# Patent

## Title
Inbred corn line LH287

## Abstract

This application claims the benefit of U.S. Provisional Application No. 60/116,439, filed Jan. 20, 1999, and U.S. Provisional Application No. 60/126,433, filed Feb. 20, 1998 now abandoned.

## Background
The invention relates to novel toxins from Photorhabdus luminescens, nucleic acid sequences whose expression results in said toxins, and methods of making and methods of using the toxins and corresponding nucleic acid sequences to control insects.

Insect pests are a major cause of crop losses. Solely in the US, about $7.7 billion are lost every year due to infestation by various genera of insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to gardeners and home owners.

Insect pests are mainly controlled by intensive applications of chemical insecticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or death of the insects. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management strategies, but there is an increasing need for alternative pest control agents. Biological insect control agents, such as Bacillus thuringiensis strains expressing insecticidal toxins like d-endotoxins, have also been applied with satisfactory results, offering an alternative or a complement to chemical insecticides. Recently, the genes coding for some of these d-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of insecticidal toxins in transgenic plants, such as Bacillus thuringiensis d-endotoxins, has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents. Yet, even in this case, the development of resistance remains a possibility and only a few specific insect pests are controllable. Consequently, there remains a long-felt but unfulfilled need to discover new and effective insect control agents that provide an economic benefit to farmers and that are environmentally acceptable.

The present invention addresses the need for novel insect control agents. Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.

In the search of novel insect control agents, certain classes of nematodes from the genera Heterorhabdus and Steinernema are of particular interest because of their insecticidal properties. They kill insect larvae and their offspring feed in the dead larvae. Indeed, the insecticidal activity is due to symbiotic bacteria living in the nematodes. These symbiotic bacteria are Photorhabdus in the case of Heterorhabdus and Xenorhabdus in the case of Steinernema.

The present invention is drawn to nucleic acid sequences isolated from Photorhabdus luminescens, and sequences substantially similar thereto, whose expression results in toxins that are highly toxic to economically important insect pests, particularly insect pests that infest plants. The invention is further drawn to the toxins resulting from the expression of the nucleic acid sequences, and to compositions and formulations containing the toxins, which are capable of inhibiting the ability of insect pests to survive, grow or reproduce, or of limiting insect-related damage or loss in crop plants. The invention is further drawn to a method of making the toxins and to methods of using the nucleic acid sequences, for example in microorganisms to control insects or in transgenic plants to confer insect resistance, and to a method of using the toxins, and compositions and formulations comprising the toxins, for example applying the toxins or compositions or formulations to insect-infested areas, or to prophylactically treat insect-susceptible areas or plants to confer protection or resistance to the insects.

The novel toxins are highly active against insects. For example, a number of economically important insect pests, such as the Lepidopterans Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Manduca sexta (Tobacco Hornworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), as well as the Coleopterans Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle) can be controlled by one or more of the toxins. The toxins can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.

According to one aspect, the present invention provides an isolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, nucleotides 4515-9269 of SEQ ID NO:1, nucleotides 15,171-18,035 of SEQ ID NO:11, and nucleotides 31,393-35,838 of SEQ ID NO:11; (b) a nucleotide sequence comprising nucleotides 23,768-31,336 of SEQ ID NO:11; or (c) a nucleotide sequence isocoding with the nucleotide sequence of (a) or (b); wherein expression of the nucleic acid molecule results in at least one toxin that is active against insects.

In one embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, or nucleotides 4515-9269 of SEQ ID NO:1. Preferably, the nucleotide sequence is substantially similar to nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, or nucleotides 4515-9269 of SEQ ID NO:1. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:2-6. Most preferably, the nucleotide sequence comprises nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, or nucleotides 4515-9269 of SEQ ID NO:1.

In another embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 15,171-18,035 of SEQ ID NO:11. Preferably, the nucleotide sequence is substantially similar to nucleotides 15,171-18,035 of SEQ ID NO:11. More preferably, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:12. Most preferably, the nucleotide sequence comprises nucleotides 15,171-18,035 of SEQ ID NO:11.

In still another embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 31,393-35,838 of SEQ ID NO:11. Preferably, the nucleotide sequence is substantially similar to nucleotides 31,393-35,838 of SEQ ID NO:11. More preferably, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:14. Most preferably, the nucleotide sequence comprises nucleotides 31,393-35,838 of SEQ ID NO:11.

In yet another embodiment of this aspect, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:13, and preferably comprises nucleotides 23,768-31,336 of SEQ ID NO:11.

In one embodiment, the nucleotide sequence of the invention comprises the approximately 9.7 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-21835.

In another embodiment, the nucleotide sequence of the invention comprises the approximately 38 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-30077.

In still another embodiment, the nucleotide sequence of the invention comprises the approximately 22.2 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-30078.

According to one embodiment of the invention, the toxins resulting from expression of the nucleic acid molecules of the invention have activity against Lepidopteran insects. Preferably, according to this embodiment, the toxins have activity against Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

According to another embodiment of the invention, the toxins resulting from expression of the nucleic acid molecule of the invention have activity against Lepidopteran and Coleopteran insects. Preferably, according to this embodiment, the toxins have insecticidal activity against Pluetlla xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, nucleotides 4515-9269 of SEQ ID NO:1, nucleotides 15,171-18,035 of SEQ ID NO:11, and nucleotides 31,393-35,838 of SEQ ID NO:11, wherein expression of the nucleic acid molecule results in at least one toxin that is active against insects.

In one embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, or nucleotides 4515-9269 of SEQ ID NO:1.

In another embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 15,171-18,035 of SEQ ID NO:11.

In still another embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 31,393-35,838 of SEQ ID NO:11.

In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence from Photorhabdus luminescens selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1, nucleotides 1686-2447 of SEQ ID NO:1, nucleotides 2758-3318 of SEQ ID NO:1, nucleotides 3342-4118 of SEQ ID NO:1, nucleotides 4515-9269 of SEQ ID NO:1, nucleotides 66-1898 of SEQ ID NO:11, nucleotides 2416-9909 of SEQ ID NO:11, the complement of nucleotides 2817-3395 of SEQ ID NO:11, nucleotides 9966-14,633 of SEQ ID NO:11, nucleotides 14,699-15,007 of SEQ ID NO:11, nucleotides 15,171-18,035 of SEQ ID NO:l1, the complement of nucleotides 17,072-17,398 of SEQ ID NO:11, the complement of nucleotides 18,235-19,167 of SEQ ID NO:11, the complement of nucleotides 19,385-20,116 of SEQ ID NO:11, the complement of nucleotides 20,217-20,963 of SEQ ID NO:11, the complement of nucleotides 22,172-23,086 of SEQ ID NO:11, nucleotides 23,768-31,336 of SEQ ID NO:11, nucleotides 31,393-35,838 of SEQ ID NO:11, the complement of nucleotides 35,383-35,709 of SEQ ID NO:11, the complement of nucleotides 36,032-36,661 of SEQ ID NO:11, and the complement of nucleotides 36,654-37,781 of SEQ ID NO:l1.

The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene. Still further, the present invention provides a host cell comprising such a chimeric gene. A host cell according to this aspect of the invention may be a bacterial cell, a yeast cell, or a plant cell, preferably a plant cell. Even further, the present invention provides a plant comprising such a plant cell. Preferably, the plant is maize.

In yet another aspect, the present invention provides toxins produced by the expression of DNA molecules of the present invention.

According to one embodiment, the toxins of the invention have activity against Lepidopteran insects, preferably against Pluetlla xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

According to another embodiment, the toxins of the invention have activity against Lepidopteran and Coleopteran insects, preferably against Pluetlla xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In one embodiment, the toxins are produced by the E. coli strain designated as NRRL accession number B-21835.

In another embodiment, the toxins are produced by E. coli strain designated as NRRL accession number B-30077.

In still another embodiment, the toxins are produced by E. coli strain designated as NRRL accession number B-30078.

In one embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2-6.

In another embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:12-14.

The present invention also provides a composition comprising an insecticidally effective amount of a toxin according to the invention.

In another aspect, the present invention provides a method of producing a toxin that is active against insects, comprising: (a) obtaining a host cell comprising a chimeric gene, which itself comprises a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention; and (b) expressing the nucleic acid molecule in the cell, which results in at least one toxin that is active against insects.

In a further aspect, the present invention provides a method of producing an insect-resistant plant, comprising introducing a nucleic acid molecule of the invention into the plant, wherein the nucleic acid molecule is expressible in the plant in an effective amount to control insects. According to one embodiment, the insects are Lepidopteran insects, preferably selected from the group consisting of: Pluetlla xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another embodiment, the insects are Lepidopteran and Coleopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa. decimlineata (Colorado Potato Beetle).

In still a further aspect, the present invention provides a method of controlling insects comprising delivering to the insects an effective amount of a toxin according to the present invention. According to one embodiment, the insects are Lepidopteran insects, preferably selected from the group consisting of: Pluetlla xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another embodiment, the insects are Lepidopteran and Coleopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle). Preferably, the toxin is delivered to the insects orally.

Yet another aspect of the present invention is the provision of a method for mutagenizing a nucleic acid molecule according to the present invention, wherein the nucleic acid molecule has been cleaved into population of double-stranded random fragments of a desired size, comprising: (a) adding to the population of double-stranded random fragments one or more single- or double-stranded oligonucleotides, wherein the oligonucleotides each comprise an area of identity and an area of heterology to a double-stranded template polynucleotide; (b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; (c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of the single-stranded fragments at the areas of identity to form pairs of annealed fragments, the areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and (d) repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and wherein the further cycle forms a further mutagenized double-stranded polynucleotide.

Other aspects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.

"Activity" of the toxins of the invention is meant that the toxins function as orally active insect control agents, have a toxic effect, or are able to disrupt or deter insect feeding, which may or may not cause death of the insect. When a toxin of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the toxin available to the insect.

"Associated with/operatively linked" refer to two nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulator nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence. The regulator nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.

A "coding sequence" is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.

To "control" insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage or loss in crop plants. To "control" insects may or may not mean killing the insects, although it preferably means killing the insects.

To "deliver" a toxin means that the toxin comes in contact with an insect, resulting in toxic effect and control of the insect. The toxin can be delivered in many recognized ways, e.g., orally by ingestion by the insect or by contact with the insect via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.

"Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.

A "gene" is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

"Gene of interest" refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability. The "gene of interest" may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.

A "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.

A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

"Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.

"Insecticidal" is defined as a toxic biological activity capable of controlling insects, preferably by killing them.

A nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

An "isolated" nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.

A "nucleic acid molecule" or "nucleic acid sequence" is a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.

"ORF" means open reading frame.

A "plant" is any plant at any stage of development, particularly a seed plant.

A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

"Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

"Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

"Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

A "promoter" is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase 11 and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

A "protoplast" is an isolated plant cell without a cell wall or with only parts of the cell wall.

"Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

In its broadest sense, the term "substantially similar"when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%. A nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

"Synthetic" refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic.

"Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest.

"Transformed/transgenic/recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore, (Xaa; X) represents any amino acid.

SEQ ID NO:1 is the sequence of the approximately 9.7 kb DNA fragment comprised in pCIB9359-7 which comprises the following ORFs at the specified nucleotide positions: Name            Start            End orf1             412             1665 orf2            1686             2447 orf3            2758             3318 orf4            3342             4118 orf5            4515             9269

SEQ ID NO:2 is the sequence of the .about.46.4 kDa protein encoded by orf1 of SEQ ID NO:1.

SEQ ID NO:3 is the sequence of the .about.28.1 kDa protein encoded by orf2 of SEQ ID NO:1.

SEQ ID NO:4 is the sequence of the .about.20.7 kDa protein encoded by orf3 of SEQ ID NO:1.

SEQ ID NO:5 is the sequence of the .about.28.7 kDa protein encoded by orf4 of SEQ ID NO:1.

SEQ ID NO:6 is the sequence of the .about.176 kDa protein encoded by orf5 of SEQ ID NO:1.

SEQ ID NOs:7-10 are oligonucleotides.

SEQ ID NO:11 is the sequence of the approximately 38 kb DNA fragment comprised in pNOV2400, which comprises the following ORFs at the specified nucleotide positions (descending numbers and "C" indicates that the ORF is on the complementary strand): Name        Start         End orf7         66          1898      (partial sequence) hph3        2416         9909 orf18       3395         2817              C orf4        9966        14,633 orf19      14,699       15,007 orf5       15,171       18,035 orf22      17,398       17,072             C orf10      19,167       18,235             C orf14      20,116       19,385             C orf13      20,963       20,217             C orf11      23,086       22,172             C hph2       23,768       31,336 orf2       31,393       35,838 orf21      35,709       35,383             C orf16      36,661       36,032             C orf8       37,781       36,654             C

SEQ ID NO:11 also includes the following restriction sites, some of which are used in the subcloning steps set forth in Example 17: Restriction Site     Nucleotide Position(s) AccIII               2835 BamHI                18,915 BsmBI                11,350 Bst11071             29,684 EagI                 13,590; 31,481 Eco721               34,474 MluI                 2444; 5116; 9327; 26,204 NotI                 13,589 PacI                 9915; 23,353; 37,888 PvuI                 8816 SapI                 35,248 SexAI                28,946 Sgfl                 8815 SpeI                 2157; 3769; 7831; 11,168 SphI                 755 StuI                 35,690 Tth111I              21,443

SEQ ID NO:12 is the sequence of the protein encoded by orf5 of SEQ ID NO:11.

SEQ ID NO:13 is the sequence of the protein encoded by hph2 of SEQ ID NO:11.

SEQ ID NO:14 is the sequence of the protein encoded by orf2 of SEQ ID NO:11.

SEQ ID NOs:15-22 are oligonucleotides.

