 phenanthroline and phosphoramidon, respectively, with final concentrations of inhibitor at 0.5-1.0 mg/ml per reaction. Treatment of three day W-14 crude broth with 1,10 phenanthroline, an inhibitor of all zinc metalloproteases, resulted in complete elimination of all protease activity while treatment with phosphoramidon, an inhibitor of thermolysin-like proteases (Weaver, L.H., Kester, W.R., and Matthews, B.W. 1977.
J. Mol. Biol. 114, 119-132), resulted in -56% reduction of protease activity. The residual proteolytic activity could not be further reduced with additional phosphoramidon.
The proteases of three day W-14 Photorhabdus broth were purified as follows: 4.0 liters of broth were concentrated using an Amicon spiral ultra filtration cartridge Type SlYlOO attached to an Amicon M-12 filtration device. The flow-through material having native proteins less than 100 kDa in size (3.8 L) was concentrated to 0.375 L using an Amicon spiral ultra filtration cartridge Type S1Y10 attached to an Amicon M-12 filtration device. The retentate material contained proteins ranging in size from 10-100 kDa. This material was loaded onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem (Framington, MA) Poros 50 HQ strong anion exchange packing that had been equilibrated in 10 mM sodium phosphate buffer (pH 7.0).
Proteins were loaded on the column at a flow rate of 5 ml/min, followed by washing unbound protein with buffer until A280 =
0.00. Afterwards, proteins were eluted using a NaCl gradient of 0-1.0 M NaCl in 40 min at a-flow rate of 7.5 ml/min. Fractions were assayed for protease activity, supra., and active fractions were pooled. Proteolytically active fractions were diluted with 50% (v/v) 10 mM sodium phosphate buffer (pH 7.0) and loaded onto a Pharmacia HR 10/10 Mono Q column equilibrated in 10 mM sodium phosphate. After washing the column with buffer until A280 -SUBSTITUTE SHEET (RULE 26) 0.00, proteins were eluted using a NaCl gradient of 0-0.5 c4 tIaC1 for I h at a flow rate of 2.0 ml/min. Fractions were assayed for procease activity. Those fractions having the greatest amount of phosphoramidon-sensitive protease activity, the phosphoramidon sensitive activity being due to the 41/38 kDa protease, infra., were pooled. These fractions were found to elute at a range of 0.15-0.25 M NaCl. Fractions containing a predominance of phosphoramidon-insensitive protease activity, the 58 kDa protease, were also pooled. These fractions were found to elute at a range of 0.25-0.35 M NaC1. The phosphoramidon-sensitive procease fractions were then concentrated to a final volume of 0.75 ml using a Millipore Ultrafree -15 centrifugal filter device Bioma:c 5K NMWL membrane. This material was applied at a flow rate of 0.5 ml/min to a Pharmacia HR 10/30 column that had been packed with Pharmacia Sephadex G-50 equilibrated in 10 mM sodium phosphate buffer (pH 7.0)/ 0.1 H NaC1. Fractions having the maximal phosphoramidon-sensitive protease activity were then pooled and centrifuged over a Millipore Ultrafree4D-15 centrifugal filter device Biomax-50K NMWL membrane. Proteolytic activity analysis, supra., indicated this macerial to have only phosphoramidon-sensitive protease activity. Pooling of the phosphoramidon-insensitive protease, the 58 kDa protein, was followed by concentrating in a Millipore Ultrafree -15 centrifugal filter device Biomax-S0K NMWL membrane and further separation on a Pharmacia Superdex-75 column. Fractions containing the protease were pooled.
Analysis of purified 58- and 41/38 kDa purified proteases re-fealed that, while both types of protease were completely inhibited with 1,10 phenanthroline, only the 41/38 kDa protease was inhibited with phosphoramidon. Further analysis of crude broth indicated that protease activity of day 1 w-14 broth has 23% of the total protease activity due to the 41/38 kDa protease, increasing to 44% in day three W-14 broth.
Standard SDS-PAGE analysis for examining protein purity and obtaining amino terminal sequence was performed using 4-20%
gradient MiniPlus SepraGels purchased from Integrated Separation Systems (Natick, MA). Proteins to be amino-terminal sequenced were blotted onto PVDF membrane following purification, infra., (ProBlottr" Membranes; Applied Siosystems, Foster City, CA), Trade-mark :Tisualized with 0.1% amido black, excised, and sent to cambridge Prochem; Cambridge, b1A, for sequencing.
Deduced amino terminal sequence of the 58- (SEQ ID NO:45) and 41/38 kDa (SEQ ID NO:44) proteases from three day old W-14 broth were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ
ID NO:44), respectively. Sequencing of the 41/38 kDa protease revealed several amino termini, each one having an additional amino acid removed by proteolysis. Examination of the primary, secondary, tertiary and quartenary sequences for the 38 and 41 kDa polypeptides allowed for deduction of the sequence shown above and revealed that these two proteases are homologous.

Example 11, Part A
Screening of Photorhabdus Genomic Library via use of Antibodies for Genes encoding TcbA Peptide In parallel to the sequencing described above, suitable probing and sequencing was done based on the TcbAii peptide (SEQ
ID NO:1). This sequencing was performed by preparing bacterial culture broths and purifying the toxin as described in Examples 1 and 2 above.
Genomic DNA was isolated from the Photorhabdus luminescens strain W-14 grown in Grace's insect tissue culture medium. The bacteria were grown in 5 ml of culture medium in a 250 ml Erlenmeyer flask at 28 C and 250 rpm for approximately 24 hours.
Bacterial cells from 100 ml of culture medium were pelleted at 5000 x g for 10 minutes. The supernatant was discarded, and the cell pellets then were used for the genomic DNA isolation.
The genomic DNA was isolated using a modification of the CTAB method described in Section 2.4.3 of Ausubel (supra.). The section entitled "Large Scale CsCl prep of bacterial genomic DT1A"
was followed through step 6. At this point, an additional chloroform/isoamyl alcohol (24:1) extraction was performed followed by a phenol/chloroform/isoamyl (25:24:1) extraction step and a final chloroform/isoamyl/alcohol (24:1) extraction. The DNA was precipitated by the addition of a 0.6 volume of isopropanol. The precipitated DNA was hooked and wound around the end of a bent glass rod, dipped briefly into 70% ethariol as a final wash, and dissolved in 3 ml of TE buffer. -77-SUBSTITUTE SHEET (RULE 26) The DNA concentration, estimated by optical density at 280/260 nm, was approximately 2 mg/ml.
Using this genomic DNA, a library was prepared.
Approximately 50 ug of genomic DNA was partly digested with Sau3 Al. Then NaCl density gradient centrifugation was used to size ' fractionate the partially digested DNA fragments. Fractions ~ containing DtIA fragments with an average size of 12 kb, or larger, as determined by agarose gel electrophoresis, were ligated into the plasmid BluScript, Stratagene, La Jolla, California, and transformed into an E. coli DH5a or DHBIO strain.
Separately, purified aliquots of the protein were sent to the biotechnology hybridoma center at the University of Wisconsin, Madison for production of monoclonal antibodies to the proteins. The material that was sent was the HPLC purified fraction containing native bands 1 and 2 which had been denatured at 65 C, and 20 g of which was injected into each of four mice.
Stable monoclonal antibody-producing hybridoma cell lines were recovered after spleen cells from unimmunized mouse were fused with a stable myeloma cell line. Monoclonal antibodies were recovered from the hybridomas.
Separateiy, polyclonal antibodies were created by taking native agarose gel purified band 1 (see Example 1) protein which was then used to immunize a New Zealand white rabbit. The protein was prepared by excising the band from the native agarose gels, briefly heating the gel pieces to 65 C to melt the agarose, and immediately emulsifying with adjuvant. Freund's complete adjuvant was used for the primary immunizations and Freund's incomplete was used for 3 additional injections at monthly intervals. For each injection, approximately 0.2 ml of emulsified band 1, containing 50 to 100 micrograms of protein, was delivered by multiple subcontaneous injections into the back of the rabbit. Serum was obtained 10 days after the final injection and additional bleeds were performed at weekly intervals for 3 weeks. The serum complement was inactivated by heating to 56 C for 15 minutes and then stored at -20 C.
The monoclonal and polyclonal antibodies were then used to screen the genomic library for the expression of antigens which could be detected by the epitope. Positive clones were detected on nitrocellulose filter colony lifts. An immunoblot analysis of the positive clones was undertaken.

SUBSTITUTE SHEET (RULE 26) An analysis of the clones as defined by both immunoblot and Southern analysis resulted in the tentative identification of five classes of clones.

In the first class of clone was a gene encoding the peptide designated here as TcbAii. Full DNA sequence of this gene (TcbA) was obtained. It is set forth as SEQ ID NO:11.
Confirmation that the sequence encodes the internal sequence of SEQ ID NO:l is demonstrated by the presence of SEQ ID NO:1 at amino acid number 88 from the deduced amino acid sequence created by the open reading frame of SEQ ID NO:11. This can be confirmed by referring to SEQ ID NO:12, which is the deduced amino acid sequence created by SEQ ID NO:11.

The second class of toxin peptides contains the segments referred to above as TcaBi, TcaBii and TcaC (Fig.
6A). Following the screening of the library with the polyclonal antisera, this second class of toxin genes was identified by several clones which produced different size proteins, all of which cross-reacted with the polyclonal antibody on an immunoblot and were also found to share DNA

homology on a Southern Blot. Sequence comparison revealed that they belonged to the gene complex designated TcaB and TcaC above.

Three other classes of antibody toxin clones were also isolated in the polyclonal screen. These classes produced proteins that cross-react with a polyclonal antibody and also shared DNA homology with the classes as determined by Southern blotting. The classes have been designated Class III, Class IV and Class V. It was also possible to identify monoclonals that cross-reacted with Class I, II, III, and IV.
This suggests that all have regions of high protein homology.
Thus, it appears that the P. luminescens extracellular protein genes represent a family of genes which are evolutionarily related.