The following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent. Clone        Accession Number     Date of Deposit pCIB9359-7       NRRL B-21835      September 17, 1997 pNOV2400        NRRL B-30077       December 3, 1998 pNOV1001        NRRL B-30078       December 3, 1998 Toxins

This invention relates to nucleic acid sequences whose expression results in novel toxins, and to the making and using of the toxins to control insect pests. The nucleic acid sequences are derived from Photorhabdus luminescens, a member of the Enterobacteriaceae family. P. luminescens is a symbiotic bacterium of nematodes of the genus Heterorhabditis. The nematodes colonize insect larva, kill them, and their offspring feed on the dead larvae. The insecticidal activity is actually produced by the symbiotic P. luminescens bacteria. The inventors are the first to isolate the nucleic acid sequences of the present invention from P. luminescens (ATCC strain number 29999). The expression of the nucleic acid sequences of the present invention results in toxins that can be used to control Lepidopteran insects such as Pluetlla xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Manduca sexta (Tobacco Hornworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), as well as Coleopteran insects such as Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), Diabrotica longicornis barberi (Northern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In one preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the approximately 9.7 kb nucleic acid sequence set forth in SEQ ID NO:1, whose expression results in insect control activity (further illustrated in Examples 1-11). Five open reading frames (ORFs) are present in the nucleic acid sequence set forth in SEQ ID NO:1, coding for proteins of predicted sizes 45 kDa, 28 kDa, 21 kDA, 29 kDa, and 176 kDa. The five ORFs are arranged in an operon-like structure. When expressed in a heterologous host, the .about.9.7 kb DNA fragment from P. luminescens results in insect control activity against Lepidopterans such as Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm),Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), showing that expression of the .about.9.7 kb nucleotide sequence set forth in SEQ ID NO:1 is necessary and sufficient for such insect control activity. In a preferred embodiment, the invention encompasses a DNA molecule, whose expression results in an insecticidal toxin, which is deposited in the E. coli strain pCIB9359-7 (NRRL accession number B-21835).

In another preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the approximately 38 kb nucleic acid fragment set forth in SEQ ID NO:11 and deposited in the E. coli strain pNOV2400 (NRRL accession number B-30077), whose expression results in insect control activity (see Examples 12-18). In a more preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the .about.22 kb DNA fragment deposited in the E. coli strain pNOV1001 (NRRL accession number B-30078), whose expression results in insect control activity. In a most preferred embodiment, the invention encompasses isolated nucleic acid molecules comprising nucleotide sequences substantially similar to the three ORFs corresponding to nucleotides 23,768-31,336 (hph2), 31,393-35,838 (orf2), and 15,171-18,035 (orf5) of the DNA fragment set forth in SEQ ID NO:11, as well as the proteins encoded thereby. When co-expressed in a heterologous host, these three ORFs result in insect control activity against Lepidopterans such as Pluetlla xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), as well as against Coleopterans such as Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle), showing that co-expression of these three ORFs (hph2, orf2, and orf5) is necessary and sufficient for such insect control activity.

The present invention also encompasses recombinant vectors comprising the nucleic acid sequences of this invention. In such vectors, the nucleic acid sequences are preferably comprised in expression cassettes comprising regulatory elements for expression of the nucleotide sequences in a host cell capable of expressing the nucleotide sequences. Such regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences of the present invention. Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cells, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host cells. In one embodiment, host cells for such vectors are microorganisms, such as bacteria, in particular E.coli. In another embodiment, host cells for such recombinant vectors are endophytes or epiphytes. A preferred host cell for such vectors is a eukaryotic cell, such as a yeast, a plant cell, or an insect cell. Plant cells such as maize cells are most preferred host cells. In another preferred embodiment, such vectors are viral vectors and are used for replication of the nucleotide sequences in particular host cells, e.g. insect cells or plant cells. Recombinant vectors are also used for transformation of the nucleotide sequences of this invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells. In one, such host cells are prokaryotic cells. In a preferred embodiment, such host cells are eukaryotic cells, such as yeast cells, insect cells, or plant cells. In a most preferred embodiment, the host cells are plant cells, such as maize cells.

In preferred embodiments, the insecticidal toxins of the invention each comprise at least one polypeptide encoded by a nucleotide sequence of the invention. In another preferred embodiment, the insecticidal toxins are produced from a purified strain of P. luminescens, such the strain with ATTC accession number 29999. The toxins of the present invention have insect control activity when tested against insect pests in bioassays; and these properties of the insecticidal toxins are further illustrated in Examples 1-18. The insecticidal toxins desribed in the present invention are further characterized in that their molecular weights are larger than 6,000, as found by size fractionation experiments. The insecticidal toxins retain full insectidical activity after being stored at 4.degree. C. for 2 weeks. One is also shown to retain its full insecticidal activity after being freeze-dried and stored at 22.degree. C. for 2 weeks. However, the insecticidal toxins of the invention lose their insecticidal activity after incubation for 5 minutes at 100.degree. C.

In further embodiments, the nucleotide sequences of the invention can be modified by incorporation of random mutations in a technique known as in-vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and U.S. Pat. No. 5,605,793, which are incorporated herein by reference. Millions of mutant copies of a nucleotide sequence are produced based on an original nucleotide sequence of this invention and variants with improved properties, such as increased insecticidal activity, enhanced stability, or different specificity or range of target insect pests are recovered. The method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising a nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another preferred embodiment, the size of the double-stranded random fragments is from about 5 bp to 5 kb. In a further preferred embodiment, the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.

As biological insect control agents, the insecticidal toxins are produced by expression of the nucleotide sequences in heterologous host cells capable of expressing the nucleotide sequences. In a first embodiment, P. luminescens cells comprising modifications of at least one nucleotide sequence of this invention at its chromosomal location are described. Such modifications encompass mutations or deletions of existing regulatory elements, thus leading to altered expression of the nucleotide sequence, or the incorporation of new regulatory elements controlling the expression of the nucleotide sequence. In another embodiment, additional copies of one or more of the nucleotide sequences are added to P. luminescens cells either by insertion into the chromosome or by introduction of extrachromosomally replicating molecules containing the nucleotide sequences.

In another embodiment, at least one of the nucleotide sequences of the invention is inserted into an appropriate expression cassette, comprising a promoter and termination signals. Expression of the nucleotide sequence is constitutive, or an inducible promoter responding to various types of stimuli to initiate transcription is used. In a preferred embodiment, the cell in which the toxin is expressed is a microorganism, such as a virus, a bacteria, or a fungus. In a preferred embodiment, a virus, such as a baculovirus, contains a nucleotide sequence of the invention in its genome and expresses large amounts of the corresponding insecticidal toxin after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleotide sequence. The insecticidal toxin thus produced is used as an insecticidal agent. Alternatively, baculoviruses engineered to include the nucleotide sequence are used to infect insects in-vivo and kill them either by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.

Bacterial cells are also hosts for the expression of the nucleotide sequences of the invention. In a preferred embodiment, non-pathogenic symbiotic bacteria, which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive nucleotide sequences for the same purpose.

Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli , either in transcriptional or translational fusion, behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector such as pKK223-3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al. In.: Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993)). Alternate systems for overexpression rely for example, on yeast vectors and include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12:173-177 (1994); van den Berg et aL, Biotechnology 8:135-139 (1990)).

In another preferred embodiment, at least one of the described nucleotide sequences is transferred to and expressed in Pseudomonas fluorescens strain CGA267356 (described in the published application EU 0 472 494 and in WO 94/01561) which has biocontrol characteristics. In another preferred embodiment, a nucleotide sequence of the invention is transferred to Pseudomonas aureofaciens strain 30-84 which also has biocontrol characteristics. Expression in heterologous biocontrol strains requires the selection of vectors appropriate for replication in the chosen host and a suitable choice of promoter. Techniques are well known in the art for expression in gram-negative and gram-positive bacteria and fungi.

In a particularly preferred embodiment, at least one of the insecticidal toxins of the invention is expressed in a higher organism, e.g., a plant. In this case, transgenic plants expressing effective amounts of the toxins protect themselves from insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed toxins. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect. A nucleotide sequence of the present invention is inserted into an expression cassette, which is then preferably stably integrated in the genome of said plant. In another preferred embodiment, the nucleotide sequence is included in a non-pathogenic self-replicating virus. Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.

Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

A nucleotide sequence of this invention is preferably expressed in transgenic plants, thus causing the biosynthesis of the corresponding toxin in the transgenic plants. In this way, transgenic plants with enhanced resistance to insects are generated. For their expression in transgenic plants, the nucleotide sequences of the invention may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences that have at least 35% about GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. Microbial nucleotide sequences which have low GC contents may express poorly in plants due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation. Although preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17. 477-498 (1989)). In addition, the nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, and WO 93/07278 (to Ciba-Geigy).

For efficient initiation of translation, sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleotide sequences of this invention. The sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).

Expression of the nucleotide sequences in transgenic plants is driven by promoters shown to be functional in plants. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. Thus, expression of the nucleotide sequences of this invention in leaves, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.

Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters. The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the insecticidal toxins to be synthesized only when the crop plants are treated with the inducing chemicals. Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

A preferred category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the insecticidal toxins only accumulate in cells which need to synthesize the insecticidal toxins to kill the invading insect pest. Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).

Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific expression is that described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy). A preferred stem specific promoter is that described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.

Especially preferred embodiments of the invention are transgenic plants expressing at least one of the nucleotide sequences of the invention in a root-preferred or root-specific fashion. Further preferred embodiments are transgenic plants expressing the nucleotide sequences in a wound-inducible or pathogen infection-inducible manner.

In addition to the selection of a suitable promoter, constructions for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence. Several such terminators are available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of this invention.

Numerous other sequences can be incorporated into expression cassettes described in this invention. These include sequences which have been shown to enhance expression such as intron sequences (e.g. from Adhl and bronze1) and viral leader sequences (e.g. from TMV, MCMV and AMV).

It may be preferable to target expression of the nucleotide sequences of the present invention to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression of the nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host cells. Techniques to achieve this are well-known in the art.

Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta). The choice of selectable marker is not, however, critical to the invention.

In another preferred embodiment, a nucleotide sequence of the present invention is directly transformed into the plastid genome. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et a/ (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.

The invention also includes compositions comprising at least one of the insecticidal toxins of the present invention. In order to effectively control insect pests such compositions preferably contain sufficient amounts of toxin. Such amounts vary depending on the crop to be protected, on the particular pest to be targeted, and on the environmental conditions, such as humidity, temperature or type of soil. In a preferred embodiment, compositions comprising the insecticidal toxins comprise host cells expressing the toxins without additional purification. In another preferred embodiment, the cells expressing the insecticidal toxins are lyophilized prior to their use as an insecticidal agent. In another embodiment, the insecticidal toxins are engineered to be secreted from the host cells. In cases where purification of the toxins from the host cells in which they are expressed is desired, various degrees of purification of the insecticidal toxins are reached.

The present invention further embraces the preparation of compositions comprising at least one insecticidal toxin of the present invention, which is homogeneously mixed with one or more compounds or groups of compounds described herein. The present invention also relates to methods of treating plants, which comprise application of the insecticidal toxins or compositions containing the insecticidal toxins, to plants. The insecticidal toxins can be applied to the crop area in the form of compositions or plant to be treated, simultaneously or in succession, with further compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

A preferred method of applying insecticidal toxins of the present invention is by spraying to the environment hosting the insect pest like the soil, water, or foliage of plants. The number of applications and the rate of application depend on the type and intensity of infestation by the insect pest. The insecticidal toxins can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application). The insecticidal toxins may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing insecticidal toxins, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds. The insecticidal toxins can also be provided as bait located above or below the ground.

The insecticidal toxins are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.

The formulations, compositions or preparations containing the insecticidal toxins and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogeneously mixing and/or grinding the insecticidal toxins with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).

Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or water.

The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.

Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil. The fatty acid methyltaurin salts may also be used.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.

The fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which have, as N-substituent, at least one C8-C22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing Corp. Ringwood, N.J., 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents," Chemical Publishing Co., Inc. New York, 1980.

The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989); and by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984). Active Against Lepidopteran Insects

Photorhabdus luminescens strain ATCC 29999 is grown in nutrient broth at 25.degree. C. for three days as described in the ATCC protocol for bioassay. The culture is grown for 24 hours for DNA isolation. Total DNA is isolated by treating freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg/ml lysozyme for 30 minutes at 37.degree. C. Proteinase K is added to a final concentration of 100 mg/ml, SDS is added to a final concentration of 0.5% SDS and the sample is incubated at 45.degree. C. After the solution becomes clear and viscous, the SDS concentration is raised to 1%, and 300 mM NaCI and an equal volume of phenol-chloroform-isoamyl alcohol are added, mixed gently for 5 minutes and centrifuged at 3K. The phenol-chloroform-isoamyl alcohol extraction is repeated twice. The aqueous phase is mixed with 0.7 volumes isopropanol, and the sample is centrifuged. The pellet is washed 3 times with 70% ethanol and the nucleic acids are gently resuspended in 0.5.times.TE.

The DNA is treated with 0.3 units of Sau3A per mg DNA at 37.degree. C. for 3.5 minutes in 100 ml volume containing a total of 6 mg DNA. The reaction is then heated for 30 minutes at 65.degree. C. to inactivate the enzyme. Then 2 units of Calf Intestinal Alkaline Phosphatase are added and incubated for 30 minutes at 37.degree. C. The sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed, precipitated with 0.7 volume isopropanol and centrifuged. The supernatant is transferred to a fresh tube, precipitated with ethanol, and the nucleic acids are resuspended in 0.5.times.TE at a concentration of 100 hg/ml.

SuperCos cosmid vector (Stratagene, La Jolla, Calif.) is prepared as described by the supplier utilizing the BamHI cloning site. Prepared SuperCos at 100 hg/ml is ligated with the Sau3A digested P. luminescens DNA at a molar ratio of 2:1 in a 5 ml volume overnight at 6.degree. C. The ligation mixture is packaged using Gigapack XL III (Stratagene), as described by the supplier. Packaged phages are used to infect XL-1 MR (Stratagene) cells as described by the supplier. The cosmid library is plated on L-agar with 50 mg/ml kanamycin and incubated 16 hours at 37.degree. C. 500 colonies are patched onto fresh L-kan plates at 50 colonies per plate. From the other plates the cells are washed off with L broth and mixed with 20% glycerol and frozen at -80.degree. C.

Plutella xylostella bioassays are performed by aliquoting of 50 .mu.l of the E. coli culture on the solid artificial Pluetlla xylostella diet (Biever and Boldt, Annals of Entomological Society of America, 1971; Shelton et al., J. Ent. Sci. 26:17). 4 ml of the diet is poured into 1 oz. clear plastic cups (Bioserve product #9051). 5 neonate P. xylostella from a diet adapted lab colony are placed in each diet-containing cup and then covered with a white paper lid (Bioserve product #9049). 10 larvae are assayed per concentration. Trays of cups are placed in an incubator for 3 days at 72.degree. F. with a 14:10 (hours) light:dark cycle. Then, the number of live larvae in each cup is recorded. Bioassays for other insects are performed as described for Pluetlla xylostella , but using the diet required by the insect to be tested.

The broth of P. luminescens undiluted and diluted 1:100 gives 100% mortality against P. xylostella. The broth of P. luminescens also gives 100% mortality against Diabrotica virgifera virgifera. Three clones with activity against P. xylostella and Heliothis virescens are obtained after screening 500 E. coli clones by insect bioassay. These cosmid clones are given the numbers pCIB9349, pCIB9350, and pCIB9351. Activity from Clones pCIB9349, pCIB9350, and pCIB9351

The three clones pCIB9349, pCIB9350 and pCIB9351 are found to be overlapping cosmids by restriction enzyme mapping. After digestion with Pacl, clones pCIB9349 and pCIB9351 give two DNA fragments each, and pCIB9350 gives three DNA fragments. Each fragment is isolated and is self-ligated. The enzyme Pacl does not cut the SuperCos vector; therefore, only fragments linked to it are re-isolated. The ligation mixtures are transformed into DH5.alpha. E. coli cells. Isolated transformed bacterial colonies are grown in L broth with 50 .mu.g/ml kanamycin, and plasmid DNA is isolated by using the alkaline miniprep protocol as described in Sambrook, et al. DNA is digested with Notl/Pacl and two clones, pCIB9355 and pCIB9356, are found by bioassay to still contain the insecticidal activity. Clone pCIB9355 is digested with Notl and a 17 kb and a 4 kb DNA fragment are generated. The 17 kb fragment is isolated and ligated into Bluescript vector previously cut with Notl and transformed into DH5.alpha. E. coli cells. The isolated transformed bacterial colonies are grown as described and plasmid DNA is isolated by the alkaline miniprep protocol. A clone containing the 17 kb insert is named pCIB9359 and tested by bioassay. The results are shown in Example 5. 3 .mu.g of the 17 kb insert is isolated and treated with 0.3 unit of Sau3A per .mu.g DNA for 4, 6, and 8 minutes at 37.degree. C., heated at 75.degree. C. for 15 minutes. The samples are pooled and ligated into pUC19 previously cut with BamHI and treated with calf intestinal alkaline phosphatase. The ligation is transformed into DH5.alpha. cells and plated on L agar with Xgal/Amp as described in Sambrook et al. and grown overnight at 37.degree. C. White colonies are picked and grown in L broth with 100 .mu./ml and plasmid DNA is isolated as previously described. DNA is digested with EcoRI/HindIII and novel restriction patterns are sequenced. Sequencing primers are ordered from Genosys Biotechnologies (Woodlands, Tex.). Sequencing is performed using the dideoxy chain-termination method. Sequencing is completed using Applied Biosystems Inc. model 377 automated DNA sequencer (Foster City, Calif.). Sequence is assembled using 3.0 from Gene Codes Corporation (Ann Arbor, Mich.).

pCIB9359 is digested with EcoRI and Xbal and the DNA is run on a 0.8% Seaplaque/TBE gel. The 9.7 kb fragment (SEQ ID NO:1) is isolated and ligated into pUC19 previously digested with EcoRI and Xbal. The ligation mixture is transformed into DH5.alpha. E. coli cells. Transformed bacteria are grown and plasmid DNA is isolated as previously described. The vector containing the 9.7 kb fragment in pUC19 is designated pCIB9359-7 and bioassay results are shown in Example 5.

Cultures of E. coli strains 9359 and 9359-7 containing clones pCIB9359 and pCIB9359-7, respectively, are tested for insecticidal activity against the following insects in insect bioassays: Clones Insects                                         pCIB9359 and pCIB9359-7 Plutella xylostella (Diamondback Moth (DBM))             +++ Hellothis virescens (Tobacco Budworm (TBW))               ++ Helicoverpa zea (Corn Earworm (CEW))                     +++ Spodoptera exigua (Beet Armyworm (BAW))                   + Spodoptera frugiperda (Fall Armyworm (FAW))               + Trichoplusia ni (Cabbage Looper (CL))                    +++ Ostrinia nubilalis (European Corn Borer (ECB))            ++ Manduca sexta (Tobacco Hornworm (THW)                     na Diabrotica virgifera (Western Corn Rootworm (WCR))           na Agrotis ipsilon (Black Cutworm (BCW))                     na na = not active + = significant growth inhibition ++ = >40% mortality, but less than 100% +++ = 100% mortality

The clones show insecticidal activity against P. xylostella, H. virescens, H. zea, T. ni, and O. nubilalis, and significant insect control activity against S. exigua and S. frugiperda.

Cultures of E. coli strains containing subclones of pCIB9359-7 are tested for insecticidal activity in insect bioassays against P. xylostella. Nucleotide Position Relative to 9.7 kb Restriction   EcoRI/Xbal fragment (SEQ ID NO:1) Insecticidal Activity Against Fragment       from pC1B9539-7 and Size in kb    Plutella xylostella EcoRI/Xbal          1 to 9712        9.7 kb              +++ EcORV           (-912) to 2309       3.2 kb               na HindIII            665 to 5438       4.7 kb               na KpnI              1441 to 8137       6.9 kb               na Sacl/Xbal         2677 to 9712       7.0 kb               na na = not active + = significant growth inhibition ++ = >40% mortality, but less than 100% +++ = 100% mortality

Dilutions of a culture of E.coli strain 9359-7 containing pCIB9359-7 are tested for insecticidal activity in insect bioassays. Dilutions are prepared in a culture of E.coli XL-1 in a total volume of 100 .mu.l and are transferred to diet cups with 5 insects per cup. The results show the percentage (%) of insect mortality. .mu.l 9359-7 Culture    Px       Hv      Hz       Tn 100             100      72      48       100 50             100      84      68       92 25             100      52      32       100 12.5            96       52      36       68 6.25           88       20       4       32 0              36       20      24        0 Px = P. xylostella, Hv = H. virescens, Hz = H. zea, Tn = T ni.

Cultures of E. coli 9359-7 still show substantial insecticidal activity after dilution.

The stability of the toxins is tested after storage for 2 weeks at different temperatures and conditions. 300 ml of Luria broth containing 100 (.mu.g/ml ampicillin is inoculated with E. coli strain 9359-7 and grown overnight at 37.degree. C. Samples are placed in sterile 15 ml screw cap tubes and stored at 22.degree. C. and 4.degree. C. Another sample is centrifuged; the supernatant is removed, freeze dried and stored at 22.degree. C. The samples are stored under these conditions for 2 weeks and then a bioassay is conducted against P. xylostella. The freeze dried material is resuspended in the same volume as before. All samples are resuspended by vortexing. Conditions                     Results 22.degree. C. (2weeks)             +++ 4.degree. C. (2 weeks)             +++ Freeze Dried (2 weeks)             +++ na = not active; + = significant growth inhibition; ++ = >40% mortality, but less than 100%; +++ = 100% mortality

This demonstrates that the toxins retain their activity for at least two weeks at 22.degree. C., 4.degree. C., and freeze-dried, and are therefore very stable.

The approximate sizes of the insecticidal toxins are determined. P. luminescens cosmid clones pCIB9359-7 and pUC19 in E. coli host DH5.alpha. are grown in media consisting of 50% Terrific broth and 50% Luria broth, supplemented with 50 .mu.g/ml ampicillin. Cultures (three tubes of each strain) are inoculated into 3 ml of the above media in culture tubes and incubated on a roller wheel overnight at 37.degree. C. Cultures of each strain are combined and sonicated using a Branson Model 450 Sonicator, micro tip, for approximately six 10 second cycles with cooling on ice between cycles. The sonicates are centrifuged in a Sorvall SS34 rotor at 6000 RPM for 10 minutes. The resultant supernatants are filtered through a 0.2 .mu. filter. The 3 ml fractions of the filtrates are applied to Bio-Rad Econo-Pac 10DG columns that have been previously equilibrated with 10 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. The flow through collected during sample loading is discarded. The samples are fractionated with two subsequent additions of 4 ml each of the NaCl--Tris equilibration buffer. The two four ml fractions are saved for testing. The first fraction contains all material above about 6,000 mol. wt; the second fraction contains material smaller than 6,000 mol. wt. A sample of the whole culture broth, the sonicate, and the filtered supernatant on the sonicate are tested along with the three fractions from the 1ODG column for activity on P. xylostella neonates in bioassays.

The culture, the sonicate, and the filtered supernatant of the sonicate, and the first column fraction from the 9359-7 sample are highly active on P. xylostella. The second column fraction from 9359-7 is slightly active (some stunting only). No activity is found in the third fraction from 9359-7. The sample from DH5-pUC19 does not have any activity. This indicates that the molecular weights of the toxins are above 6,000.

The heat stability of the toxins is determined. Overnight cultures of the E. coli strain pCIB9359-7 are grown in a 50:50 mixture of Luria broth and Terrific broth. Cultures are grown at 37.degree. C. in culture tubes on a tube roller. A one ml sample of the culture is placed in a 1.5 ml eppendorf tube and placed in a boiling water bath. The sample is removed after five minutes and allowed to cool to room temperature. This sample along with an untreated portion of the culture is assayed on P. xylostella. 50 .mu.l of sample of sample is spread on diet, allowed to dry and neonate larvae P. xylostella applied to the surface. The assay is incubated for 5 days at room temperature.

The untreated sample causes 100% mortality. The heat treated sample and a diet alone control do not cause any observable mortality, showing the toxins are heat sensitive.

Insecticidal activity of the toxins is tested in a leaf dip bioassay. Six leaves approximately 2 cm in diameter each are cut from seedlings of turnip and placed in a 1 oz. plastic cup (Jet Plastica) with 4 ml-5 ml of the resuspended toxin, covered tightly, and shaken until thoroughly wetted. The treated leaves are placed in 50 mm petri dishes (Gelman Sciences) on absorbent pads moistened with 300 .mu.l of water. The dish covers are left open until the leaf surface appears dry and then placed on tightly so that the leaves do not dry out.

Ten neonate P. xylostella larvae are placed in each petri dish arena. Also, a treatment of 0.1% Bond spreader/sticker with no toxin is set up as a control. The arenas are monitored daily for signs of drying leaves, and water is added or leaves replaced if necessary. After 3 days the leaves and arenas are examined under a dissecting microscope, and the number of live larvae in each arena is recorded.

100% mortality is found for 9359-7 and none in the no-toxin control, showing that the toxins are also insecticidal in a leaf dip assay. Active Against Lepidopteran and Coleopteran Insects

Photorhabdus luminescens strain ATCC 29999 is grown 14-18 hours in L broth. Total DNA is isolated from 1.5 mis of culture resuspended in 0.5% SDS, 100 .mu.g/ml proteinase K, TE to a final volume of 600 .mu.l. After a 1 hour incubation at 37.degree. C., 100 .mu.l 5M NaCl and 80 .mu.l CTAB/NaCl are added and the culture is incubated at 65.degree. C. for 10 minutes. An equal volume of chloroform is added; the culture is mixed gently and spun. The aqueous phase is extracted once with phenol and once with chloroform. The nucleic acids are treated with 10 .mu.g RNase A for 30 minutes at room temperature. The aqueous phase is mixed with 0.6 volumes isopropanol and the sample is centrifuged. The pellet is washed once with 70% ethanol and the nucleic acids are gently resuspended in 100-200 ul TE.

Two probes are PCR amplified from Photorhabdus luminescens strain ATCC 29999 genomic DNA using oligos 5'-ACACAGCAGGTTCGTCAG-3' (SEQ ID NO:7) and 5'-GGCAGAAGCACTCAACTC-3' (SEQ ID NO:8) to amplify probe #1 and oligos 5'-ATTGATAGCACGCGGCGACC-3' (SEQ ID NO:9) and 5'-TTGTAACGTGGAGCCGMCTGG-3' (SEQ ID NO:10) to amplify probe #2. The oligos are ordered from Genosys Biotechnologies, Inc. (Texas). Approximately 10-50 ng of genomic DNA is used as the template. 0.8 .mu.M of oligos, 200 .mu.M of dNTPs, 1.times. Taq DNA Polymerase buffer and 2.5 units of Taq DNA Polymerase are included in the reaction. The reaction conditions are as follows: (25 cycles)

The reactions are preferably carried out in a PCR System 9600 (Perkin Elmer) thermocycler.

600 clones from the P. luminescens cosmid library described in Example 1 are patched to L-amp plates in duplicate. The colonies are grown overnight then moved to 4.degree. C. The colonies are lifted onto Colony/Plaque Screen Hybridization Transfer Membranes (Biotechnology Systems NEN Research Products). The membranes are incubated 2-3 minutes in 0.75 ml 0.5N NaOH twice. The membranes are then incubated 2-3 minutes in 0.75 ml 1.0M Tris-HCl, pH 7.5 twice. The membranes are allowed to dry at room temperature.

Probe #1 and probe #2 described in Example 13 are labeled using the DECAprime II Kit as described by the manufacturer (Ambion cat#1455). Unincorporated nucleotides are removed from the labeled probes using Quick Spin Columns as described by the manufacturer (Boehringer Mannheim cat#1273973). The labeled probes are measured for incorporated radioactivity and the specific activity is 10,000,000 cpm. Membranes are prewetted with 2.times. SSC and hybridized with the probes for 12-16 hours at 65.degree. C. One set of colony lifts is hybridized with probe #1 and the other set is hybridized with probe #2. The membranes are washed with wash CHURCH solutions 1 and 2 (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984)) and exposed to Kodak film.

Twenty one clones are identified that hybridize to probe #1 and seven clones are identified that hybridize to probe #2. The gene in the clones isolated with probe #1 is named hphl and the gene in the clones isolated with probe #2 is named hph2.

The clones identified in Example 14 are tested for insecticidal activity against the following insects in insect bioassays: Diabrotica virgifera virgifera (Western Corn Rootworm (WCR)),Diabrotica undecimpunctata howardi (Southern Corn Rootworm (SCR)), Ostrinia nubilalis (European Corn Borer (ECB)), and Pluetlla xylostella (Diamondback Moth (DBM)).

Diabrotica virgifera virgifera (Western Corn Rootworm) and Diabrotica undecimpunctata howardi (Southern Corn Rootworm) assays are performed using a diet incorporation method. 500 .mu.l of an overnight culture of the cosmid library in XL-1 Blue MR cells (Stratagene) is sonicated and then mixed with 500 .mu.l of diet. Once the diet solidifies, it is dispensed in a petri dish and 20 larvae are introduced over the diet. Trays of dishes are placed in an incubator for 3-5 days, and percent mortality is recorded at the end of the assay period.

Ostrinia nubilalis (European Corn Borer) and Pluetlla xylostella (Diamondback Moth) assays are performed by a surface treatment method. The diet is poured in the petri dish and allowed it to solidify. The E. coli culture of 200 -300 .mu.l volume is dispensed over the diet surface and entire diet surface is covered to spread the culture with the help of bacterial loop. Once the surface is dry, 10 larvae are introduced over the diet surface. Trays of dishes are placed in an incubator for 3-5 days. The assay with European Corn Borer is incubated at 30.degree. C. in complete darkness; the assay with Diamondback Moth is incubated at 72.degree. F. with a 14:10 (hours) light:dark cycle. Percent mortality is recorded at the end of the assay period.