To further pursue the concept that there might be evolutionarily related variations in the toxin peptides contained within this organism, two approaches have been undertaken to examine other strains of P. luminescens for the presence of related proteins. This was done both by PCR
amplification of genomic DNA and by immunoblot analysis using the polyclonal and monoclonal antibodies.

- 79a -The results indicate that related proteins are produced b,=
P. luminescens strains WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, 'r7X-?, WX-11, WX-12, WX-15 and W-14.

Example 11, Part B
Sequence and anaylsis of C1ass III toxin clones - rcc Further DNA sequencing was performed on plasmids isolated from Class III E. coli clones described in Example 11, Part A.
The nucleotide sequence was shown to be three closely linked open reading frames at this genomic locus. This locus was designated tcc with the three open reading frames designated tccA SEQ ID
NO:56, tccB SEQ ID NO:58 and tccC SEQ ID NO:60 (Fig. 6B).
The deduced amino acid from the tccA open reading frame indicates the gene encodes a protein of 105,459 Da. This protein was designated TccA. The first 12 amino acids of this protein match the N-terminal sequence obtained from a 108 kDa protein, SEQ ID NO:7, previously identified as part of the toxin complex.
The deduced amino acid from the tccB open reading frame indicates this gene encodes a protein of 175,716 Da. This protein was designated TccB. The first 11 amino acids of this protein match the N-terminal sequence obtained from a protein with estimated molecular weight of 185 kDa, SEQ ID NO:8.
The deduced amino acid sequence of tccC indicated that this open reading frame encodes a protein of 111,694 Da and the protein product was designated TccC.

Example 12 Characterization of Photorhabdus Strains In order to establish that the collection described herein was comprised of Photorhabdus strains, the strains herein were assessed in terms of recognized microbiological traits that are characteristic of Photorhabdus and which differentiate it from other Enterobacteriaceae and Xenorhabdus spp. (Farmer, J.J. 1984.
Bergey's Manual of Systemic Bacteriology, vol 1. pp. 510-511.
(ed. Kreig N.R. and Holt, J.G.). Williams & Wilkins, Baltimore.;
, Akhurst and Boemare, 1988, Boemare et al., 1993). These characteristic traits are as follows: Gram's stain negative SUBSTITUTE SHEET (RULE 26) roo+=s, .~rganism size of 0.5-2 um in width and 2-10 um in !_ngth, red;yellow colcny pigmencacicn, presence of cr-/scaliine incluslOri bodies, presence of cacalase, inability to reduce nitrate, presence of bioluminescence, abilicy to take up dye Erom growth media, posicive for procease production, grewth-temceraturs range below 3% C, survival under anaerobic conditior.s and pesici=:e1v mocile. (Table 18). Reference Escherichia coli, Xenorrcaodus and Phocorhabdus strains were included in all tescs for ccmparison.
The overall results are consistent with all strains being part ot the family Encerobacceriaceae and the genus Phocorhabdus.
A luminometer was used to establish the bioluminescence of each strain and provide a quantitative and relative measurement of light production. For measurement of relative light emitting units, the broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (6, 12, and 24 hr) and compared to background luminosity (uninoculated media and water). Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlin, OH) spectrephotometer using a sipper cell. Apprcrriace dilutions were then made (to normalize optical density to 1.0 unit) before measuring luminosity. Aliquots of the diluted broths were then placed into cuvettes (300 ul each) and read in a Bio-Orbit* 1251 Luminometer (Bio-Orbit Oy, Twiku, Finland) . The integration period for each sample was 45 seconds. The samples were continuously mixed (spun in baffled cuvettes) while being read to provide oxygen availability. A positive test was determined as being >_ 5-fold background luminescence (-5-10 units). In addition, colony luminosity was detected with photographic film overlays and visually, after adaptation in a darkroom. The Gram's staining characteristics of each strain were established with a commercial Gram's stain kit (BBL, Cockeysville, MD) used in conjunction with Gram's stain control slides (Fisher Scientific, Pittsburgh, PA). Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) 100X oil immersion objective lens (with lOX
ocular and 2X body magnification). Microscopic examination of individual scrains for organism size, cellular descripticn and inclusion bodies (the lacter after logarithmic growth) was *Trade-mark performed using wet mcunt siides !10j ocular, 2:{ body and 40Z
objec_ive magnification) with oil immersion and phase contrast microscopy with a micromecer (Akhurst, R.J. and Boemare, N.E.
1990. EncomooachoQenic Nematodes in Biological Control (ed.
Gaugler, R. and Kaya. H.). pp. 75-?0. CRC Press, Boca Raton, USA.; Bachdiguian S., Boyer-Giglio M.H., Thaler, J.O., Bonnct G., Boemare U. 1993. Biol. Cell 7?, 177-185.). Colony pigmentation was observed after inoculacion on Bacto nutrient agar, (Difco Laboratories, Detroit, MI) prepared as per label instructions.
Incubation occurred at 28 C and descripcions were produced after 5-7 days. To test for the presence of the enzyme catalase, a colony of the test organism was removed on a small plug fr::~ a nutrient agar plate and placed into the bottom of a glass tesc tube. One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or within 5 seconds. Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined.
To test for nitrate reduction, each culture was inoculated inco 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, :=II).
After 24 hours incubation at 28 C, nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984). The generation of a distinct pink or red color indicates the formation of nitrite from nitrate. The ability of each strain to uptake dye from growth media was tested with Bacto MacConkey agar containiiig the dye neutral red; Bacto Tergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agar containing the dye eosin-Y
(agars from Difco Laboratories, Detroit, MI, all prepared according to label instructions). After inoculation on these media, dye uptake was recorded after incubation at 28 C for 5 days. Growth on these latter media is characteristic for members of the family EnceroDacceriaceae. Motility of each strain was tested using a solution of Bacto Motility Test Medium (Difco Laboratories, Detroit, MI) prepared as per label inscructions. A
butt-stab inoculation was performed with each strain and motility was judged macroscopically by a diffuse zone of growth spreading from the line of inoculum. In many c3ses, motility was also *Trade-mark observed microscopically from liquid culture under wet mount slides. Biochemical nutrient evaluation for each strain was performed using BBL Enterotube II (Benton, Dickinson, Germany).
Product instructions were followed with the exception that incubation was carried out at 28 C for 5 days. Results were consistent with previously cited reports for Photorhabdus. The production of protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, MI) plates made as power label instructions. Cultures were inoculated and the plates were incubated at 28 C for 5 days. To assess growth at different temperatures, agar plates [2%
proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, MI) in deionized water] were streaked from a common source of inoculum. Plates were sealed with NescoO film and incubated at 20, 28 and 37 C for up to three weeks. Plates showing no growth at 37 C showed no cell viability after transfer to a 28 C incubator for one week. Oxygen requirements for Photorhabdus strains were tested in the following manner.
A butt-stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, MI) was made. The tubes were incubated at room temperature for one week and cultures were then examined for type and extent of growth. The indicator resazurin demonstrates the level of medium oxidation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism.

Table 18 Taxonomic Traits of Photorhabdus Strains Traits Assessed*

Strain A B C D E F G H I J K L M N 0 P Q
W-14 _t + + rd S + - + + + 0 + + + + + + -wx-i - + + rd S + - + + + 0 + + + + + + -WX-2 - + + rd S + - + + + 0 + + + + + + -WX-3 - + + rd S + - + + + YT + + + + + + -WX-4 - + + rd S + - + + + YT + + + + + +
wX-5 - + + rd S + - + + + LO + + + + + + -WX-6 - + + rd S + - + + + LY + + + + + + -WX-7 - + + rd S + - + + + R + + + + + +
WX-8 - + + rd S + - + + + 0 + + + + + +
WX-9 - + + rd S + - + + + YT + + + + + + -wx-io - + + rd S - + + + Ro + + + + + + -WX-11 - + + rd S + - + + + Ro + + + + + + -WX-12 - + + rd S + - + + + 0 + + + + + + -WX-14 - + + rd S + - + + + LR + + + + + + -WX-15 - + + rd S + - + + + LR + + + , + +
H9 - + + rd S + - + + + LY + + + + + +
Hb - + + rd S + - + + + YT + + + + + + -Hm - + + rd S + - + + + TY + + + + + + -HP88 - + + rd S + - + + + LY + + + + + + -NC-1 - + + rd S -~ - + + + 0 + + + + + + -W30 - + + rd S + - + + + YT + + + + +
WIR - + + rd S + - + + + RO + + + + +
B2 - + + rd S + - + + + R + + + + + + -43948 - + + rd S - + + + 0 + + + + + +
43949 - + + rd S + - + + +
-4 0 + + + + + + -43950 - + + rd S + - + + + 0 + + + + + + -43951 - + + rd S + - + + + 0 + + + + + + -43952 - + + rd S + - + + + 0 + J + + + + + -* - A Gram's stain, B=Crystaline inclusion bodies, C=Bioluminescence, D=Cell form, E=Motility, F=Nitrate reduction, G=Presence of catalase, H=Gelatin hydrolysis, I=Dye uptake, J=Pigmentation, K=Growth on EMB agar, L=Growth on MacConkey agar, M=Growth on Tergitol-7 agar, N=Facultative anaerobe, O=Growth at 20 C, P=Growth at 28 C, Q=Growth at 37 C, '29355-1 t: + = positive for trait, -= negative for trait, rd=rod, S=sized within Genus descriptors, RO=red-orange, LR= light red, R= red, 0= orange, Y= yellow, T= tan, LY= light yellow, YT=
yellow tan, and LO = light orange.