Cosmids containing hph2 are identified with a range of activities, including: WCR only; SCR only; WCR and SCR; SCR and ECB; WCR, SCR, and ECB; or WCR, SCR, ECB, and DBM activity.

In addition to probing the P. luminescens cosmid library with DNA probes, 600 clones are screened by Western Corn Rootworm bioassay. A clone is identified with activity against Western Corn Rootworm. This clone hybridizes with probe #2.

From these bioassays, cosmid 514, having activity against WCR, SCR, ECB, and DBM, is selected for sequencing.

Cosmid 514 is sequenced using dye terminator chemistry on an ABI 377 instrument. The nucleotide sequence of cosmid 514 is set forth as SEQ ID NO:11. Cosmid 514 is designated pNOV2400 and deposited with the NRRL in E. coli DH5.alpha. and assigned accession no. B-30077.

514a

An 9011 base pair fragment within cosmid 514 (SEQ ID NO:11) is removed by digesting the cosmid with the restriction endonuclease Spel (New England Biolabs (Massachusetts), and ligating (T4 DNA Ligase, NEB) the remainder of 514. Subclone 514a consists of cosmid 514 DNA from base pairs 1-2157 ligated to base pairs 11,169-37,948.

H202/pET34

hph2 and orf2 (SEQ ID NO:11, base pairs 23,768-35,838) are cloned into pET34b (Novagen, Wis.). Restriction sites are engineered on both ends of each gene to facilitate cloning. PCR is used to add the restriction sites to the genes. A BamHI site is on the 5' end of hph2 immediately upstream of the ATG of hph2, and a Sacl site is added to the 3' end of hph2 immediately following the DNA triplet encoding the stop codon. A guanidine is added between the BamHI site and the start codon of hph2 to put the hph2 gene in frame with the Cellulose Binding Domain tag in pET34b. Orf2 has a Sacl site upstream of the 56 base pairs between the stop codon of hph2 and the start codon of off2. The 56 base pairs are included in the hph2-orf2 construct to mimic their setup in the 514 cosmid. Orf2 has an Xhol site on the 3' end immediately following the stop codon. The oligos used to add the restriction sites to hph2 and orf2 are as follows: hph2-A      5'-CGGGATCCGATGATTTTAAAAGG-3' (SEQ ID NO:16) hph2-B      5'-GCGCCATTGATTTGAG-3' (SEQ ID NO:17) hph2-C      5'-CATTAGAGGTCGAACGTAC-3' (SEQ ID NO:18) hph2-D      5'-GAGCGAGCTCTTACTTAATGGTGTAG-3' (SEQ ID NO:19) orf2-A3     5'-CAGCGAGCTCCATGCAGAATTCACAGAC-3' (SEQ ID NO:20) orf2-B      5'-GGCAATGGCAGCGATAAG-3' (SEQ ID NO:21) orf2-C      5'-CATTAACGCAGGAAGAGC-3' (SEQ ID NO:22) orf2-D      5'-GACCTCGAGTTACACGAGCGCGTCAG-3'

The BamHI-Sacl 7583 base pair fragment, corresponding to the hph2 gene, and the Saci-Xhol 4502 base pair orf2 (including the 56 base pairs between hph2 and orf2 open reading frames), corresponding to orf2, are ligated with BamHI-Xhol-digested vector DNA pET34b.

Orf5/pBS (Notl-BamHI)

The 5325 base pair Notl-BamHI fragment of cosmid 514 is cloned into pBS-SK using AfIIII-Notl (415 bp) and BamHI-AfIIII (2530 bp) fragments of pBS-SK.

O5-H2-O2

The 12,031 base pair BamHI-Xhol fragment of H2O2/pET34 is cloned into the 8220 base pair Xhol-BamHI fragment of Orf5/pBS.

051011H2)2

A 7298 base pair BamHI-MluI fragment from subclone 514a is ligated (T4 DNA Ligase, NEB) with 9588 bp MluI-XhoI and 8220 bp XhoI-BamHI fragments of subclone O5-H2O2. The resulting .about.22 kb subclone 051011H2O2, which has activity against WCR and ECB, is designated pNOV1001 and deposited with the NRRL in E. coli DH5.alpha. and assigned accession no. B-30078.

AKH2O2

A 12,074 base pair BamHI-AvrII fragment of H2O2/pET34 is ligated (T4 DNA Ligase, NEB) into pK184 Nhel-BamHI fragment (2228 bp), generating a clone containing hph2 and orf2 in a p15a origin of replication, kanamycin-resistant vector.

Bioassays as described above are performed with E. coli cultures that express the above subclones, both singly and in combination. Coexpressing AKH2O2 and Orf5/pBS in E. coli, for example in DH5.alpha. or HB101, is found to give insecticidal activity against the Lepidopterans Pluetlla xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), as well as against the Coleopterans Diabrotica virgifera virgifera (Western Corn Rootworm),Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle). Thus, coexpression of hph2 (SEQ ID NO:11, base pairs 23,768-31,336), orf2 (SEQ ID NO:11, base pairs 31,393-35,838), and orf5 (SEQ ID NO:11, base pairs 15,171-18,035) is sufficient to control these insects. In addition, expression of each of these three ORFs on separate plasmids gives insect control activity, demonstrating that they do not have to be genetically linked to be active, so long as all three gene products are present. Heterologous Microbial Hosts

Microorganisms which are suitable for the heterologous expression of the nucleotide sequences of the invention are all microorganisms which are capable of colonizing plants or the rhizosphere. As such they will be brought into contact with insect pests. These include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, the gram-positive microorganism Bacillus and the fungi Trichoderma, Gliocladium, and Saccharomyces cerevisiae. Particularly preferred heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, Bacillus cereus, Trichoderma viride, Trichoderma harzianum, Gliocladium virens, and Saccharomyces cerevisiae. Bacteria

Many genes have been expressed in gram-negative bacteria in a heterologous manner. Expression vector pKK223-3 (Pharmacia catalogue #27-4935-01) allows expression in E. coli. This vector has a strong tac promoter (Brosius, J. et al., Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A number of other expression systems have been developed for use in E. coli The thermoinducible expression vector pP.sub.L (Pharmacia #27-4946-01) uses a tightly regulated bacteriophage .lambda. promoter which allows for high level expression of proteins. The lac promoter provides another means of expression but the promoter is not expressed at such high levels as the tac promoter. With the addition of broad host range replicons to some of these expression system vectors, expression of the nucleotide sequence in closely related gram negative-bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia is possible. For example, pLRKD211 (Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820 (1984)) contains the broad host range replicon ori Twhich allows replication in many gram-negative bacteria.

In E. coli, induction by IPTG is required for expression of the tac (i.e. trp-lac) promoter. When this same promoter (e.g. on wide-host range plasmid pLRKD21 1) is introduced into Pseudomonas it is constitutively active without induction by IPTG. This trp-lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacterium for the purposes of the constitutive expression of such a gene. Thus, a nucleotide sequence whose expression results in an insecticidal toxin can therefore be placed behind a strong constitutive promoter, transferred to a bacterium which has plant or rhizosphere colonizing properties turning this organism to an insecticidal agent. Other possible promoters can be used for the constitutive expression of the nucleotide sequence in gram-negative bacteria. These include, for example, the promoter from the Pseudomonas regulatory genes gafA and lemA (WO 94/01561) and the Pseudomonas savastanoi IAA operon promoter (Gaffney et al., J. Bacteriol. 172: 5593-5601 (1 990).

Heterologous expression of the nucleotides sequence in gram-positive bacteria is another means of producing the insecticidal toxins. Expression systems for Bacillus and Streptomyces are the best characterized. The promoter for the erythromycin resistance gene (ermR) from Streptococcus pneumoniae has been shown to be active in gram-positive aerobes and anaerobes and also in E. coli (Trieu-Cuot et al., Nuci Acids Res 18: 3660 (1990)). A further antibiotic resistance promoter from the thiostreptone gene has been used in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). The shuttle vector pHT3101 is also appropriate for expression in Bacillus (Lereclus, FEMS Microbiol Lett 60: 211-218 (1989)). A significant advantage of this approach is that many gram-positive bacteria produce spores which can be used in formulations that produce insecticidal agents with a longer shelf life. Bacillus and Streptomyces species are aggressive colonizers of soils

Trichoderma harzianum and Gliocladium virens have been shown to provide varying levels of biocontrol in the field (U.S. Pat. Nos. 5,165,928 and 4,996,157, both to Cornell Research Foundation). A nucleotide sequence whose expression results in an insecticidal toxin could be expressed in such a fungus. This could be accomplished by a number of ways which are well known in the art. One is protoplast-mediated transformation of the fungus by PEG or electroporation-mediated techniques. Alternatively, particle bombardment can be used to transform protoplasts or other fungal cells with the ability to develop into regenerated mature structures. The vector pAN7-1, originally developed for Aspergillus transformation and now used widely for fungal transformation (Curragh et al., Mycol. Res. 97(3): 313-317 (1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 56: 117-124 (1987)) is engineered to contain the nucleotide sequence. This plasmid contains the E. coli the hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter and the trpC terminator (Punt et al., Gene 56: 117-124 (1987)).

In a preferred embodiment, the nucleic acid sequences of the invention are expressed in the yeast Saccharomyces cerevisiae. Each of the three ORF's of SEQ ID NO:11 (hph2, orf2 and orf5), which together confer insecticidal activity, are cloned into individual vectors with the GALL inducible promoter and the CYC1 terminator. Each vector has ampicillin resistance and the 2 micron replicon. The vectors differ in their yeast growth markers. hph2 is cloned into p424 (TRP1, ATCC 87329), orf2 into p423 (HIS3, ATCC 87327), and orf5 into p425 (LEU2, ATCC 87331). The three constructs are transformed into S. cerevisiae independently and together. The three ORFs are expressed together and tested for protein expression and insecticidal activity.

Insecticidal formulations are made using active ingredients which comprise either the isolated toxin or alternatively suspensions or concentrates of cells which produce it and which are described in the examples above. For example, E. coli cells expressing the insecticidal toxin may be used for the control of the insect pests. Formulations are made in liquid or solid form and are described below.

In the following examples, percentages of composition are given by weight: 1. Emulsifiable concentrates:     a       b        c Active ingredient               20%     40%      50% Calcium dodecylbenzenesulfonate    5%      8%       6% Castor oil polyethlene glycol    5%      --       -- ether (36 moles of ethylene oxide) Tributylphenol polyethylene glyco    --      12%      4% ether (30 moles of ethylene oxide) Cyclohexanone                   --      15%      20% Xylene mixture                  70%     25%      20%

Emulsions of any required concentration can be produced from such concentrates by dilution with water. 2. Solutions:                   a       b       c       d Active ingredient              80%     10%     5%      95% Ethylene glycol monomethyl ether   20%     --      --      -- Polyethylene glycol 400        --      70%     --      -- N-methyl-2-pyrrolidone         --      20%     --      -- Epoxidised coconut oil         --      --      1%      5% Petroleum distillate           94%     -- (boiling range 160-1900.degree.)

These solutions are suitable for application in the form of microdrops. 3. Granulates:                   a           b Active ingredient               5%          10% Kaolin                          94%         -- Highly dispersed silicic acid   1%          -- Attapulgit                      --          90%

The active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier, and the solvent is subsequently evaporated off in vacuo. 4. Dusts:                        a           b Active ingredient               2%          5% Highly dispersed silicic acid   1%          5% Talcum                          97%         -- Kaolin                          --          90%

Ready-to-use dusts are obtained by intimately mixing the carriers with the active ingredient.

In the following examples, percentages of compositions are by weight. 1. Wettable powders:                 a       b      c Active ingredient                   20%     60%    75% Sodium lignosulfonate               5%      5%     -- Sodium lauryl sulfate               3%      --     5% Sodium diisobutylnaphthalene sulfonate    --      6%     10% Octylphenol polyethylene glycol ether    --      2%     -- (7-8 moles of ethylene oxide) Highly dispersed silicic acid       5%      27%    10% Kaolin                              67%     --     --

The active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations. 2. Emulsifiable concentrate: Active ingredient                       10% Octylphenol polyethylene glycol ether         3% (4-5 moles of ethylene oxide) Calcium dodecylbenzenesulfonate          3% Castor oil polyglycol ether              4% (36 moles of ethylene oxide) Cyclohexanone                           30% Xylene mixture                          50%

Emulsions of any required concentration can be obtained from this concentrate by dilution with water. 3. Dusts:                   a            b Active ingredient          5%            8% Talcum                     95%           -- Kaolin                     --           92%

Ready-to-use dusts are obtained by mixing the active ingredient with the carriers, and grinding the mixture in a suitable mill. 4. Extruder granulate: Active ingredient                       10% Sodium lignosulfonate                    2% Carboxymethylcellulose                   1% Kaolin                                  87%

The active ingredient is mixed and ground with the adjuvants, and the mixture is subsequently moistened with water. The mixture is extruded and then dried in a stream of air. 5. Coated granulate: Active ingredient                        3% Polyethylene glycol 200                  3% Kaolin                                  94%

The finely ground active ingredient is uniformly applied, in a mixer, to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner. 6. Suspension concentrate: Active ingredient                       40% Ethylene glycol                         10% Nonylphenol polyethylene glycol          6% (15 moles of ethylene oxide) Sodium lignosulfonate                   10% Carboxymethylcellulose                   1% 37% aqueous formaldehyde solution       0.2% Silicone oil in 75% aqueous emulsion       0.8% Water                                   32%

The finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desire concentration can be obtained by dilution with water.

The insecticidal formulations described above are applied to the plants according to methods well known in the art, in such amounts that the insect pests are controlled by the insecticidal toxin.

The nucleic acid sequences described in this application can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting a coding sequence of the invention into an expression system to which the coding sequence is heterologous (i.e., not normally present) using standard cloning procedures known in the art. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems .lambda.gtl1, .lambda.gtl0 and Charon 4; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, PCDNAII; and other similar systems. The components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. The expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the nucleotide sequence of the invention confer insect resistance to the transgenic plants.

The nucleotide sequences described in this application can be modified for expression in transgenic plant hosts. A host plant expressing the nucleotide sequences and which produces the insecticidal toxins in its cells has enhanced resistance to insect attack and is thus better equipped to withstand crop losses associated with such attack.

The transgenic expression in plants of genes derived from microbial sources may require the modification of those genes to achieve and optimize their expression in plants. In particular, bacterial ORFs which encode separate enzymes but which are encoded by the same transcript in the native microbe are best expressed in plants on separate transcripts. To achieve this, each microbial ORF is isolated individually and cloned within a cassette which provides a plant promoter sequence at the 5' end of the ORF and a plant transcriptional terminator at the 3' end of the ORF. The isolated ORF sequence preferably includes the initiating ATG codon and the terminating STOP codon but may include additional sequence beyond the initiating ATG and the STOP codon. In addition, the ORF may be truncated, but still retain the required activity; for particularly long ORFs, truncated versions which retain activity may be preferable for expression in transgenic organisms. By "plant promoter" and "plant transcriptional terminator" it is intended to mean promoters and transcriptional terminators which operate within plant cells. This includes promoters and transcription terminators which may be derived from non-plant sources such as viruses (an example is the Cauliflower Mosaic Virus).

In some cases, modification to the ORF coding sequences and adjacent sequence is not required. It is sufficient to isolate a fragment containing the ORF of interest and to insert it downstream of a plant promoter. For example, Gaffney et al. (Science 261: 754-756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants under the control of the CaMV 35S promoter and the CaMV tml terminator successfully without modification of the coding sequence and with x bp of the Pseudomonas gene upstream of the ATG still attached, and y bp downstream of the STOP codon still attached to the nahG ORF. Preferably as little adjacent microbial sequence should be left attached upstream of the ATG and downstream of the STOP codon. In practice, such construction may depend on the availability of restriction sites.

In other cases, the expression of genes derived from microbial sources may provide problems in expression. These problems have been well characterized in the art and are particularly common with genes derived from certain sources such as Bacillus. These problems may apply to the nucleotide sequence of this invention and the modification of these genes can be undertaken using techniques now well known in the art. The following problems may be encountered:

1. Codon Usage.

The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) will enable an identification of the codons within the ORF which should preferably be changed. Typically plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides A or T at this position. By modifying a gene to incorporate preferred codon usage for a particular target transgenic species, many of the problems described below for GC/AT content and illegitimate splicing will be overcome.

2. GC/AT Content.

Plant genes typically have a GC content of more than 35%. ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of messages and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice sites (see below).

3. Sequences Adjacent to the Initiating Methionine.

Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that ribosomes attach to the 5' end of the message and scan for the first available ATG at which to start translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210, incorporated herein by reference) have suggested one sequence as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (NAR 15: 6643-6653 (1987), incorporated herein by reference) has compared many plant sequences adjacent to the ATG and suggests another consensus sequence. In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. A survey of 14 maize genes located in the GenBank database provided the following results: Position Before the Initiating ATG in 14 Maize Genes: -10   -9   -8   -7    -6    -5   -4   -3    -2    -1 C     3     8    4    6     2     5    6    0    10     7 T     3     0    3    4     3     2    1    1     1     0 A     2     3    1    4     3     2    3    7     2     3 G     6     3    6    0     6     5    4    6     1     5

This analysis can be done for the desired plant species into which the nucleotide sequence is being incorporated, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotides.

4. Removal of Illegitimate Splice Sites.

Genes cloned from non-plant sources and not optimized for expression in plants may also contain motifs which may be recognized in plants as 5' or 3' splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques well known in the art.

Techniques for the modification of coding sequences and adjacent sequences are well known in the art. In cases where the initial expression of a microbial ORF is low and it is deemed appropriate to make alterations to the sequence as described above, then the construction of synthetic genes can be accomplished according to methods well known in the art. These are, for example, described in the published patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which are incorporated herein by reference. In most cases it is preferable to assay the expression of gene constructions using transient assay protocols (which are well known in the art) prior to their transfer to transgenic plants.

Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described below. The following is a description of various components of typical expression cassettes.

1. Promoters

The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non-limiting examples of promoters that may be used in expression cassettes.

a. Constitutive Expression, the Ubiquitin Promoter:

Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower--Binet et al. Plant Science 79: 87-94 (1991); maize--Christensen etal. Plant Molec. Biol. 12: 619-632 (1989); and Arabidopsis--Norris et al., Plant Mol. Biol. 21:895-906 (1993)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The Arabidopsis ubiquitin promoter is ideal for use with the nucleotide sequences of the present invention. The ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.

b. Constitutive Expression, the CaMV 35S Promoter:

Construction of the plasmid pCGN1761 is described in the published patent application EP 0 392 225 (Example 23), which is hereby incorporated by reference. pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence-tml terminator cassette of such a construction can be excised by HindIII, Sphl, Sall, and Xbal sites 5' to the promoter and Xbal, BamHI and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described below. Furthermore, the double 35S promoter fragment can be removed by 5' excision with HindIII, Sphl, Sall, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Pat. No. 5,639,949, incorporated herein by reference.

c. Constitutive Expression, the Actin Promoter:

Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Actl gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). A 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the Actl-intron 1, Adhl 5' flanking sequence and Adhl-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

d. Inducible Expression, the PR-1 Promoter:

The double 35S promoter in pCGN1761ENX may be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, one of the chemically regulatable promoters described in U.S. Pat. No. 5,614,395 may replace the double 35S promoter. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al., 1992). pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with HindIII and the resultant PR-1a promoter-containing fragment is gel purified and cloned into pCGN1761ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with HindIII and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1a promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites. The selected coding sequence can be inserted into this vector, and the fusion products (i.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described infra. Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395.

e. Inducible Expression, an Ethanol-Inducible Promoter:

A promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention. Such a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat. Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer. For the purposes of the present invention, the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) are replaced by a coding sequence of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.

f. Inducible Expression, a Glucocorticoid-lnducible Promoter:

Induction of expression of a nucleic acid sequence of the present invention using systems based on steroid hormones is also contemplated. For example, a glucocorticoid-mediated induction system is used (Aoyama and Chua (1997) The Plant Journal 11: 605-612) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to 1 mM, more preferably from 10 mM to 100 mM. For the purposes of the present invention, the luciferase gene sequences are replaced by a nucleic acid sequence of the invention to form an expression cassette having a nucleic acid sequence of the invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods well known in the art. The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al. (1986) Science 231: 699-704) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080). The expression of the fusion protein is controlled by any promoter suitable for expression in plants known in the art or described here. This expression cassette is also comprised in the plant comprising a nucleic acid sequence of the invention fused to the 6xGAL4/minimal promoter. Thus, tissue- or organ-specificity of the fusion protein is achieved leading to inducible tissue- or organ-specificity of the insecticidal toxin.

g. Root Specific Expression:

Another pattern of gene expression is root expression. A suitable root promoter is described by de Framond (FEBS 290: 103-106 (1991)) and also in the published patent application EP 0 452 269, which is herein incorporated by reference. This promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.

h. Wound-Inducible Promoters:

Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et aL Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.

i. Pith-Preferred Expression:

Patent Application WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to -1726 bp from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.

j. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.

k. Pollen-Specific Expression:

WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells. The gene sequence and promoter extend up to 1400 bp from the start of transcription. Using standard molecular biological techniques, this promoter or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a nucleic acid sequence of the invention in a pollen-specific manner.

2. Transcriptional Terminators

A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used.

3. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski etal. Plant Molec. Biol. 15: 65-79 (1990)).

4. Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting" in Example 37 of U.S. Pat. No. 5,639,949.

Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et aL Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

In addition, sequences have been characterized which cause the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.

Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.

a. pCIB200 and pCIB2001:

The binary vectors pcIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Acci fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the Xhol-digested fragment are cloned into Sail-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sall. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pCIB10 and Hygromycin Selection Derivatives thereof:

The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et aL (Gene 53: 153-161 (1987)). Various derivatives of pCIB10 are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et aL (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors Suitable for non-Agrobacterium Transformation

Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.

a. pCIB3064:

pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 355 transcriptional terminator and is described in the PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction sites are 96 and 37 bp away from the unique Sall site and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sall and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et a/. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, HindIII, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pSOG19 and pSOG35:

pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pBl221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.

3. Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention in plant plastids, plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.

Once a nucleic acid sequence of the invention has been cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants.

1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art and include grobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et aL, Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.

Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil etal. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics.RTM. helium device using a burst pressure of .about.1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as "GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

Tranformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of which are incorporated herein by reference.

3. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. `Xanthi nc` are germinated seven per plate in a 1" circular array on T agar medium and bombarded 12-14 days after sowing with 1 .mu.m tungsten particles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 .mu.mol photons/m.sup.2 /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 .mu.g/ml spectinomycin dihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5, 346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with .sup.32 P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment from pC8 containing a portion of the rps7/12 plastid targeting sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the greenhouse.

The plants obtained via tranformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, N.Y. (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, N.Y. (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.

In seed production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD.RTM.), methalaxyl (Apron.RTM.), and pirimiphos-methyl (Actellic.RTM.). If desired, these compounds are formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

It is a further aspect of the present invention to provide new agricultural methods, such as the methods examplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.