Cellular fatty acid analysis is a recognized tool for bacterial characterization at the genus and species level (Tornabene, T.G. 1985. Lipid Analysis and the Relationship to 84a Chemccaxcncr: iz Methods in Microbiolog'r,Vol 13, 20'1-2:4. , Goodfellow, t4. and O'Donnell, A.G. 1993. P.oocs of Bacterial Systemacics in Handbock of t)ew Bacterial Systemacics (ed.
Goodfellow, M. & O'Donnell, A.G.) pp. 3-54. London: acaciemic Press Ltd.) and were used to confirm that our collection was related at the genus level. Cultures were shipped to an external, contract laboratory for fatty acid methyl ester analysis (FAME) using a Microbial ID (MIDI, Newark, DE, USa) Microbial Identification System (MIS). The MIS system consists of a Hewlett Packard HP5890A gas chromatograph with a 25mm x 0.2mm 5% methylphenyl silicone fused silica capillary column. Hydrogen is used as the carrier gas and a flame-ionizacion detector functions in conjunction with an automatic sampler, incegrator and compucer. The computer compares the sample fatty acid methyl esters to a microbial fatty acid library and against a calibration mix of known fatty acids. As selected by the contract laboratory, strains were grown for 24 hours at 23 C on trypticase soy agar prior to analysis. Extraction of samples was performed by the contract lab as per standard FAME methodology.
There was no direct identification of the strains to any luminescent bacterial group other than Photorhabdus. wh,~n the cluster analysis was performed, which ccmpares the fatty acid profiles of a group of isolates, the strain fatty acid profiles were related at the genus level.
The evolutionary diversity of the Photorhabdus strains in our collection was measured by analysis of PCR (Polymerase Chain Reaction) mediated genomic fingerprinting using genamic DNA from each strain. This technique is based on families of repetitive DNA sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic, J., Schneider, M., DE Bruijn, F.J. and Lupski, J.R. 1994. Methods Mol. Cell. Biol., 5, 25-40.).
Three of these, repetitive extragenic palindromic sequence (REP), enterobacterial repetitive intergenic consensus (ERIC) arid the BOX element are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elemencs within the genome of closely related bacterial strains can be used to discriminate these scrains (e.g.

L 7~~~- 1 CA 02209659 2001-01-19 LJ'14iS. F.J. , =~ilbriyht, O.W. . Stephens, C.T. and DE Brui7r.. , .,, .
1994 . Appl. Environ. t4icro. 60, 2286-2295.). Rep-PCR utilizes oligonucleocide primers :omplemencary to these repecitive sequences to amplify the variably sized DNA fragments lying between them. The resulcing products are separated by eleccrophoresis to establish the DtdA Lingerprint" for each strain.
To isolace genomic DNA from our strains, cell pellets were resuspended in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and 12 ml of 5 M NaC1 was then added. This mixture was centrifuged 20 min. at 15,000 x g. The resulting pellet was resuspended in 5.7 ml of TE and 300 ul of 10% SDS and 60 ul 20 mg/ml proteinase K (Gibco BP.L Products, Grand Island, NY) were added. This mixture was incubated at 37 C for 1 hr, approximately 10 mg of lysoz=yme was then added and the mixture was incubaced for an additional 45 min. One milliliter of 5M NaC1 and 800 ul of CTAB/D1aC1 solution (10% w/v CTAB, 0.7 M NaC1) were then added and the mixture was incubated 10 min. at 65 C, gently agitated, then incubated and agitated for an additional 20 min.
to aid in clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently then centrifuged. Two extractions were then performed with an equal volume of phenol /chloroform/ isoamyl alcohol (50:49:1) Gencmic DNA was precipitated with 0.6 volume of isopropanol.
Precipitated DNA was removed with a glass rod, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HC1 pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by optical density at 260 nm. To perform rep-PCR analysis of Photorhabdus genomic DNA the following primers were used, REP1P.-I; 5'-IIIICGICGICATCIGGC-3' and REP2-I; 5'-ICGICTTATCIGGCCTAC-3'.
PCR was performed using the following 25ul reaction: 7.75 ul H20, 2.5 ul lOX LA buffer (PanVera Corp., Madison, WI), 16 ul dNTP mix (2.5 mM each), 1 ul of each primer at 50 pM/ul, 1 ul DMSO, 1.5 u1 genomic DNA (concentrations ranged from 0.075-0.480 ug/ul) and 0.25 ul TaKaRa EY Taq (PanVera Corp., Madison, WI). The PCR
amplification was performed in a Perkin Elmer DNA Thermal Cycler (Norwalk, CT) using the following conditions: 95 C/7 min. then 35 cycles of; 94 C/1 min.,44 C/1 min., 65 C/8 min., followed by 15 min. at 65 C. After cycling, the 25 ul reaction was added to 5 ul *Trade-mark of 6X gel loading buffer (0.25o bromophenol blue, 40o w-r sucrose in H20). A 15x20cm 1%-agarose gel was then run in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 ul of each reaction.
The gel was run for approximately 16 hours at 45v. Gels were then stained in 20 ug%'ml ethidium bromide for 1 hour and destained in TBE buffer for approximately 3 hours. PolaroidO photographs of i the gels were then taken under UV illumination.
The presence or absence of bands at specific sizes for each strain was scored from the photographs and entered as a similarity matrix in the numerical taxonomy software program, NTSYS-pc (Exeter Software, Setauket, NY). Controls of E. coli strain HB101 and Xanthomonas oryzae pv. oryzae assayed at the same time produced PCR "fingerprints" corresponding to published reports (Versalovic, J., Koeuth, T. and Lupski, J.R. 1991.
Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C.M., Halda-Alija, L., Louws, F., Skinner, D.Z., George, M.L., Nelson, R.J., DE
Bruijn, F.J., Rice, C. and Leach, J.E. 1995. Int. Rice Res.
Notes, 20, 23-24.; Vera Cruz, C.M., Ardales, E.Y., Skinner, D.Z., Talag, J., Nelson, R.J., Louws, F.J., Leung, H., Mew, T.W. and Leach, J.E. 1996. Phytopathology (in press, respectively). The data.from Photorhabdus strains were then analyzed with a series of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data) to generate a matrix of similarity coefficients (using the Jaccard coefficient) and SAHN (Sequential, Agglomerative, Heirarchical and Nested) clustering [using the UPGMA (Unweighted Pair-Group Method with Arithmetic Averages) method] which groups related strains and can be expressed as a phenogram (Figure 5).
The COPH (cophenetic values) and MXCOMP (matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based. A resulting normalized Mantel statistic (r) was generated which is a measure of the goodness of fit for a cluster analysis (r=0.8-0.9 represents a very good fit). In our case r = 0.919. Therefore, our collection is comprised of a diverse group of easily distinguishable strains representative of the Photorhabdus genus.

SUBSTITUTE SHEET (RULE 26) Example 13 Insecticidal Utility of Toxin(s) Produced by Various Photorhabdus Strains Initial "seed" cultures of the various Photorhabdus strains were produced by inoculating 175 ml of 2% Proteose Peptone #3 (PP3) (Difco Laboratories, Detroit, MI) liquid media with a primary variant subclone in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput. Inoculum for each seed culture was derived from oil-overlay agar slant cultures or plate cultures. After inoculation, these flasks were incubated for 16 hrs at 28 C on a rotary shaker at 150 rpm. These seed cultures were then used as uniform inoculum sources for a given fermentation of each strain. Additionally, overlaying the post-log seed culture with sterile mineral oil, adding a sterile magnetic stir bar for future resuspension and storing the culture in the dark, at room temperature provided long-term preservation of inoculum in a toxin-competent state. The production broths were inoculated by adding 1% of the actively growing seed culture to fresh 2% PP3 media (e.g. 1.75 ml per 175 ml fresh media).
Production of broths occurred in either 500 ml tribaffled flasks (see above), or 2800 ml baffled, convex bottom flasks (500 ml volume) covered by a silicon foam closure. Production flasks were incubated for 24-48 hrs under the above mentioned conditions. Following incubation, the broths were dispensed into sterile 1 L polyethylene bottles, spun at 2600 x g for 1 hr at 10 C and decanted from the cell and debris pellet. The liquid broth was then vacuum filtered through Whatman GF/D (2.7 uM
retention) and GF/B (1.0 i1M retention) glass filters to remove debris. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron, Northborough, MA) using a 0.5 pM open-channel filter. When necessary, additional clarification could be obtained by chilling the broth (to 4 C) and centrifuging for several hours at 2600 x g. Following these procedures, the broth was filter sterilized using a 0.2 uM nitrocellulose membrane filter. Sterile broths were then used directly for biological assay, biochemical analysis or concentrated (up to 15-fold) using a 10,000 tnJ cut-off, M12 ultra-filtration device (Amicon, Beverly MA) or SUBSTITUTE SHEET (RULE 26) centrifugal concentrators (Millipore, Bedford, MA and Pall Filtron, Northborougii, MA) with a 10,000 MW pore size. In the case of centrifugal concentrators, the broth was spun at 2000 x g for approximately 2 hr. The 10,000 MW permeate was added to the ' corresponding retentate to achieve the desired concentration of components greater than 10,000 MW. Heat inactivation of processed broth samples was acheived by heating the samples at 100 C in a sand-filled heat block for 10 minutes.