The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants. <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 22 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 9717 <212> TYPE: DNA <213> ORGANISM: Photorhabdus luminescens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (412)..(1665) <223> OTHER INFORMATION: orf1 ~46.4 kDa <400> SEQUENCE:  1 gaattcatat gctatgaaat aaacagttgg cgcaataatt aaagctatta tttttatttt     60 gtttttatac aatgatatgc tttattaaac agaataatga gttaatgata aataaatcct    120 cgggatttat catgatatta tggccgaatg tgatgtgaac aattatttta taattagatt    180 aataatataa tggtattaaa ataacaatat atttattcat gggtatttat catcggtttt    240 attacatggg gaataatcta taaattagtt ttacataatt cacaaatagc gattccatta    300 accaggaata ttaaaaatac ttatttatga ttatggtgat atatcttcat tagcctactt    360 ttataactag aaaaattgac attttcaatc catgtataaa tggtaaccaa t atg cag     417 Met Gln 1 aga gct caa cga gtt gtt att aca ggt atg ggt gcc gta aca ccg att      465 Arg Ala Gln Arg Val Val Ile Thr Gly Met Gly Ala Val Thr Pro Ile 5                  10                  15 ggt gaa gat gtt gaa tca tgt tgg caa agt att att gaa aaa caa cat      513 Gly Glu Asp Val Glu Ser Cys Trp Gln Ser Ile Ile Glu Lys Gln His 20                  25                  30 cga ttt cac aga att gaa ttt cct gac tca ttc att aat tcg cgt ttc      561 Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe Ile Asn Ser Arg Phe 35                  40                  45                  50 ttt tct ttc ctt gca cca aac cca tcc cgc tat cag tta tta cca aaa      609 Phe Ser Phe Leu Ala Pro Asn Pro Ser Arg Tyr Gln Leu Leu Pro Lys 55                  60                  65 aag ttg act cat aca ctt tct gac tgc gga aaa gca gca ttg aag gcg      657 Lys Leu Thr His Thr Leu Ser Asp Cys Gly Lys Ala Ala Leu Lys Ala 70                  75                  80 act tat caa gct ttt acc caa gca ttc ggc gtg aat ata tca cct gtt      705 Thr Tyr Gln Ala Phe Thr Gln Ala Phe Gly Val Asn Ile Ser Pro Val 85                  90                  95 gaa tat tac gat aaa tac gaa tgt ggc gta att ctt ggc agt ggt tgg      753 Glu Tyr Tyr Asp Lys Tyr Glu Cys Gly Val Ile Leu Gly Ser Gly Trp 100                 105                 110 gga gct att gat aat gcc gga gat cat gct tgc caa tat aag caa gca      801 Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cys Gln Tyr Lys Gln Ala 115                 120                 125                 130 aaa tta gct cat cct atg agt aat ctt att acc atg cca agc tcc atg      849 Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr Met Pro Ser Ser Met 135                 140                 145 acg gct gca tgt tcg att atg tat gga cta cgt ggt tat caa aat acc      897 Thr Ala Ala Cys Ser Ile Met Tyr Gly Leu Arg Gly Tyr Gln Asn Thr 150                 155                 160 gtt atg gct gcc tgc gca acg ggc aca atg gcg ata ggc gat gcc ttt      945 Val Met Ala Ala Cys Ala Thr Gly Thr Met Ala Ile Gly Asp Ala Phe 165                 170                 175 gaa att att cgc tca ggg cgg gca aaa tgt atg att gcc gga gcc gct      993 Glu Ile Ile Arg Ser Gly Arg Ala Lys Cys Met Ile Ala Gly Ala Ala 180                 185                 190 gaa tca ctc acg cgg gaa tgt aat att tgg agt att gat gta ctg aat     1041 Glu Ser Leu Thr Arg Glu Cys Asn Ile Trp Ser Ile Asp Val Leu Asn 195                 200                 205                 210 gca tta tcg aaa gaa caa gcg gac cca aat ctt gca tgt tgt cca ttt     1089 Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu Ala Cys Cys Pro Phe 215                 220                 225 agc ctt gat cgc tct gga ttt gta tta gcc gaa gga gcg gcg gta gtt     1137 Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu Gly Ala Ala Val Val 230                 235                 240 tgt ctg gaa aat tat gat tca gcc atc gcg cgt ggt gca acg att tta     1185 Cys Leu Glu Asn Tyr Asp Ser Ala Ile Ala Arg Gly Ala Thr Ile Leu 245                 250                 255 gcg gaa att aaa ggt tac gcc caa tat tca gat gcc gtt aat tta acc     1233 Ala Glu Ile Lys Gly Tyr Ala Gln Tyr Ser Asp Ala Val Asn Leu Thr 260                 265                 270 cgg cca aca gaa gat att gaa cct aaa ata tta gcg ata act aaa gcc     1281 Arg Pro Thr Glu Asp Ile Glu Pro Lys Ile Leu Ala Ile Thr Lys Ala 275                 280                 285                 290 att gag cag gca cag att tcg ccg aaa gat att gac tac att aat gct     1329 Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile Asp Tyr Ile Asn Ala 295                 300                 305 cat ggt act tct aca ccg tta aat gat ctt tat gaa act cag gca att     1377 His Gly Thr Ser Thr Pro Leu Asn Asp Leu Tyr Glu Thr Gln Ala Ile 310                 315                 320 aaa gca gca ctg ggc caa tat gct tat cag gta cct ata tca agc aca     1425 Lys Ala Ala Leu Gly Gln Tyr Ala Tyr Gln Val Pro Ile Ser Ser Thr 325                 330                 335 aaa tct tat acc ggc cac ctt att gct gcc gcc ggt agt ttt gaa acg     1473 Lys Ser Tyr Thr Gly His Leu Ile Ala Ala Ala Gly Ser Phe Glu Thr 340                 345                 350 att gta tgt gtg aaa gca tta gct gaa aat tgc ttg cca gca aca ttg     1521 Ile Val Cys Val Lys Ala Leu Ala Glu Asn Cys Leu Pro Ala Thr Leu 355                 360                 365                 370 aat tta cac cgg gcc gat cca gat tgc gat ctc aat tat ttg cct aat     1569 Asn Leu His Arg Ala Asp Pro Asp Cys Asp Leu Asn Tyr Leu Pro Asn 375                 380                 385 caa cat tgc tac acc gct caa cca gag gtg aca ctc aat att agc gca     1617 Gln His Cys Tyr Thr Ala Gln Pro Glu Val Thr Leu Asn Ile Ser Ala 390                 395                 400 ggt ttc ggc ggg cat aac gct gcg ttg gtt atc gct aag gta agg taa     1665 Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile Ala Lys Val Arg 405                 410                 415 ctgatatgtt gatttttgca atg gaa gat att gaa cat tgg tcg aat ttc tct   1718 Met Glu Asp Ile Glu His Trp Ser Asn Phe Ser 420                 425 ggg gat ttt aac ccc atc cat tat tcg gcg aaa agc gag tct ttg cgc     1766 Gly Asp Phe Asn Pro Ile His Tyr Ser Ala Lys Ser Glu Ser Leu Arg 430                 435                 440                 445 aat ata cag caa cac ccg gtg cag gga atg ttg agt ttg ctc tat gta     1814 Asn Ile Gln Gln His Pro Val Gln Gly Met Leu Ser Leu Leu Tyr Val 450                 455                 460 cgg caa cag ttt tct caa tta act tcc gct ttt aca acg gga ata ttg     1862 Arg Gln Gln Phe Ser Gln Leu Thr Ser Ala Phe Thr Thr Gly Ile Leu 465                 470                 475 aac att gat gcc tct ttc cgc cag tat gtt tat acc gca tta ccc cat     1910 Asn Ile Asp Ala Ser Phe Arg Gln Tyr Val Tyr Thr Ala Leu Pro His 480                 485                 490 caa ctg agg att aat act aaa aac aaa acg ttt aaa tta gaa aat ccc     1958 Gln Leu Arg Ile Asn Thr Lys Asn Lys Thr Phe Lys Leu Glu Asn Pro 495                 500                 505 agt aaa gaa aac acg ttg ttc ggc aat acc agc gta gag aat aca atg     2006 Ser Lys Glu Asn Thr Leu Phe Gly Asn Thr Ser Val Glu Asn Thr Met 510                 515                 520                 525 gag tca att gaa gat tgg atc gtt cag gat aat tgt caa aaa cta acg     2054 Glu Ser Ile Glu Asp Trp Ile Val Gln Asp Asn Cys Gln Lys Leu Thr 530                 535                 540 ata aca ggg gag gaa gtt tgt gaa aag tat gct gtc ttt aga tac tat     2102 Ile Thr Gly Glu Glu Val Cys Glu Lys Tyr Ala Val Phe Arg Tyr Tyr 545                 550                 555 ttc cca agt gtc act tct att gga tgg ttc ctg gat gcg ctt gct ttt     2150 Phe Pro Ser Val Thr Ser Ile Gly Trp Phe Leu Asp Ala Leu Ala Phe 560                 565                 570 cat ctt att att aat tcg aca gga ttt ctt aat ttt gag cac tac cat     2198 His Leu Ile Ile Asn Ser Thr Gly Phe Leu Asn Phe Glu His Tyr His 575                 580                 585 ttt aac caa tta cag gat tat ctg agt caa tct ttt act ttg cat act     2246 Phe Asn Gln Leu Gln Asp Tyr Leu Ser Gln Ser Phe Thr Leu His Thr 590                 595                 600                 605 ggg caa gcg att aaa atc agg aag gag att gtt aat agt aca gta tta     2294 Gly Gln Ala Ile Lys Ile Arg Lys Glu Ile Val Asn Ser Thr Val Leu 610                 615                 620 tta tct tca ccg gat atc tgt gtt gaa tta aat cct cct tta ttg att     2342 Leu Ser Ser Pro Asp Ile Cys Val Glu Leu Asn Pro Pro Leu Leu Ile 625                 630                 635 aag aat ggc gat aaa gat tat att cgt att ttc tat tat cga tgt tta     2390 Lys Asn Gly Asp Lys Asp Tyr Ile Arg Ile Phe Tyr Tyr Arg Cys Leu 640                 645                 650 tat gat aaa aaa cct att ttt gta tca aag act tca att atc tct aag     2438 Tyr Asp Lys Lys Pro Ile Phe Val Ser Lys Thr Ser Ile Ile Ser Lys 655                 660                 665 atg aaa taa aaggaaagcg aaatgccaac acaaagtgat attttcactg             2487 Met Lys 670 aaataaagaa tagaatatta atgatgaagg atatagaaga tgaagaaata acaccagagt   2547 cctcttttgt ttcgcttgaa tttgatagtc ttgactatgt ggaaatccaa gtttttgtgt   2607 tggaagcgta tggtattgtg cttaaagccg aacttttttc aaatcattct atttcaacat   2667 taaatgagct cactgactat ttaaaatcaa aattgtaatc tgaattttta cttaattatg   2727 ttttttcacc attaacatta agaggttata atg aac gtt tta gaa caa ggt aag    2781 Met Asn Val Leu Glu Gln Gly Lys 675                 680 gtt gct gct tta tat tca gcc tat tcg gaa aca gaa ggt tct tcg tgg     2829 Val Ala Ala Leu Tyr Ser Ala Tyr Ser Glu Thr Glu Gly Ser Ser Trp 685                 690                 695 gtg gga aac ttg tgc tgt ttt tca agt gat cgg gag cat ttg cct att     2877 Val Gly Asn Leu Cys Cys Phe Ser Ser Asp Arg Glu His Leu Pro Ile 700                 705                 710 atc gtg aat ggg cgt cgt ttc ttg att gaa ttt gtt att cca gat cat     2925 Ile Val Asn Gly Arg Arg Phe Leu Ile Glu Phe Val Ile Pro Asp His 715                 720                 725 tta ctt gat aaa acg gtt aaa ccc aga gta ttc gat ttg gat atc aat     2973 Leu Leu Asp Lys Thr Val Lys Pro Arg Val Phe Asp Leu Asp Ile Asn 730                 735                 740 aaa caa ttt tta ctg cgt cgt gac cat cgt gag ata aat att tat ctt     3021 Lys Gln Phe Leu Leu Arg Arg Asp His Arg Glu Ile Asn Ile Tyr Leu 745                 750                 755                 760 tta ggt gaa gga aat ttt atg gat agg acg acg aca gat aaa aat cta     3069 Leu Gly Glu Gly Asn Phe Met Asp Arg Thr Thr Thr Asp Lys Asn Leu 765                 770                 775 ttc gag tta aat gag gat ggt tca cta ttt att aag acg tta cgc cat     3117 Phe Glu Leu Asn Glu Asp Gly Ser Leu Phe Ile Lys Thr Leu Arg His 780                 785                 790 gct ctt ggt aaa tat gtt gct att aat cct tca act acg caa ttt atc     3165 Ala Leu Gly Lys Tyr Val Ala Ile Asn Pro Ser Thr Thr Gln Phe Ile 795                 800                 805 ttc ttt gca caa gga aag tac agt gaa ttt atc atg aat gcc tta aag     3213 Phe Phe Ala Gln Gly Lys Tyr Ser Glu Phe Ile Met Asn Ala Leu Lys 810                 815                 820 aca gtt gaa gac gaa tta tca aaa cgt tat cga gtc aga att att cct     3261 Thr Val Glu Asp Glu Leu Ser Lys Arg Tyr Arg Val Arg Ile Ile Pro 825                 830                 835                 840 gaa ttg caa ggg ccg tat tat ggc ttt gaa ctt gat att ctt tct att     3309 Glu Leu Gln Gly Pro Tyr Tyr Gly Phe Glu Leu Asp Ile Leu Ser Ile 845                 850                 855 aca gct taa ttcacaatat tatggagagt gtt atg gaa aag aaa ata aca aca   3362 Thr Ala                               Met Glu Lys Lys Ile Thr Thr 860                 865 ttt acc att gag aaa act gat gac aat ttt tat gct aat ggg cgt cat     3410 Phe Thr Ile Glu Lys Thr Asp Asp Asn Phe Tyr Ala Asn Gly Arg His 870                 875                 880 caa tgt atg gta aaa atc tct gta ctt aaa caa gaa tat agg aat ggt     3458 Gln Cys Met Val Lys Ile Ser Val Leu Lys Gln Glu Tyr Arg Asn Gly 885                 890                 895 gat tgg ata aaa tta gca ctt agt gag gct gaa aaa aga tcg att cag     3506 Asp Trp Ile Lys Leu Ala Leu Ser Glu Ala Glu Lys Arg Ser Ile Gln 900                 905                 910 gtg gcg gca tta agt gat agc ctc ata tat gac caa tta aaa atg cct     3554 Val Ala Ala Leu Ser Asp Ser Leu Ile Tyr Asp Gln Leu Lys Met Pro 915                 920                 925                 930 tca ggt tgg aca acg aca gat gca aga aat aaa ttt gat ctt ggg tta     3602 Ser Gly Trp Thr Thr Thr Asp Ala Arg Asn Lys Phe Asp Leu Gly Leu 935                 940                 945 tta aat ggt gtt tat cat gct gat gct ttt att gac gaa cag gta aca     3650 Leu Asn Gly Val Tyr His Ala Asp Ala Phe Ile Asp Glu Gln Val Thr 950                 955                 960 gat cgt gcg gga gat tgc tgc aca aat gaa aac tat cag aac agt gtg     3698 Asp Arg Ala Gly Asp Cys Cys Thr Asn Glu Asn Tyr Gln Asn Ser Val 965                 970                 975 aaa agt gtt cct gaa att atc tat cgt tat gtc agt agt aat aga aca     3746 Lys Ser Val Pro Glu Ile Ile Tyr Arg Tyr Val Ser Ser Asn Arg Thr 980                 985                 990 agc aca gaa tac cta atg gca aaa atg aca ttt gaa gat acg gat ggg     3794 Ser Thr Glu Tyr Leu Met Ala Lys Met Thr Phe Glu Asp Thr Asp Gly 995                1000                1005                1010 aaa cgc aca tta aca acg aat atg tca gtt ggt gat gaa gtt ttt gac     3842 Lys Arg Thr Leu Thr Thr Asn Met Ser Val Gly Asp Glu Val Phe Asp 1015                1020                1025 agc aag gtt tta tta aaa gcc att gct cct tat gca att aat aca aat     3890 Ser Lys Val Leu Leu Lys Ala Ile Ala Pro Tyr Ala Ile Asn Thr Asn 1030                1035                1040 caa ttg cat gaa aac atc aat aca ttg ttt gat aaa aca gaa gag ccg     3938 Gln Leu His Glu Asn Ile Asn Thr Leu Phe Asp Lys Thr Glu Glu Pro 1045                1050                1055 aca aaa tcc gat act cat cat caa ata att aat ctt tat cgc tgg aca     3986 Thr Lys Ser Asp Thr His His Gln Ile Ile Asn Leu Tyr Arg Trp Thr 1060                1065                1070 ttg cca tat cat ttg agg att ctt gaa ggg aat gac agt act gtt aat     4034 Leu Pro Tyr His Leu Arg Ile Leu Glu Gly Asn Asp Ser Thr Val Asn 1075               1080                1085                1090 aga ata tat gtc ctt ggt aaa gag cca tca aat gat aga ttc ctg aca     4082 Arg Ile Tyr Val Leu Gly Lys Glu Pro Ser Asn Asp Arg Phe Leu Thr 1095                1100                1105 aga gga agg gta ttt aaa cga gga act cat atg tga atgcacgtga          4128 Arg Gly Arg Val Phe Lys Arg Gly Thr His Met 1110                1115 taatgtgagt ggaggatgtg ttatggacta tgcttatacc gtaactattc cggacacgca   4188 gcttgctgct gaagtgcttc atgtgacagg gtgttcgtgg acgagtggtt attatgatgg   4248 atatcatgat gtcacaatca ttgataacta cggttgtcag cataaattta gaatttcttc   4308 ggttaatatt ggacgtgcgc taagcatagc gagaataagt tgattttcct tagtaaaaaa   4368 cctttgttta tgctggtaaa cgcatgtgcg tttgccagca attaatatat tccattattg   4428 aaataggaat atagccatat ctgtaattat acataaacga atttttactc gaatataatt   4488 ttaattgatc aaacaggaaa tttaaa atg aaa gct acc gat ata tat tcc aat    4541 Met Lys Ala Thr Asp Ile Tyr Ser Asn 1120                1125 gct ttt aat ttc ggt tct tat att aat act ggt gtc gat ccc aga aca     4589 Ala Phe Asn Phe Gly Ser Tyr Ile Asn Thr Gly Val Asp Pro Arg Thr 1130                1135                1140 ggt caa tat agt gca aat att aat att atc acg tta aga cct aat aat     4637 Gly Gln Tyr Ser Ala Asn Ile Asn Ile Ile Thr Leu Arg Pro Asn Asn 1145                1150                1155 gtg ggt aat tcg gaa caa aca ttg agc cta tca ttc tcg cca tta aca     4685 Val Gly Asn Ser Glu Gln Thr Leu Ser Leu Ser Phe Ser Pro Leu Thr 1160               1165                1170                1175 acg tta aac aat ggc ttt ggt att ggc tgg cgc ttt tca tta aca aca     4733 Thr Leu Asn Asn Gly Phe Gly Ile Gly Trp Arg Phe Ser Leu Thr Thr 1180                1185                1190 tta gat ata aaa aca ctt aca ttt agc cga gca aat ggg gag caa ttt     4781 Leu Asp Ile Lys Thr Leu Thr Phe Ser Arg Ala Asn Gly Glu Gln Phe 1195                1200                1205 aaa tgt aag cca ttg ccg cct aat aat aat gat ctt agt ttt aaa gat     4829 Lys Cys Lys Pro Leu Pro Pro Asn Asn Asn Asp Leu Ser Phe Lys Asp 1210                1215                1220 aaa aaa cta aaa gat ttg cgc gta tat aag ctc gat agc aat act ttt     4877 Lys Lys Leu Lys Asp Leu Arg Val Tyr Lys Leu Asp Ser Asn Thr Phe 1225                1230                1235 tat gtt tat aac aaa aac ggc att ata gag ata ctt aaa cga att ggg     4925 Tyr Val Tyr Asn Lys Asn Gly Ile Ile Glu Ile Leu Lys Arg Ile Gly 1240               1245                1250                1255 tcg agt gat att gca aaa aca gtt gca ctt gaa ttt cct gat ggt gaa     4973 Ser Ser Asp Ile Ala Lys Thr Val Ala Leu Glu Phe Pro Asp Gly Glu 1260                1265                1270 gca ttt gat tta att tat aat tca aga ttt gca ttg tcc gaa ata aaa     5021 Ala Phe Asp Leu Ile Tyr Asn Ser Arg Phe Ala Leu Ser Glu Ile Lys 1275                1280                1285 tac cgt gtg aca ggt aaa act tat ctt aaa ctc aat tac tct gga aat     5069 Tyr Arg Val Thr Gly Lys Thr Tyr Leu Lys Leu Asn Tyr Ser Gly Asn 1290                1295                1300 aac tgt aca tca gtg gaa tac cct gat gat aat aat att tct gcg aaa     5117 Asn Cys Thr Ser Val Glu Tyr Pro Asp Asp Asn Asn Ile Ser Ala Lys 1305                1310                1315 ata gca ttc gat tat cgt aac gat tac ctt att acg gtg act gta cct     5165 Ile Ala Phe Asp Tyr Arg Asn Asp Tyr Leu Ile Thr Val Thr Val Pro 1320               1325                1330                1335 tac gat gct tct ggt cct att gat tct gcc cga ttt aag atg acc tat     5213 Tyr Asp Ala Ser Gly Pro Ile Asp Ser Ala Arg Phe Lys Met Thr Tyr 1340                1345                1350 cag aca tta aaa ggc gta ttt cca gtt atc agc acc ttc cgt aca cca     5261 Gln Thr Leu Lys Gly Val Phe Pro Val Ile Ser Thr Phe Arg Thr Pro 1355                1360                1365 acc ggt tat gtt gag ctg gtg agt tat aaa gag aat ggg cat aaa gtg     5309 Thr Gly Tyr Val Glu Leu Val Ser Tyr Lys Glu Asn Gly His Lys Val 1370                1375                1380 acg gac acg gaa tat att cct tat gcg gct gca ctc act att caa ccc     5357 Thr Asp Thr Glu Tyr Ile Pro Tyr Ala Ala Ala Leu Thr Ile Gln Pro 1385                1390                1395 ggc aat gga caa cct gcg gtc agc aaa tcc tat gaa tat agt tca gta     5405 Gly Asn Gly Gln Pro Ala Val Ser Lys Ser Tyr Glu Tyr Ser Ser Val 1400               1405                1410                1415 cat aac ttc ttg ggc tat tct tct ggc cgg aca agc ttt gat tcc agt     5453 His Asn Phe Leu Gly Tyr Ser Ser Gly Arg Thr Ser Phe Asp Ser Ser 1420                1425                1430 caa gat aat ttg tat ttg gtc aca ggg aaa tac act tat tca tcc att     5501 Gln Asp Asn Leu Tyr Leu Val Thr Gly Lys Tyr Thr Tyr Ser Ser Ile 1435                1440                1445 gaa cgg gtt tta gat ggt caa agt gtg gtt tca gta ata gaa cga gta     5549 Glu Arg Val Leu Asp Gly Gln Ser Val Val Ser Val Ile Glu Arg Val 1450                1455                1460 ttt aat aaa ttc cat tta atg acc aaa gaa gca aaa aca caa gat aat     5597 Phe Asn Lys Phe His Leu Met Thr Lys Glu Ala Lys Thr Gln Asp Asn 1465                1470                1475 aag aga att aca aca gaa att act tac aat gag gat cta tca aaa agt     5645 Lys Arg Ile Thr Thr Glu Ile Thr Tyr Asn Glu Asp Leu Ser Lys Ser 1480               1485                1490                1495 ttc tca gag caa cca gaa aat tta caa caa cct tct cgc gtg tta acc     5693 Phe Ser Glu Gln Pro Glu Asn Leu Gln Gln Pro Ser Arg Val Leu Thr 1500                1505                1510 cgt tat acg gat ata caa aca aat act tca cga gaa gag act gtc aat     5741 Arg Tyr Thr Asp Ile Gln Thr Asn Thr Ser Arg Glu Glu Thr Val Asn 1515                1520                1525 att aaa agt gat gat tgg gga aat act cta ctt att act gag acc agt     5789 Ile Lys Ser Asp Asp Trp Gly Asn Thr Leu Leu Ile Thr Glu Thr Ser 1530                1535                1540 ggg ata cag aaa gaa tac gtt tat tat ccg gtc aat ggc gaa ggt aat     5837 Gly Ile Gln Lys Glu Tyr Val Tyr Tyr Pro Val Asn Gly Glu Gly Asn 1545                1550                1555 agt tgc cct gcc gat ccc ttg ggt ttt tct cgg ttc tta aaa tca gtt     5885 Ser Cys Pro Ala Asp Pro Leu Gly Phe Ser Arg Phe Leu Lys Ser Val 1560               1565                1570                1575 acg caa aaa gga tcg cct gat gct gct caa agt gtc gca aat aaa gtg     5933 Thr Gln Lys Gly Ser Pro Asp Ala Ala Gln Ser Val Ala Asn Lys Val 1580                1585                1590 att cat tat aca tat caa aaa ttt cct act ttt acc ggc gct tat gtt     5981 Ile His Tyr Thr Tyr Gln Lys Phe Pro Thr Phe Thr Gly Ala Tyr Val 1595                1600                1605 aag gaa tat gtc agt aaa gtc tca gag acg ata gac aat aaa ata gcg     6029 Lys Glu Tyr Val Ser Lys Val Ser Glu Thr Ile Asp Asn Lys Ile Ala 1610                1615                1620 aga acc ttt agc tat gtt aac tca ccg acg agt aaa tct cat ggt tcg     6077 Arg Thr Phe Ser Tyr Val Asn Ser Pro Thr Ser Lys Ser His Gly Ser 1625                1630                1635 tta gca aaa ata acg tca gtg atg aat aac cag caa acg gtc acc aca     6125 Leu Ala Lys Ile Thr Ser Val Met Asn Asn Gln Gln Thr Val Thr Thr 1640               1645                1650                1655 ttt aaa tat gaa tat tca gaa agt gag atg acc aca aat gct acg gtg     6173 Phe Lys Tyr Glu Tyr Ser Glu Ser Glu Met Thr Thr Asn Ala Thr Val 1660                1665                1670 acc ggt ttt gat ggc gca cat atg gaa tcg aaa aat gtg acg tct att     6221 Thr Gly Phe Asp Gly Ala His Met Glu Ser Lys Asn Val Thr Ser Ile 1675                1680                1685 tat acc cat cgg caa ctt cgt aaa gtt gat gta aac cac gtg att acc     6269 Tyr Thr His Arg Gln Leu Arg Lys Val Asp Val Asn His Val Ile Thr 1690                1695                1700 gat cag tct tat gat ctt ttg ggt cgc att aca ggg caa att att gat     6317 Asp Gln Ser Tyr Asp Leu Leu Gly Arg Ile Thr Gly Gln Ile Ile Asp 1705                1710                1715 ccc ggc acg gca aga gaa att aaa cgt aat tac gtt tat caa tat ccc     6365 Pro Gly Thr Ala Arg Glu Ile Lys Arg Asn Tyr Val Tyr Gln Tyr Pro 1720               1725                1730                1735 ggc ggt gac gaa aat gat ttt tgg ccg gtg atg ata gaa gtt gat tct     6413 Gly Gly Asp Glu Asn Asp Phe Trp Pro Val Met Ile Glu Val Asp Ser 1740                1745                1750 caa ggc gtc aga cgt aaa acc cat tac gat gga atg gga cgt att tgt     6461 Gln Gly Val Arg Arg Lys Thr His Tyr Asp Gly Met Gly Arg Ile Cys 1755                1760                1765 tcg att gaa gaa caa gat gat gat ggc gcc tgg ggc aca tcg ggg att     6509 Ser Ile Glu Glu Gln Asp Asp Asp Gly Ala Trp Gly Thr Ser Gly Ile 1770                1775                1780 tat caa ggc aca tat cga aaa gtt ctt gcc aga caa tat gat gtt ttg     6557 Tyr Gln Gly Thr Tyr Arg Lys Val Leu Ala Arg Gln Tyr Asp Val Leu 1785                1790                1795 ggg cag ttg agc aag gaa att tca aat gat tgg tta tgg aat tta tct     6605 Gly Gln Leu Ser Lys Glu Ile Ser Asn Asp Trp Leu Trp Asn Leu Ser 1800               1805                1810                1815 gcc aat cct ttg gtt cgt ctt gct acc ccg ttg gtt aca acg aaa acc     6653 Ala Asn Pro Leu Val Arg Leu Ala Thr Pro Leu Val Thr Thr Lys Thr 1820                1825                1830 tat aaa tat gat ggt tgg gga aat ctt tac agc acg gaa tac agt gat     6701 Tyr Lys Tyr Asp Gly Trp Gly Asn Leu Tyr Ser Thr Glu Tyr Ser Asp 1835                1840                1845 ggt cgg ata gag ctg gaa atc cat gat cct att acg agg aca att act     6749 Gly Arg Ile Glu Leu Glu Ile His Asp Pro Ile Thr Arg Thr Ile Thr 1850                1855                1860 caa ggg gtc aaa gga tta ggg atg tta aat att cag caa aat aat ttt     6797 Gln Gly Val Lys Gly Leu Gly Met Leu Asn Ile Gln Gln Asn Asn Phe 1865                1870                1875 gag caa ccg gct tcg atc aaa gct gtg tat cct gat ggt acg ata tat     6845 Glu Gln Pro Ala Ser Ile Lys Ala Val Tyr Pro Asp Gly Thr Ile Tyr 1880               1885                1890                1895 agc acc cgt act tat cgt tat gat gga ttt ggt cgt aca gtg acg gaa     6893 Ser Thr Arg Thr Tyr Arg Tyr Asp Gly Phe Gly Arg Thr Val Thr Glu 1900                1905                1910 aca gat gca gaa ggt cat gct acc caa att gga tat gat gtg ttt gat     6941 Thr Asp Ala Glu Gly His Ala Thr Gln Ile Gly Tyr Asp Val Phe Asp 1915                1920                1925 cgt ata gtg aaa aaa acg ttg cca gac gga aca ata tta gaa tcc gct     6989 Arg Ile Val Lys Lys Thr Leu Pro Asp Gly Thr Ile Leu Glu Ser Ala 1930                1935                1940 tat gca agc ttt agc cat gaa gaa tta att tcg gca ctg aac gtg aat     7037 Tyr Ala Ser Phe Ser His Glu Glu Leu Ile Ser Ala Leu Asn Val Asn 1945                1950                1955 ggc aca cag ttg ggg gca tta gtt tat gat ggt ctt ggg cgg gta ata     7085 Gly Thr Gln Leu Gly Ala Leu Val Tyr Asp Gly Leu Gly Arg Val Ile 1960               1965                1970                1975 agt gat acg gtg ggt ggt cgc aaa acg gaa tat tta tat ggg cct caa     7133 Ser Asp Thr Val Gly Gly Arg Lys Thr Glu Tyr Leu Tyr Gly Pro Gln 1980                1985                1990 ggt gac aaa ccg att cag tca att act cct tcg cat aat aag caa aat     7181 Gly Asp Lys Pro Ile Gln Ser Ile Thr Pro Ser His Asn Lys Gln Asn 1995                2000                2005 atg gat tac ctc tac tat ctt ggt agt gtg atg tcc aaa ttt acc acg     7229 Met Asp Tyr Leu Tyr Tyr Leu Gly Ser Val Met Ser Lys Phe Thr Thr 2010                2015                2020 ggg aca gac caa caa aac ttt cgt tat cat tcg aaa acg gga aca tta     7277 Gly Thr Asp Gln Gln Asn Phe Arg Tyr His Ser Lys Thr Gly Thr Leu 2025                2030                2035 tta tct gcg tca gaa ggc gta tct cag act aat tac agt tat ttc cca     7325 Leu Ser Ala Ser Glu Gly Val Ser Gln Thr Asn Tyr Ser Tyr Phe Pro 2040               2045                2050                2055 tcg ggt gta tta cag cga gaa tca ttt tta cgg gat aat aaa ccg att     7373 Ser Gly Val Leu Gln Arg Glu Ser Phe Leu Arg Asp Asn Lys Pro Ile 2060                2065                2070 tca tcg ggc gag tac ctt tat acg atg tcc ggt ttg att caa cgt cat     7421 Ser Ser Gly Glu Tyr Leu Tyr Thr Met Ser Gly Leu Ile Gln Arg His 2075                2080                2085 aaa gat agt ttt ggt cat aat cat gtt tat agt tac gat gct cag gga     7469 Lys Asp Ser Phe Gly His Asn His Val Tyr Ser Tyr Asp Ala Gln Gly 2090                2095                2100 aga ttg gtc aaa aca gaa cag gat gca caa tac gct aca ttt gaa tat     7517 Arg Leu Val Lys Thr Glu Gln Asp Ala Gln Tyr Ala Thr Phe Glu Tyr 2105                2110                2115 gac aat gtt ggg cga ttg ata aca acg acg acc aaa gac acg acg tca     7565 Asp Asn Val Gly Arg Leu Ile Thr Thr Thr Thr Lys