The broth(s) and toxin complex(es) from different Photorhabdus strains are useful for reducing populations of insects and were used in a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described. A
demonstration of the breadth of insecticidal activity observed from broths of a selected group of Photorhabdus strains fermented as described above is shown in Table 19. It is possible that additional insecticidal activities could be detected with these strains through increased concentration of the broth or by employing different fermentation methods. Consistent with the activity being associated with a protein, the insecticidal activity of all strains tested was heat labile (see above).
Culture broth(s) from diverse Photorhabdus strains show differential insecticidal activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects.
More specifically, the activity is seen against corn rootworm larvae and boll weevil larvae which are members of the insect order Coleoptera. Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle. Activity is also observed against aster leafhopper and corn plant hopper, which are members of the order Homoptera. Other members of the Homoptera include planthoppers, pear psylla, apple sucker, scale insects, whiteflies, spittle bugs as well as numerous host specific aphid species. The broths and purified toxin complex(es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera. Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent SUBSTITUTE SHEET (RULE 26) caterpillar, sod webworm and fall armyworm. Activit_v is also seen against fruitfly and mosquito larvae which are members of the order Diptera. Other members of the order Diptera are, pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly and house fly and various mosquito species. Activity with broth(s) and toxin complex(es) is also seen against two-spotted spider mite which is a member of the order Acarina which includes strawberry spider mites, broad mites, citrus red mite, European red mite, pear rust mite and tomato russet mite.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth(s) (0-15 fold concentrated, filter sterilized), 2% Proteose Peptone #3, purified toxin complex(es) [0.23 mg/mll or 10 mM sodium phosphate buffer , pH 7.0 were applied directly to the surface (about 1.5 cm2) of artificial diet (Rose, R. I. and McCabe, J. M. (1973). J. Econ. Entomol. 66, (398-400) in 40 ul aliquots. Toxin complex was diluted in 10 mM
sodium phosphate buffer, pH 7Ø The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber and maintained at 27 C for the appropriate period (3-5 days). Mortality and larval weight determinations were then scored. Generally, 16 insects per treatment were used in all studies. Control mortality was generally less than 5%.
Activity against boll weevil (Anthomonas grandis) was tested as follows. Concentrated (1-10 fold) Photorhabdus broths, control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 mg/mll or 10 mM sodium phosphate buffer, pH 7.0 were applied in 60 ul aliquots to the surface of 0.35 g of artificial diet (Stoneville Yellow lepidopteran diet) and allowed to dry. A single, 12-24 hr boll weevil larva was placed on the diet, and the wells were sealed and held at 25 C, 50% RH for 5 days. Mortality and larval weights were then assessed. Control mortality ranged between 0-13%.
Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well = contained 200 ul of aqueous solution (10-fold concentrated Photorhabdus culture broth(s), control medium (2% Proteose SUBSTITUTE SHEET (RULE 26) Peptone #3), 10 mM sodium phosphate buffer, toxin complex(es) q 0.23 mg/ml or H20) and approximately 20, 1-day old larvae (Aedes aegypti). There were 6 wells per treatment. The results were read at 3-4 days after infestation. Control mortality was between 0-20%.
Activity against fruitflies was tested as follows.
Purchased Drosophila melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium (2%

Proteose Peptone #3), 10-fold concentrated Photorhabdus culture broth(s), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer , pH 7Ø This was accomplished by placing 4.0 ml of dry medium in each of 3 rearing vials per treatment and adding 4.0 ml of the appropriate liquid. Ten late instar Drosophila melanogaster maggots were then added to each 25 ml vial. The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 15 days of exposure. Adult emergence as compared to water and control medium (0-16% reduction).
Activity against aster leafhopper adults (Macrosteles severirni) and corn planthopper nymphs (Peregrinus maidis) was tested with an ingestion assay designed to allow ingestion of the active without other external contact. The reservoir for the active/"food" solution is made by making 2 holes in the center of the bottom portion of a 35X10 mm Petri dish. A 2 inch Parafilm M square is placed across the top of the dish and secured with an "0" ring. A 1 oz. plastic cup is then infested with approximately 7 hoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes. In tests using 10-fold concentrated Photorhabdus culture broth(s), the broth and control medium (2%
Proteose Peptone #3) were dialyzed against 10 mM sodium phosphate buffer, pH 7.0 and sucrose (to 5%) was added to the resulting solution to reduce control mortality. Purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 was also tested. Mortality is reported at day 3. The assay was held in an incubator at 28 C, 70% RH with a 16/8 photoperiod. The assays were graded for mortality at 72 hours. Control mortality was less than 6%.

SUBSTITUTE SHEET (RULE 28) Activity against lepidopteran larvae was tested as follo=Ns.
Concentrated (10-fold) Photorhabdus culture broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 mgimi] or 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (-1.5 cm2) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 tll aliquots.
The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva. European corn borer (Ostrinia nubilalis) and tobacco hornworm (Manduca sexta) eggs were obtained from commercial sources and hatched in-house, whereas tobacco budworm (Heliothis virescens) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 270C for the appropriate period.
P4ortality and weight determinations were scored at day 5.
Generally, 16 insects per treatment were used in all studies.
Control mortality generally ranged from 4-12.5% for control medium and was less than 10% for phosphate buffer.
Activity against two-spotted spider mite (Tetranychus urticae) was determined as follows. Young squash plants were trimmed to a single cotyledon and sprayed to run-off with 10-fold concentrated broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7Ø After drying, the plants were infested with a mixed population of spider mites and held at lab temperature and humidity for 72 hr. Live mites were then counted to determine levels of control.

SUBSTITUTE SHEET (RULE 28) Table 19 Observed Insecticidal Spectrum of Broths From Different Photorhabdus Strains Phocorhabdus Strain Sensitive* Insect Species wx-1 3**, 4, 5, 6, 7, 8 WX-2 2, 4 =
WX-3 1, 4 WX-4 1, 4 WX-7 3, 4, 5, 6, 7, 8 WX-8 1, 2, 4 WX-9 1, 2, 4 wx-1o 4 WX-11 1, 2, 4 WX-12 2, 4, 5, 6, 7, 8 WX-14 1, 2, 4 WX-15 1, 2, 4 W30 3, 4, 5, 8 NC=1 1, 2, 3, 4, 5, 6, 7, 8, 9 WIR 2, 3, 5, 6, 7, 8 HP88 1, 3, 4, 5, 7, 8 Hb 3, 4, 5, 7, 8 Hm 1, 2, 3, 4, 5, 7, 8 H9 1, 2, 3, 4, 5, 6, 7, 8 W-14 1, 2, 3, 4, 5, 6, 7, 8, 10 >_ 25% mortality and/or growth inhibition vs. control ** = 1; Tobacco budworm, 2; European corn borer, 3;
Tobacco hornworm, 4; Southern corn rootworm, 5;
Boll weevil, 6; Mosquito, 7; Fruit Fly, 8;
Aster Leafhopper, 9; Corn planthopper, 10;
Two-spotted spider mite.

SUBSTITUTE SHEET (RULE 26) Example 14 Non ,,1-14 Photorhabdus Strains:
Purification, Characteri?at-ion and Activity Spectrum Purification The protocol, as follows, is similar to that developed for + the purification of W-14 and was established based on purifying those fractions having the most activity against Southern corn root worm (SCR), as determined in bioassays (see Example 13).
Typically, 4-20 L of broth that had been filtered, as described in Example 13, were received and concentrated using an Amicon spiral ultra filtration cartridge Type S1Y100 attached to an Amicon M-12 filtration device. The retentate contained native proteins consisting of molecular sizes greater than 100 kDa, whereas the flow through material contained native proteins less than 100 kDa in size. The majority of the activity against SCR
was contained in the 100 kDa retentate. The retentate was then continually diafiltered with 10 mM sodium phosphate (pH = 7.0) until the filtrate reached an A280 < 0.100. Unless otherwise stated, all procedures from this point were performed in buffer as defined by 10 mM sodium phosphate (pH 7.0). The retentate was then concentrated to a final volume of approximately 0.20 L and filtered using a 0.45 mm NalgeneT" Filterware sterile filtration unit. The filtered material was loaded at 7.5 ml/min onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem PorosO 50 HQ strong anion exchange matrix equilibrated in buffer using a PerSeptive Biosystem Sprint HPLC system.
After loading, the column was washed with buffer until an A280 0.100 was achieved. Proteins were then eluted from the column at 2.5 ml/min using buffer with 0.4 M NaCl for 20 min for a total volume of 50 ml. The column was then washed using buffer with 1.0 M NaCl at the same flow rate for an additional 20 min (final volume = 50 ml). Proteins eluted with 0.4 M and 1.0 M NaCl were placed in separate dialysis bags (Spectra/Por Membrane MWCO:
2,000) and allowed to dialyze overnight at 4 C in 12 L buffer.
The majority of the activity against SCR was contained in the 0.4 M fraction. The 0.4 M fraction was further purified by application of 20 ml to a Pharmacia XK 26/100 column that had been prepacked with Sepharose CL4B (Pharmacia) using a flow rate SUBSTITUTE SHEET (RULE 26) of 0.75 ml/min. Fractions were pooled based cn A280 peak profiie and concentrated to a final volume of 0.75 ml using a Millipore Ultrafree -15 centrifugal filter device Biomax-50K LiMWL membrane.
Protein concentrations were determined using a Biorad Protein 5 Assay Kit with bovine gamma globulin as a standard.

Characterization The native molecular weight of the SCR toxin complex was determined using a Pharmacia HR 16/50 that had been prepacked with Sepharose CL4B in buffer. The column was then calibrated using proteins of known molecular size thereby allowing for calculation of the toxin approximate native molecular size. As shown in Table 20, the molecular size of the toxin complex ranged from 777 kDa with strain Hb to 1,900 kDa with strain WX-14. The yield of toxin complex also varied, from strain WX-12 producing 0.8 mg/L to strain Hb, which produced 7.0 mg/L.
Proteins found in the toxin complex were examined for individual polypeptide size using SDS-PAGE analysis. Typically, mg protein of the toxin complex from each strain was loaded 20 onto a 2-15% polyacrylamide gel (Integrated Separation Systems) and electrophoresed at 20 mA in Biorad SDS-PAGE buffer. After completion of electrophoresis, the gels were stained overnight in Biorad Coomassie blue R-250 (0.2% in methanol: acetic acid:
water; 40:10:40 v/v/v). Subsequently, gels were destained in methanol:acetic acid: water; 40:10:40 (v/v/v). The gels were then rinsed with water for 15 min and scanned using a Molecular Dynamics Personal Laser Densitometer . Lanes were quantitated and molecular sizes were calculated as compared to Biorad high molecular weight standards, which ranged from 200-45 kDa.
Sizes of the individual polypeptides comprising the SCR
toxin complex from each strain are listed in Table 21. The sizes of the individual polypeptides ranged from 230 kDa with strain WX-1 to a size of 16 kDa, as seen with strain WX-7. Every strain, with the exception of strain Hb, had polypeptides comprising the toxin complex that were in the 160-230 kDa range, the 100-160 kDa range, and the 50-80 kDa range. These data indicate that the toxin complex may vary in peptide composition and components from strain to strain, however, in all cases the SUBSTITUTE SHEET (RULE 26) toxin attributes appears to consist of a large, oligomeric protein complex.

Table 20 Characterization of a Toxin Complex From Non W-14 Photorhabdus Strains Strain Approx. Yield Native Active Molecular Wt.a Fraction (mg/L)b H9 972,000 1.8 Hb 777,000 7.0 Hm 1,400,000 1.1 HP88 813,000 2.5 NC1 1,092,000 3.3 WIR 979,000 1.0 wx-1 973,000 0.8 WX-2 951,000 2.2 WX-7 1,000,000 1.5 WX-12 898,000 0.4 WX-14 1,900,000 1.9 W-14 860,000 7.5 a Native molecular weight determined using a Pharmacia HP.

16/50 column packed with Sepharose CL4B
b Amount of toxin complex recovered from culture broth.
Activity Spectrum As shown in Table 21, the toxin complexes purified from strains Hm and H9 were tested for activity against a variety of insects, with the toxin complex from strain W-14 for comparison.
The assays were performed as described in Example 13. The toxin complex from all three strains exhibited activity against tobacco bud worm, European corn borer, Southern corn root worm, and aster leafhopper. Furthermore, the toxin complex from strains Hm and W-14 also exhibited activity against two-spotted spider mite. In addition, the toxin complex from W-14 exhibited activity against mosquito larvae. These data indicate that the toxin complex, while having similarities in activities between certain orders of insects, can also exhibit differential activities against other orders of insects.

SUBSTITUTE SHEET (RULE 26) Table 21 The Approximate Sizes (in kDa) of Peptides in a Purified Toxin Complex From Non W-14 Photorhabdus H9 Hb Hm HP NC-1 WIR WX-1 WX-2 WX-7 WX-12 WX-14 W-11 84 88 46 86 62 58 37 28 87 80 111-) SUBSTITUTE SHEET (RULE 26) Table 22 Observed Insecticidal Spectrum of a Purified Toxin Complex from Photorhabdus Strains Phocorhabdus Strain Sensitive* Insect Species Hm Toxin Complex 1**, 2, 3, 5, 6, 7, 8 H9 Toxin Complex 1, 2, 3, 6, 7, 8 w-14 Toxin Complex 1, 2, 3, 4, 5, 6, 7, 8 *=> 25% mortality or growth inhibition *=> 25% mortality or growth inhibition ** = 1; Tobacco bud worm, 2; European corn borer, 3; Southern corn root worm, 4; Mosquito, 5; Two-spotted spider mite, 6; Aster Leafhopper, 7; Fruit Fly, 8; Boll Weevil Example 15 Sub-Fractionation of Photorhabdus Protein Toxin Complex The Photorhabdus protein toxin complex was isolated as described in Example 14. Next, about 10 mg toxin was applied to a MonoQ 5/5 column equilibrated with 20 mM Tris-HC1, pH 7.0 at a flow rate of lml/min. The column was washed with 20 cnM Tris-HC1, pH 7.0 until the optical density at 280 nm returned to baseline absorbance. The proteins bound to the column were eluted with a linear gradient of 0 to 1.0 M NaCl in 20 mM Tris-HC1, pH 7.0 at 1 ml/min for 30 min. One ml fractions were collected and subjected to Southern corn rootworm (SCR) bioassay (see Example 13). Peaks of activity were determined by a series of dilutions of each fraction in SCR bioassays. Two activity peaks against SCR were observed and were named A (eluted at about 0.2-0.3 M NaCl) and B
(eluted at 0.3-0.4 M NaCl). Activity peaks A and B were pooled separately and both peaks were further purified using a 3-step procedure described below.
Solid (NH4)2SO4 was added to the above protein fraction to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 5/5 column equilibrated with 1.7 M(NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M
(NH4)2SO4, 0% ethylene glycol, 50 mM potassium phosphate, pH 7.0 to 25% ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no (NH4)2SO4) at 0.5 ml/min. Fractions were dialyzed overnight SUBSTITUTE SHEET (RULE 26) against 10 mM sodium phosphate buffer, pH 7Ø Acti-:ities in each fraction against SCR were determined by bioassay.
The fractions with the highest activity were pooled and applied to a MonoQ 5/5 column which was equilibrated with 20 mM
Tris-HC1, pH 7.0 at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to 1P=I NaCl in 20 mM Tris-HC1, pH 7Ø
For the final step of purification, the most active fractions above (determined by SCR bioassay) were pooled and subjected to a second phenyl-Superose 5/5/ column. Solid (NH4)2SO4 was added to a final concentration of 1.7 M. The solution was then loaded onto the column equilibrated with 1.7 M
(NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at lml/min.
Proteins bound to the column were eluted with a linear gradient of 1.7 M(NH4)2SO4, 50 mM potassium phosphate, pH 7.0 to 10 mM
potassium phosphate, pH 7.0 at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7Ø
Activities in each fraction against SCR were determined by bioassay.
The final purified protein by the above 3-step procedure from peak A was named toxin A and the final purified protein from peak B was named toxin B.

Characterization and Amino Acid Sequencing of Toxin A and Toxin B
In SDS-PAGE, both toxin A and toxin B contained two major (>
90% of total Commassie stained protein) peptides: 192 kDa (named Al and B1, respectively) and 58 kDa (named A2 and B2, respectively). Both toxin A and toxin B revealed only one major band in native PAGE, indicating Al and A2 were subunits of one protein complex, and El and B2 were subunits of one protein complex. Further, the native molecular weight of both toxin A
and toxin B were determined to be 860 kDa by gel filtration chromatography. The relative molar concentrations of Al to A2 was judged to be a 1 to 1 equivalence as determined by densiometric analysis of SDS-PAGE gels. Similarly, Bl and B2 peptides were present at the same molar concentration.
Toxin A and toxin B were electrophoresed in 10% SDS-PAGE and transblotted to PVDF membranes. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal -99-SUBSTITUTE SHEET (RULE 26) amino sequence of B1 was determined to be identical to SEK ID
11O:1, the TcbAii region of the tcbA gene (SEQ ID NO:12, pcsition 87 to 99). A unique N-terminal sequence was obtained for peptide B2 (SEQ ID NO:40). The 11-terminal amino acid sequence of peptide B2 was identical to the TcbAiii region of the derived amino acid sequence for the tcbA gene (SEQ ID NO:12, position 1935 to 1945).
Therefore, the B toxin contained predominantly two peptides, TcbAii and TcbAiii, that were observed to be derived from the same gene product, TcbA.
The N-terminal sequence of A2 (SEQ ID NO:41) was unique in comparison to the TcbAiii peptide and other peptides. The A2 peptide was denoted TcdAiii (see Example 17). SEQ ID NO:6 was determined to be a mixture of amino acid sequences SEQ ID NO:40 and 41.
Peptides Al and A2 were further subjected to internal amino acid sequencing. For internal amino acid sequencing, 10 ug of toxin A was electrophoresized in 10% SDS-PAGE and transblotted to PVDF membrane. After the blot was stained with amido black, peptides Al and A2, denoted TcdAii and TcdAiii, respectively, were excised from the blot and sent to Harvard MicroChem and Cambridge ProChem. Peptides were subjected to trypsin digestion followed by HPLC chromatography to separate individual peptides.
N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal amino acid sequences of peptide Al (TcdAii-PK71, SEQ ID NO:38 and TcdAii-PK44, SEQ ID NO:39) were found to have significant homologies with deduced amino acid sequences of the TcbAii region of the tcbA gene (SEQ ID 11O:12).
Similarly, the N-terminal sequence (SEQ ID NO:41) and two internal sequences of peptides A2 (TcdAiii-PK57, SEQ ID NO:42 and TcdAiii-PK20, SEQ ID NO.43) also showed significant homology with deduced amino acid sequences of TcbAiii region of the tcbA gene (SEQ ID NO:12).
In summary of above results, the toxin complex has at least two active protein toxin complexes against SCR; toxin A and toxin B. Toxin A and toxin B are similar in their native and subunits molecular weight, however, their peptide compositions are different. Toxin A contained peptides TcdAii and TcdAiii as the major peptides and the toxin B contains TcbAii and TcbAiii as the major peptides.

SUBSTITUTE SHEET (RULE 26) Example 16 Cleavage and Activation of TcbA Peptide In the toxin B complex, peptide TcbAii and TcbAiii originate from the single gene product TcbA (Example 15). The processing of TcbA peptide to TcbAii and TcbAiii is presumably by the action of Photorhabdus protease(s), and most likely, the metalloproteases described in Example 10. In some cases, it was noted that when Photorhabdus W-14 broth was processed, TcbA peptide was present in toxin B complex as a major component, in addition to peptides TcbAii and TcbAiii= Identical procedures, described for the purification of toxin B complex (Example 15), were used to enrich peptide TcbA from toxin complex fraction of W-14 broth. The final purified material was analyzed in a 4-20% gradient SDS-PAGE and major peptides were quantified by densitometry. It was determined that TcbA, TcbAii and TcbAiii comprised 58%, 36%, and 6%, respectively, of total protein. The identities of these peptides were confirmed by their respective molecular sizes in SDS-PAGE and Western blot analysis using monospecific antibodies. The native molecular weight of this fraction was determined to be 860 kDa.
The cleavage of TcbA was evaluated by treating the above purified material with purified 38 kDa and 58 kDa W-14 .25 Photorhabdus metalloproteases (Example 10), and Trypsin as a control enzyme (Sigma, MO). The standard reaction consisted 17.5 ug the above purified fraction, 1.5 unit protease, and 0.1 M Tris buffer, pH 8.0 in a total volume of 100 ul. For the control reaction, protease was omitted. The reaction mixtures were incubated at 37 C for 90 min. At the end of the reaction, 20 ul was taken and boiled with SDS-PAGE sample buffer immediately for electrophoresis analysis in a 4-20% gradient SDS-PAGE. It was determined from SDS-PAGE that in both 38 kDa and 58 kDa protease treatments, the amount of peptides TcbAii and TcbAiii increased about 3-fold while the amount of TcbA peptide decreased proportionally (Table 23). The relative reduction and augmentation of selected peptides was confirmed by Western blot analyses. Furthermore, gel filtration of the cleaved material revealed that the native molecular size of the complex remained the same. Upon trypsin treatment, peptides TcbA and TcbAii were SUBSTITUTE SHEET (RULE 26) nonspecifically digested into small peptides. This indicated that 38 kDa and 58 kDa Photorhabdus proteases can specifically process peptide TcbA into peptides TcbAii and TcbAiii= Protease treated and untreated control of the remaining 80 jil reaction mixture were serial diluted with 10 mP4 sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay. By comparing activity in several dilution, it was determined that the 38 kDa protease treatment increased SCR insecticidal activity-approximately 3 to 4 fold.
The growth inhibition of remaining insects in the protease treatment was also more severe than control (Table 23).
Table 23 Conversion and activation of peptide TcbA into peptides TcbAii and TcbAiii by protease treatment.
Control 38 kDa protease treatment SO (% of totai protein) 58 18 Sl (% of total protein) 36 64 S9 (% of total protein) 6 18 LD50 (ug protein) 2.1 0.52 SCR Weight (mg/insect)* 0.2 0.1 *: an indication of growth inhibition by measuring the average weight of live insect after 5 days on diet in the assay.

Example 17 Screening of the library for a gene encoding the TcdAii Peptide The cloning and characterization of a gene encoding the TcdAii peptide, described as SEQ ID NO:17 (internal peptide TcdAii-PT111 N-terminal sequence) and SEQ ID NO:18 (internal peptide TcdAii-PT79 N-terminal sequence) was completed. Two pools of degenerate oligonucleotides, designed to encode the amino acid sequences of SEQ ID NO:17 (Table 24) and SEQ ID NO:18 (Table 25), and the reverse complements of those sequences, were synthesized as described in Example B. The DNA sequence of the oligonucleotides is given below:

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E E a n \7 C\7 N r+ E E E~ E+ E U
uu ~ ~a d QQ 4 U U U U U U U U U >4, '14, 0 d C9 C7 C9 ~~ U a) ~i C7 r+ -- - - - - a r+ rC ~n in ~n ~n - - - - -en in in tn Ln C U
V rt r1 (7 U N~ 01 i e1 f'''1 n ~~ m ~o ut vai uai E+ A+b q a- o- a nG 0 0 ~
i i rn rn rai ~ b ~ a, a' a q ~of a r ;t' ~ z 4 4 4 a1 ou a a a a 4 a U a a a a SUBSTITUTE SHEET (RULE 26) Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using as forward primers P2.3.5.CB or P2.3.5, and as reverse primers P2.79.R.1 or P2.79R.CB, in all forward/reverse combinations, using Photorhabdus W-14 genomic DtIA
as template. In another set of reactions, primers P2.79.2 or P2.79.3 were used as forward primers, and P2.3.5R, P2.3.5RI, and P2.3R.CB were used as reverse primers in all forward/reverse combinations. Only in the reactions containing P2.3.6.CB as the forward primers combined with P2.79.R.1 or P2.79R.CB as the reverse primers was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 2500 base pairs.
The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdAii-PT111 lies amino-proximal to the peptide fragment TcdAii-PT79.
The 2500 bp PCR products were ligated to the plasmid vector pCRTMII (Invitrogen, San Diego, CA) according to the supplier's instructions, and the DNA sequences across the ends of the insert fragments of two isolates (HS24 and HS27) were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of both isolates was the same. New primers were synthesized based on the determined sequence, and used to prime additional sequencing reactions to obtain a total of 2557 bases of the insert [SEQ ID NO:361.
Translation of the partial peptide encoded by SEQ ID No: 36 yields the 845 amino acid sequence disclosed as SEQ ID NO:37.
Protein homology analysis of this portion of the TcdAii peptide fragment reveals substantial amino acid homology (68% similarity;
53% identity) to residues 542 to 1390 of protein TcbA [SEQ ID
NO:12]. It is therefore apparent that the gene represented in part by SEQ ID NO:36 produces a protein of similar, but not identical, amino acid sequence as the TcbA protein, and which likely has similar, but not identical biological activity as the TcbA protein.
In yet another instance, a gene encoding the peptides TcdAii-PK44 and the TcdAiii 58 kDa N-terminal peptide, described as SEQ ID NO:9 (internal peptide TcdAii-PK44 sequence), and SEQ ID
NO:41(TcdAiii 58 kDa N-terminal peptide sequence) was isolated.
Two pools of degenerate oligonucleotides, designed to encode the amino acid sequences described as SEQ ID NO:39 (Table 27) and SEQ

SUBSTITUTE SHEET (RULE 26) ID NO:41 !Table 26), and the reverse complements ot those sequences, were synthesized as described in Example 8, and their DNA sequences.

SUBSTITUTE SHEET (RULE 26) MM

pj ~ G1 M M M M
ft HE~ UE+U
z 2 a uau a H (1.' H H
A A ao rl U 0 U 0 c~uc~
a a W W
En H u ?4 H H
FC7+ W H Q Q a ' ~EE
rts E+ N C! ?+
~ U

0 0 -4 nHRC aaQ
=~ r-ro M>tE 7 CE7t E &4 tn tr- tri Oa a HH
cv N U U U U
~n W U C7 U C7 >+ H H H
U1 Lf1 lf1 ifl H N N
= . .

_ ~ ,~ t = = ~C ~ 4 SUBSTITUTE SHEET (RULE 26) Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using as forward primers A1.44.1 or A1.44.2, and reverse primers A2.3R or A2.4R, in all forward/reverse combinations, using Photorhabdus W-14 genomic DNA
as template. In another set of reactions, primers A2.1 or A2.2 were used as forward primers, and A1.44.1R, and A1.44.2R were used as reverse primers in all forward/reverse combinations. Only in the reactions containing A1.44.1 or A1.44.2 as the forward primers combined with A2.3R as the reverse primer was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 1400 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdAii-PK44 lies amino-proximal to the 58 kDa peptide fragment of TcdAiii-The 1400 bp PCR products were ligated to the plasmid vector pCRT"II according to the supplier's instructions. The DNA
sequences across the ends of the insert fragments of four isolates were determined using primers similar in sequence to the supplier's recommended primers and using sequencing methods described previously. The nucleic acid sequence of all isolates differed as expected in the regions corresponding to the degenerate primer sequences, but the amino acid sequences deduced from these data were the same as the actual amino acid sequences for the peptides determined previously, (SEQ ID NOS:41 and 39).
Screening of the W-14 genomic cosmid library as described in Example 8 with a radiolabeled probe comprised of the DNA
prepared above (SEQ ID NO:36) identified five hybridizing cosmid isolates, namely 17D9, 20B10, 21D2, 27B10, and 26D1. These cosmids were distinct from those previously identified with probes corresponding to the genes described as SEQ ID NO:11 or SEQ ID NO:25. Restriction enzyme analysis and DNA blot hybridizations identified three EcoR I fragments, of approximate sizes 3.7, 3.7, and 1.1 kbp, that span the region comprising the DNA of SEQ ID NO:36. Screening of the W-14 genomic cosmid library using as probe the radiolabeled 1.4 kbp DNA fragment prepared in this example identified the same five cosmids (17D9, 20B10, 21D2, 27B10, and 26D1). DNA blot hybridization to EcoR I-digested cosmid DNAs also showed hybridization to the same subset -107-SUBSTITUTE SHEET (RULE 26) of EcoR I fragments as seen with the 2.5 kbp TcdAii gene probe, indicating that both fragments are encoded on the genomic DNA.
DNA sequence determination of the cloned EcoR I fragments revealed an uninterrupted reading frame of 7551 base pairs (SEQ
ID NO:46), encoding a 282.9 kDa protein of 2516 amino acids (SEQ
ID NO:47). Analysis of the amino acid sequence of this protein revealed all expected internal fraaments of peptides TcdAii(SEQ
ID NOS:17, 18, 37, 38 and 39) and the TcdAiii peptide N-terminus (SEQ ID NO:41) and all TcdAiii internal peptides (SEQ ID NOS:42 and 43). The peptides isolated and identified as TcdAii and TcdAiii are each products of the open reading frame, denoted tcdA, disclosed as SEQ ID NO:46. Further, SEQ ID NO:47 shows, starting at position 89, the sequence disclosed as SEQ ID NO:13, which is the N-terminal sequence of a peptide of size approximately 201 kDa, indicating that the initial protein produced from SEQ ID No: 46 is processed in a manner similar to that previously disclosed for SEQ ID NO:12. In addition, the protein is further cleaved to generate a product of size 209.2 kDa, encoded by SEQ ID NO:48 and disclosed as SEQ ID NO:49 (TcdAii peptide), and a product of size 63.6 kDa, encoded by SEQ
ID NO:50 and disclosed as SEQ ID NO:51 (TcdAiii peptide). Thus, it is thought that the insecticidal activity identified as toxin A (Example 15) derived from the products of SEQ ID NO:46, as exemplified by the full-length protein of 282.9 kDa disclosed as SEQ ID N0:47, is processed to produce the peptides disclosed as SEQ ID NOS:49 and 51. It is thought that the insecticidal activity identified as toxin B (Example 15) derives from the products of SEQ ID NO:11, as exemplified by the 280.6 kDa protein disclosed as SEQ ID NO:12. This protein is proteolytically processed to yield the 207.6 kDa peptide disclosed as SEQ ID
NO:53, which is encoded by SEQ ID NO:52, and the 62.9 kDa peptide having N-terminal sequence disclosed as SEQ ID NO:40, and further disclosed as SEQ ID NO:55, which is encoded by SEQ ID NO:54.
Amino acid sequence comparisons between the proteins disclosed as SEQ ID NO:12 and SEQ ID NO:47 reveal that they have 69% similarity and 54% identity. This high degree of evolutionary relationship is not uniform throughout the entire amino acid sequence of these peptides, but is higher towards the carboxy-terminal end of the proteins, since the peptides SUBSTITUTE SHEET (RULE 26) disclosed as SEQ ID NO:51 (derived from SEQ ID r1O:47) and SEQ 1D
NO:55 (derived from SEQ ID N0:12) have 76% similarity and 64%
identity.

Example 18 Control of European Cornborer-Induced Leaf Damage on Maize Plants by Spray Application of Photorhabdus (Strain W-14) Broth The ability of Photorhabdus toxin(s) to reduce plant damage caused by insect larvae was demonstrated by measuring leaf damage caused by European corn borer (Ostrinia nubilalis) infested onto maize plants treated with Photorhabdus broth. Fermentation broth from Photorhabdus strain W-14 was produced and concentrated approximately 10-fold using ultrafiltration (10,000 MW pore-size) as described in Example 13. The resulting concentrated broth was then filter sterilized using 0.2 micron nitrocellulose membrane filters. A similarly prepared sample of uninoculated 2% proteose peptone #3 was used for control purposes. Maize plants (a DowElanco proprietary inbred line) were grown from seed to vegetative stage 7 or 8 in pots containing a soilless mixture in a greenhouse (27 C day; 22 C night, about 50%RH, 14 hr day-length, watered/fertilized as needed). The test plants were arranged in a randomized complete block design (3 reps/treatment, 6 plants/treatment) in a greenhouse with temperature about 22 C
day; 18 C night, no artificial light and with partial shading, about 50%RH and watered/fertilized as needed. Treatments (uninoculated media and concentrated Photorhabdus broth) were applied with a syringe sprayer, 2.0 mis applied from directly (about 6 inches) over the whorl and 2.0 additional mis applied in a circular motion from approximately one foot above the whorl.
In addition, one group of plants received no treatment. After the treatments had dried (approximately 30 minutes), twelve neonate European corn borer larvae (eggs obtained from commercial sources and hatched in-house) were applied directly to the whorl.
After one week, the plants were scored for damage to the leaves using a modified Guthrie Scale (Koziel, M. G., Beland, G. L., Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L., Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, M. Z., Merlin, E., Rhodes, R., SUBSTITUTE SHEET (RULE 26) Warren, G. W., Wright, M. and EVola, S. V. 1993).
Bio/Technology, 11, 194-195.) and the scores were compared statistically [T-test (LSD) p<0.05 and Tukey's Studentized Range (HSD) Test p<0.1]. The results are shown in Table 28. For reference, a score of 1 represents no damage, a score of 2 represents fine "window pane" damage on the unfurled leaf with no pinhole penetration and a score of 5 represents leaf penetration with elongated lesions and/or mid rib feeding evident on more than three leaves (lesions < 1 inch). These data indicate that broth or other protein containing fractions may confer protection against specific insect pests when delivered in a sprayable formulation or when the gene or derivative thereof, encoding the protein or part thereof, is delivered via a transgenic plant or microbe.
Table 28 Effect of Photorhabdus Culture Broth on European Corn Borer-Induced Leaf Damage on Maize Treatment Average Guthrie Score No Treatment 5.02a Uninoculated medium 5.15a Photorhabdus Broth 2.24b Means with different letters are statistically different (p<0.05 or p<0.1).

Example 19 Genetic Engineering of Genes for Expression in E. coli summary of constructions A series of plasmids were constructed to express the tcbA
gene of Photorhabdus W-14 in Escherichia coli. A list of the plasmids is shown in Table 29. A brief description of each construction follows as well as a summary of the E. coli expression data obtained.

SUBSTITUTE SHEET (RULE 26) Table 29 Expression plasmids for the tcbA gene.
Plasmid Gene Vector/Selection Compartment pDAB634 tcbA pBC/Chl Intracellular pAcGP67B/ tcbA tcbA pAcGP67B/Amp Baculovirus, secreted pDAB635 tcbA pET27b/Kan Periplasm pET15-tcbA tcbA pET15-tcbA Intracelluiar Abbreviations: Kan=kanamycin, Chl=chloramphenicol, Amp=ampicillin Construction of pDAB634 In Example 9, a large EcoR I fragment which hybridizes to the TcbAii probe is described. This fragment was subcloned into pBC (Stratagene, La Jolla CA). Sequence analysis indicates that this fragment is 8816 base pairs. The fragment encodes the tcbA
gene with the initiating ATG at position 571 and the terminating TAA at position 8086. The fragment therefore carries 570 base pairs of Photorhabdus DNA upstream of the ATG and 730 base pairs downstream of the TAA.
Construction of Plasmid pAcGP67B/tcbA
The tcbA gene was PCR amplified using the following primers;
5' primer (S1Ac51) 5' TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA
TC 3' and 31 primer (S1Ac31) 5' TTT AAA GCG GCC GCT TAA CGG ATG
GTA TAA CGA ATA TG 3'. PCR was performed using a TaKaRa LA PCR
kit from PanVera (Madison, Wisconsin) in the following reaction:
57.5 ml water, 10 ml lOX LA buffer, 16 ml dNTPs (2.5 mM each stock solution), 20 ml each primer at 10 pmoles/ml, 300 ng of the plasmid pDAB634 containing the W-14 tcbA gene and one ml of TaKaRa LA Taq polymerase. The cycling conditions were 98 C/20 sec, 68 C/5 min, 72 C/10 min for 30 cycles. A PCR product of the expected about 7526bp was isolated in a 0.8% agarose gel in TBE
(100 mM Tris, 90 mM boric acid, 1 mM EDTA) buffer and purified using a Qiaex II kit from Qiagen (Chatsworth, California). The purified tcbA gene was digested with Nco I and Not I and ligated into the baculovirus transfer vector pAcGP67B (PharMingen (San Diego, California)) and transformed into DH5a E. coli. The tcbA

gene was then cut from pAcGP67B and transferred to pET27b to create plasmid pDAB635. A missense mutation in the tcbA gene was repaired in pDAB635.

SUBSTITUTE SHEET (RULE 26) WO 97/17432 PCTiUS96/18003 The repaired tcbA gene contains two changes from the sequence shown in Sequence ID NO:11; an A>G at 212 changing an asparagine 71 to serine 71 and a G.-A at 229 changing an alanine 77 to threonine 77. These changes are both upstream of the proposed TcbAii N-term3.nus.
Construction of pET15-tcbA
The tcbA coding region of pDAB635 was transferred to vector pET15b. This was accomplished using shotgun ligations, the DNAs were cut with restriction enzymes Nco I and Xho I. The resulting recombinant is called pET15-tcbA.

Expression of TcbA in E. coli from plasmid pET15-tcbA
Expression of tcbA in E. co1.i was obtained by modification of the methods previously described by Studier et al. (Studier, F.W., Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol., 185: 60-89.). Competent E. coli cells strain BL21(DE3) were transformed with plasmid pET15-tcbA and plated on LB agar containing 100 g/ml ampicillin and 40 mM glucose. The transformed cells were plated to a density of several hundred isolated colonies/plate. Following overnight incubation at 37 C
the cells were scraped from the plates and suspended in LB broth containing 100 g /ml ampicillin. Typical culture volumes were from 200-500 ml. At time zero, culture densities (OD600) were from 0.05-0.15 depending on the experiment. Cultures were shaken at one of three temperatures (22 C, 30 C or 37 C) until a density of 0.15-0.5 was obtained at which time they were induced with 1 mM isopropylthio-p-galactoside (IPTG). Cultures were incubated at the designated temperature for 4-5 hours and then were transferred to 4 C until processing (12-72 hours).
Purification and characterization of TcbA expressed in E.co1i from Plasmid pET15-tcbA.
E. coli cultures expressing TcbA peptides were processed as follows. Cells were harvested by centrifugation at 17,000 x G and the media was decanted and saved in a separate container.
The media was concentrated about 8x using the M12 (Amicon, Beverly MA) filtration system and a 100 kD molecular mass cut-off filter. The concentrated media was loaded onto an anion exchange SUBSTiTUTE SHEET (RULE 26) column and the bound proteins were eluted with 1.0 M tIaC1. The 1.0 M NaCl elution peak was found to cause mortality against Southern corn rootworm (SCR) larvae Table 30). The 1.0 P=s NaCl fraction was dialyzed against 10 mM sodium phosphate buffer pH 5 7.0, concentrated, and subjected to gel filtration on Sepharose CL-4B (Pharmacia, Piscataway, New Jersey). The region of the CL-4B elution profile corresponding to calculated molecular weight (about 900 kDa) as the native W-14 toxin complex was collected, concentrated and bioassayed against larvae. The collected 900 kDa fraction was found to have insecticidal activity (see Table 30 below), with symptomology similar to that caused by native w-14 toxin complex. This fraction was subjected to Proteinase K
and heat treatment, the activity in both cases was either eliminated or reduced, providing evidence that the activity is proteinaceous in nature. In addition, the active fraction tested immunologically positive for the TcbA and TcbAiii peptides in immunoblot analysis when tested with an anti-TcbAiii monoclonal antibody (Table 30).

Table 30 Results of Immunoblot and SCR Bioassays.

Fraction SCR Activity Immunoblot Native Size % % Growth Peptides (CL-4B
Mortality Inhibit. Detected Estimated Size]
TcbA Media 1.0 M +++ +++ TcbA
Ion Exchange TcbA Media CL-4B +++ +++ TcbA, -900 kDa TcbAiii TcbA Media CL-4B ++ +++ NT
+ Proteinase K
TcbA Media CL-4B - - NT
+ heat treatment .... .. .:...: :.::...-.:. .
:.z:......,.: _r.w.,._.__.
.::;:.>::._..........._....... . .::::.:;:.::::.~....._...:.:..., ...:........:...: ::: .::.:.... ... :.::........ . ..,.: . . ..._ .._.. -. ... ,....._ .
TcbA Cell Sup CL-4B - +++ NT --900 kD

PK = Proteinase K treatment 2 hours; Heat treatment = 100 C for 10 minutes; ND = None Detected; NT = Not Tested. Scoring system for mortality and growth inhibition as compared to control samples; 5-240="+~, 25-49%="++", 50-100a="+++".

The cell pellet was resuspended in 10 mM sodium phosphate buffer, pH=7.0, and lysed by passage through a Bio-NebT" cell 30 nebulizer (Glas-Col Inc., Terra Haute, IN). The pellets were SUBSTITUTE SHEET (RULE 26) WO 97/17432 PCT/[TS96/18003 treated with Dtlase to remove DNA and centrifuged at 17,000 x g ro separate the cell pellet from the cell supernatant. The supernatant fraction was decanted and filtered through a 0.2 micron filter to remove large particles and subjected to anion exchange chromatography. Bound proteins were eluted with 1.0 M
NaCl, dialyzed and concentrated using BiomaxTM (Millipore Corp, Bedford, MA) concentrators with a molecular mass cut-off of 50,000 Daltons. The concentrated fraction was subjected to gel filtration chromatography using Sepharose CL-4B beaded matrix.
Bioassay data for material prepared in this way is shown in Table 30 and is denoted as " TcbA Cell Sup".
In yet another method to handle large amounts of material, the cell pellets were re-suspended in 10 mM sodium phosphate buffer, pH = 7.0 and thoroughly homogenized by using a Kontes Glass Company (Vineland, NJ) 40 ml tissue grinder. The cellular debris was pelleted by centrifugation at 25,000 x g and the cell supernatant was decanted, passed through a 0.2 micron filter and subjected to anion exchange chromatography using a Pharmacia 10/10 column packed with Poros HQ 50 beads. The bound proteins were eluted by performing a NaCl gradient of 0.0 to 1.0 M.
Fractions containing the TcbA protein were combined and concentrated using a 50 kDa concentrator and subjected to gel filtration chromatography using Pharmacia CL-4B beaded matrix.
The fractions containing TcbA oligomer, molecular mass of approximately 900 kDa, were collected and subjected to anion exchange chromatography using a Pharmacia Mono Q 10/10 column equilibrated with 20 mM Tris buffer pH = 7.3. A gradient of 0.0 to 1.0 M NaCl was used to elute recombinant TcbA protein.
Recombinant TcbA eluted from the column at a salt concentration of approximately 0.3-0.4 M NaCl, the same molarity at which native TcbA oligomer is eluted from the Mono Q 10/10 column. The recombinant TcbA fraction was found to cause SCR mortality in bioassay experiments similar to those in Table 30.

SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: WISCONSIN ALUMNI RESEARCH FOUNDATION

(ii) TITLE OF INVENTION: INSECTICIDAL PROTEIN TOXINS FROM PHOTORHABDUS
(iii) NUMBER OF SEQUENCES: 61 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: FETHERSTONHAUGH & CO.
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA

(D) STATE: ONT

(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: CA 2,209,659 (B) FILING DATE: 06-NOV-1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/007,255 (B) FILING DATE: 06-NOV-1995 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/608,423 (B) FILING DATE: 28-FEB-1996 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/705,484 (B) FILING DATE: 28-AUG-1996 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FETHERSTONHAUGH & CO.
(B) REGISTRATION NUMBER:

(C) REFERENCE/DOCKET NUMBER: 29355-1 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (613)-235-4373 (B) TELEFAX: (613)-232-8440 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Trp (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ala Gly Asp Thr Ala Asn Ile Gly Asp (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Leu Gly Gly Ala Ala Thr Leu Leu Asp Leu Leu Leu Pro Gln Ile (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Asn Leu Ala Ser Pro Leu Ile Ser (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Met Ile Asn Leu Asp Ile Asn Glu Gln Asn Lys Ile Met Val Val Ser (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Ala Ala Lys Asp Val Lys Phe Gly Ser Asp Ala Arg Val Lys Met Leu Arg Gly Val Asn (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7515 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..7515 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu GAG ACA GCC GCT ATT TTG GCT AAT ATT AAT ATC TCT CAG CAA GCT GTT.1536 Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gin Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2504 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gin Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gin Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Xaa Ala (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Gln Asn Ser Gln Thr Phe Ser Val Gly Glu Leu (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Gln Asn Ser Leu (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Phe Asn Ile Asp Asp Val Ser Leu Phe (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

40 Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 20 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6005 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: RBS
(B) LOCATION: 1..9 (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..3585 (D) OTHER INFORMATION: /product= "P8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Met Ser Glu Ser Leu Phe Thr Gin Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gin Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1189 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gin Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1881 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1881 (D) OTHER INFORMATION: /product= "P8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala Trp Leu Ser (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 627 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

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

(A) LENGTH: 1689 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1689 (D) OTHER INFORMATION: /product= "S8"

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

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 562 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg Asp Ala (2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4458 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..4458 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly Asn Gly Pro Phe Gly Ile Gly Trp Gin Cys Gly Val Met Ser Ile Ser Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gin Phe Ser Gln Ser Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro (2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1485 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly Gln Pro Asp Ile Arg Gin Asp Val Lys Thr Leu Gln Gly Val Thr Leu Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro 29355-i Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr Ala Met Met Asp Asp Thr Ser Leu Gin Ala Tyr Glu Gly Val Ile Glu Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gin Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro (2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser Gin Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gin Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1095 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn ~ . -Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala'Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gin Leu Ser Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln (2) INFORMATION FOR SEQ ID NO:35 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 603 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Arg Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Xaa Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu Asp Trp Met Ser Pro Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Xaa Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Arg Asp Asn Pro Phe Thr Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Val Ser Leu Thr Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gin Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2557 base pairs (B) TYPE: nucleic acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

GGAATACGCC AATCACCTTT GATGTCAATA AAAAAATATC CGAGCTAAAA CTGGAP,AAAA 2040 (2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 845 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (partial) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Gly Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Lys Cys Arg Pro Thr Gly Arg Phe Arg Phe Gly Arg Ala Gly Leu Tyr Ser Ile Asn Glu Arg Asp Thr Asp Leu Cys Pro Val Gly Lys Arg Gly Arg Arg Ile Asn Arg Arg Val Glu Phe Asn Asn Arg Leu Ile His Tyr Asn Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDNESS: single 40 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Ala Asn Ser Leu Tyr Ala Leu Phe Leu Pro Gln (2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Glu (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys (2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Asp Asp Ser Gly Asp Asp Asp Lys Val Thr Asn Thr Asp Ile His Arg (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Asp Val Xaa Gly Ser Glu Lys Ala Asn Glu Lys Leu Lys (2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7551 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46 Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gin Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gin Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gin Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gin Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys (2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2516 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gin Leu Gin Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gin Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5547 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gin Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1849 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gin Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu Ser (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1740 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gin Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys (2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 579 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51 Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gin Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5532 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala 29355-.1 Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1844 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu (2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1722 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His 50 Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg (2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 573 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg (2) INFORMATION FOR SEQ ID NO:56 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2898 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Giu Ile His Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu Ile Leu Ser Lys Ala Ile Gln Lys Lys Leu Ser Leu Thr Asp Leu Glu Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln Leu Asn Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val Phe Lys His Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala Gln Trp Gln Gln Gln His ASp Leu Glu Phe Ser Ala Leu Leu Leu Leu Leu Ser Asp Asn Pro Ile Ser Thr Ser Gin Gly Thr Asp Asp Gln Leu Asn Phe Ile Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly GCA ACA TTG TTG TCC CGC AGT GGG GCA CCA TTA GTC GAT ACC AAC GGC 2016.
Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln Ser Leu Pro Ala Leu Leu Leu Arg Trp Ser Gly Gln Thr Thr Tyr Gln Trp Leu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala Asp Ile Pro Ala Asp Tyr Leu Arg Gln Leu Arg Glu Val Val Arg Arg Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu Leu Leu Gin Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His Val Lys Gly Ser Asn (2) INFORMATION FOR SEQ ID NO:57 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 965 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

Claims (10)

CLAIMS:

1. A polynucleotide that is operably associated with a heterologous promoter, wherein said polynucleotide encodes a protein that has toxin activity against an insect pest wherein a nucleotide molecule that codes for said protein maintains hybridization with the complement of a nucleic acid sequence after hybridization and wash wherein said hybridization is conducted at 60°C in solution containing 10% w/v PEG
(polyethylene glycol, M.W. approximately 8000), 0.6X SSC, 7%
w/v SDS, 10 mM sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA, said wash is conducted in 0.25X SSC and 0.2% SDS at 60°C, and said nucleic acid sequence is selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:46.

2. A polynucleotide that encodes a protein having toxin activity against an insect pest wherein said protein comprises the amino acid sequence of SEQ ID NO:12, and SEQ ID NO:47, wherein said polynucleotide is operably associated with a heterologous promoter.

3. A polynucleotide comprising a nucleotide sequence that encodes the protein of SEQ ID NO: 12 operably associated with a heterologous promoter.

4. A polynucleotide comprising a nucleotide sequence that encodes the protein of SEQ ID NO:47 operably associated with a heterologous promoter.

5. A transgenic plant cell comprising a polynucleotide according to any one of claims 1 to 4.

6. A recombinant protein having toxin activity against an insect pest wherein a nucleotide sequence that codes for said protein maintains hybridization with a nucleic acid sequence under hybridization and wash wherein said hybridization is conducted at 60°C in solution containing 10%
w/v PEG (polyethylene glycol, M.W. approximately 8000), 0.6X SSC, 7% w/v SDS, 10 mM sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA, wherein said wash is conducted in 0.25X SSC and 0.2% SDS at 60°C and wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:46.

7. A recombinant protein having toxin activity against an insect pest wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:47.

8. A method of controlling an insect pest wherein said method comprises feeding a protein of claim 6 or 7 to said pest.

9. The method of claim 8 wherein said protein is produced by and is present in a transgenic plant that is accessible to said pest.

10. A substantially pure culture of a Photorhabdus strain of ATCC 553597.