Asp Thr Thr Ser 2120               2125                2130                2135 tta tcc caa tta gtg aca aaa atc gaa tat gat gct ttt gat cga gaa     7613 Leu Ser Gln Leu Val Thr Lys Ile Glu Tyr Asp Ala Phe Asp Arg Glu 2140                2145                2150 ata aaa cgc tcg cta att agt gac ttc tca ata caa gtt att acc tta     7661 Ile Lys Arg Ser Leu Ile Ser Asp Phe Ser Ile Gln Val Ile Thr Leu 2155                2160                2165 agc tat acg aag aat aat caa atc agt caa cgt atc acc tcc atc gat     7709 Ser Tyr Thr Lys Asn Asn Gln Ile Ser Gln Arg Ile Thr Ser Ile Asp 2170                2175                2180 ggg gtg gtt atg aaa aat gaa cgt tat caa tat gat aat aat caa cgc     7757 Gly Val Val Met Lys Asn Glu Arg Tyr Gln Tyr Asp Asn Asn Gln Arg 2185                2190                2195 tta agc caa tac caa tgt gag gga gaa caa tct ccg att gat cat acg     7805 Leu Ser Gln Tyr Gln Cys Glu Gly Glu Gln Ser Pro Ile Asp His Thr 2200               2205                2210                2215 ggt cgt gta tta aat cag cag att tac cat tat gac caa tgg gga aat     7853 Gly Arg Val Leu Asn Gln Gln Ile Tyr His Tyr Asp Gln Trp Gly Asn 2220                2225                2230 att aag cgg ctc gat aat aca tat cga gat ggt aag gaa acg gtg gat     7901 Ile Lys Arg Leu Asp Asn Thr Tyr Arg Asp Gly Lys Glu Thr Val Asp 2235                2240                2245 tat cat ttc agt caa gcc gat cca act caa ctt att cgt att acc agc     7949 Tyr His Phe Ser Gln Ala Asp Pro Thr Gln Leu Ile Arg Ile Thr Ser 2250                2255                2260 gac aaa cag cag ata gag tta agt tat gat gct aat ggc aac cta aca     7997 Asp Lys Gln Gln Ile Glu Leu Ser Tyr Asp Ala Asn Gly Asn Leu Thr 2265                2270                2275 cgt gac gaa aaa ggg caa acg ctc att tac gat cag aat aat cgc ttg     8045 Arg Asp Glu Lys Gly Gln Thr Leu Ile Tyr Asp Gln Asn Asn Arg Leu 2280               2285                2290                2295 gta cag gtc aaa gac cgg ttg ggc aat ctg gtg tgc agc tac cag tat     8093 Val Gln Val Lys Asp Arg Leu Gly Asn Leu Val Cys Ser Tyr Gln Tyr 2300                2305                2310 gat gca ttg aac aaa tta acc gca cag gtt ttg gcg aat ggt acc gtt     8141 Asp Ala Leu Asn Lys Leu Thr Ala Gln Val Leu Ala Asn Gly Thr Val 2315                2320                2325 aat cga cag cat tat gct tcc ggt aaa gtg acg aat att caa ttg ggt     8189 Asn Arg Gln His Tyr Ala Ser Gly Lys Val Thr Asn Ile Gln Leu Gly 2330                2335                2340 gat gaa gcg att act tgg ttg agc agt gat aag caa cga att gga cat     8237 Asp Glu Ala Ile Thr Trp Leu Ser Ser Asp Lys Gln Arg Ile Gly His 2345                2350                2355 caa agc gcc aag aat ggt caa tca gtc tac tat caa tat ggt att gac     8285 Gln Ser Ala Lys Asn Gly Gln Ser Val Tyr Tyr Gln Tyr Gly Ile Asp 2360               2365                2370                2375 cat aac agt acg gtt atc gcc agt cag aac gaa aac gag ttg atg gct     8333 His Asn Ser Thr Val Ile Ala Ser Gln Asn Glu Asn Glu Leu Met Ala 2380                2385                2390 tta tcc tat aca cct tat ggc ttt agg agt tta att tcc tca tta ccg     8381 Leu Ser Tyr Thr Pro Tyr Gly Phe Arg Ser Leu Ile Ser Ser Leu Pro 2395                2400                2405 ggt ttg aat ggc gca cag gtt gat cca gta aca ggc tgg tac ttc tta     8429 Gly Leu Asn Gly Ala Gln Val Asp Pro Val Thr Gly Trp Tyr Phe Leu 2410                2415                2420 ggt aac gga tat cgt gtt ttc aac ccg gtt ctc atg agg ttt cac agc     8477 Gly Asn Gly Tyr Arg Val Phe Asn Pro Val Leu Met Arg Phe His Ser 2425                2430                2435 ccc gat agt tgg agt cct ttt ggt cgg gga ggg att aac cct tat acc     8525 Pro Asp Ser Trp Ser Pro Phe Gly Arg Gly Gly Ile Asn Pro Tyr Thr 2440               2445                2450                2455 tat tgc caa ggc gat ccc ata aac cgg att gat ctg aac ggt cat ctt     8573 Tyr Cys Gln Gly Asp Pro Ile Asn Arg Ile Asp Leu Asn Gly His Leu 2460                2465                2470 agt gcc ggc ggg ata tta ggc att gtg cta ggg gca att ggc atc att     8621 Ser Ala Gly Gly Ile Leu Gly Ile Val Leu Gly Ala Ile Gly Ile Ile 2475                2480                2485 gtc ggg att gta tca ctg gga gcc gga gcg gcg att agc gcg ggt ctc     8669 Val Gly Ile Val Ser Leu Gly Ala Gly Ala Ala Ile Ser Ala Gly Leu 2490                2495                2500 att gct gcg ggg ggc gct ttg ggg gcg att gct tct acc agc gcg ctt     8717 Ile Ala Ala Gly Gly Ala Leu Gly Ala Ile Ala Ser Thr Ser Ala Leu 2505                2510                2515 gca gtt act gcg act gtc att gga ttg gct gcc gat tcg ata ggg att     8765 Ala Val Thr Ala Thr Val Ile Gly Leu Ala Ala Asp Ser Ile Gly Ile 2520               2525                2530                2535 gcg tca gca gca tta tcg gaa aaa gat ccg aaa aca tct ggg ata tta     8813 Ala Ser Ala Ala Leu Ser Glu Lys Asp Pro Lys Thr Ser Gly Ile Leu 2540                2545                2550 aat tgg att agt gcg gga ttg ggg gtt tta agc ttt ggt atc agc gca     8861 Asn Trp Ile Ser Ala Gly Leu Gly Val Leu Ser Phe Gly Ile Ser Ala 2555                2560                2565 ata acc ttt acc tct tcg ctg gta aaa tcg gca cgg agt ggt tct cag     8909 Ile Thr Phe Thr Ser Ser Leu Val Lys Ser Ala Arg Ser Gly Ser Gln 2570                2575                2580 gca gtc agc gcg ggt gtt atc ggg tca gtg cct ctt gaa ttt ggt gaa     8957 Ala Val Ser Ala Gly Val Ile Gly Ser Val Pro Leu Glu Phe Gly Glu 2585                2590                2595 gtt gct agc cgt tcc agc aga cga tgg gat att gcg tta tct tcg ata     9005 Val Ala Ser Arg Ser Ser Arg Arg Trp Asp Ile Ala Leu Ser Ser Ile 2600               2605                2610                2615 tcg ttg ggc gca aat gcg gcg tct ctc tct acg ggg ata gcg gcg gcg     9053 Ser Leu Gly Ala Asn Ala Ala Ser Leu Ser Thr Gly Ile Ala Ala Ala 2620                2625                2630 gcg gtt gca gac agt aat gcg aat gca gct aat att ctg gga tgg gta     9101 Ala Val Ala Asp Ser Asn Ala Asn Ala Ala Asn Ile Leu Gly Trp Val 2635                2640                2645 tcc ttt ggt ttt ggt gca gta tcg aca acc tca gga ata att gag ctt     9149 Ser Phe Gly Phe Gly Ala Val Ser Thr Thr Ser Gly Ile Ile Glu Leu 2650                2655                2660 acg cgt aca gct tat gca gtg aat cat cag act tgg gaa ctg agt tca     9197 Thr Arg Thr Ala Tyr Ala Val Asn His Gln Thr Trp Glu Leu Ser Ser 2665                2670                2675 tca gca ggt act tcg gag gaa gtg aag cct ata cgt tgt ctc gtt tca     9245 Ser Ala Gly Thr Ser Glu Glu Val Lys Pro Ile Arg Cys Leu Val Ser 2680               2685                2690                2695 cac cgc tgg aat cag aag cag tga atgttaaccc tcctcgggca gttgagttaa    9299 His Arg Trp Asn Gln Lys Gln 2700 tcaaacgttt cgaaatagta ccgggaacta tttagccaat cgtccattga aacccgtaat   9359 gtgttgcgac gtcgtttgac aatataaaga ttctgcgaac cgattggtta agtctcacga   9419 aaaataacta ttaggcgaca tttgcgtcgc cttttttaag gaactttatc aggttacatt   9479 tataagaagc tattttgttt tcgacggatg ttggtttctc tgagataaaa aatagaggga   9539 aatgatgtca agggtgataa tggttaattg taaaatatgt gatattattc gcatttatat   9599 gtcaatgtaa ttcctcttat tatttaattt tattgcattt gctacgcgaa atcgccttat   9659 aattttattt ttaataaatt attatttcat cattaaacta aaataaatta tttctaga     9717 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 417 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  2 Met Gln Arg Ala Gln Arg Val Val Ile Thr Gly Met Gly Ala Val Thr 1               5                  10                  15 Pro Ile Gly Glu Asp Val Glu Ser Cys Trp Gln Ser Ile Ile Glu Lys 20                  25                  30 Gln His Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe Ile Asn Ser 35                  40                  45 Arg Phe Phe Ser Phe Leu Ala Pro Asn Pro Ser Arg Tyr Gln Leu Leu 50                  55                  60 Pro Lys Lys Leu Thr His Thr Leu Ser Asp Cys Gly Lys Ala Ala Leu 65                  70                  75                  80 Lys Ala Thr Tyr Gln Ala Phe Thr Gln Ala Phe Gly Val Asn Ile Ser 85                  90                  95 Pro Val Glu Tyr Tyr Asp Lys Tyr Glu Cys Gly Val Ile Leu Gly Ser 100                 105                 110 Gly Trp Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cys Gln Tyr Lys 115                 120                 125 Gln Ala Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr Met Pro Ser 130                 135                 140 Ser Met Thr Ala Ala Cys Ser Ile Met Tyr Gly Leu Arg Gly Tyr Gln 145                 150                 155                 160 Asn Thr Val Met Ala Ala Cys Ala Thr Gly Thr Met Ala Ile Gly Asp 165                 170                 175 Ala Phe Glu Ile Ile Arg Ser Gly Arg Ala Lys Cys Met Ile Ala Gly 180                 185                 190 Ala Ala Glu Ser Leu Thr Arg Glu Cys Asn Ile Trp Ser Ile Asp Val 195                 200                 205 Leu Asn Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu Ala Cys Cys 210                 215                 220 Pro Phe Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu Gly Ala Ala 225                 230                 235                 240 Val Val Cys Leu Glu Asn Tyr Asp Ser Ala Ile Ala Arg Gly Ala Thr 245                 250                 255 Ile Leu Ala Glu Ile Lys Gly Tyr Ala Gln Tyr Ser Asp Ala Val Asn 260                 265                 270 Leu Thr Arg Pro Thr Glu Asp Ile Glu Pro Lys Ile Leu Ala Ile Thr 275                 280                 285 Lys Ala Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile Asp Tyr Ile 290                 295                 300 Asn Ala His Gly Thr Ser Thr Pro Leu Asn Asp Leu Tyr Glu Thr Gln 305                 310                 315                 320 Ala Ile Lys Ala Ala Leu Gly Gln Tyr Ala Tyr Gln Val Pro Ile Ser 325                 330                 335 Ser Thr Lys Ser Tyr Thr Gly His Leu Ile Ala Ala Ala Gly Ser Phe 340                 345                 350 Glu Thr Ile Val Cys Val Lys Ala Leu Ala Glu Asn Cys Leu Pro Ala 355                 360                 365 Thr Leu Asn Leu His Arg Ala Asp Pro Asp Cys Asp Leu Asn Tyr Leu 370                 375                 380 Pro Asn Gln His Cys Tyr Thr Ala Gln Pro Glu Val Thr Leu Asn Ile 385                 390                 395                 400 Ser Ala Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile Ala Lys Val 405                 410                 415 Arg <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 253 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  3 Met Glu Asp Ile Glu His Trp Ser Asn Phe Ser Gly Asp Phe Asn Pro 1               5                  10                  15 Ile His Tyr Ser Ala Lys Ser Glu Ser Leu Arg Asn Ile Gln Gln His 20                  25                  30 Pro Val Gln Gly Met Leu Ser Leu Leu Tyr Val Arg Gln Gln Phe Ser 35                  40                  45 Gln Leu Thr Ser Ala Phe Thr Thr Gly Ile Leu Asn Ile Asp Ala Ser 50                  55                  60 Phe Arg Gln Tyr Val Tyr Thr Ala Leu Pro His Gln Leu Arg Ile Asn 65                  70                  75                  80 Thr Lys Asn Lys Thr Phe Lys Leu Glu Asn Pro Ser Lys Glu Asn Thr 85                  90                  95 Leu Phe Gly Asn Thr Ser Val Glu Asn Thr Met Glu Ser Ile Glu Asp 100                 105                 110 Trp Ile Val Gln Asp Asn Cys Gln Lys Leu Thr Ile Thr Gly Glu Glu 115                 120                 125 Val Cys Glu Lys Tyr Ala Val Phe Arg Tyr Tyr Phe Pro Ser Val Thr 130                 135                 140 Ser Ile Gly Trp Phe Leu Asp Ala Leu Ala Phe His Leu Ile Ile Asn 145                 150                 155                 160 Ser Thr Gly Phe Leu Asn Phe Glu His Tyr His Phe Asn Gln Leu Gln 165                 170                 175 Asp Tyr Leu Ser Gln Ser Phe Thr Leu His Thr Gly Gln Ala Ile Lys 180                 185                 190 Ile Arg Lys Glu Ile Val Asn Ser Thr Val Leu Leu Ser Ser Pro Asp 195                 200                 205 Ile Cys Val Glu Leu Asn Pro Pro Leu Leu Ile Lys Asn Gly Asp Lys 210                 215                 220 Asp Tyr Ile Arg Ile Phe Tyr Tyr Arg Cys Leu Tyr Asp Lys Lys Pro 225                 230                 235                 240 Ile Phe Val Ser Lys Thr Ser Ile Ile Ser Lys Met Lys 245                 250 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 186 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  4 Met Asn Val Leu Glu Gln Gly Lys Val Ala Ala Leu Tyr Ser Ala Tyr 1               5                  10                  15 Ser Glu Thr Glu Gly Ser Ser Trp Val Gly Asn Leu Cys Cys Phe Ser 20                  25                  30 Ser Asp Arg Glu His Leu Pro Ile Ile Val Asn Gly Arg Arg Phe Leu 35                  40                  45 Ile Glu Phe Val Ile Pro Asp His Leu Leu Asp Lys Thr Val Lys Pro 50                  55                  60 Arg Val Phe Asp Leu Asp Ile Asn Lys Gln Phe Leu Leu Arg Arg Asp 65                  70                  75                  80 His Arg Glu Ile Asn Ile Tyr Leu Leu Gly Glu Gly Asn Phe Met Asp 85                  90                  95 Arg Thr Thr Thr Asp Lys Asn Leu Phe Glu Leu Asn Glu Asp Gly Ser 100                 105                 110 Leu Phe Ile Lys Thr Leu Arg His Ala Leu Gly Lys Tyr Val Ala Ile 115                 120                 125 Asn Pro Ser Thr Thr Gln Phe Ile Phe Phe Ala Gln Gly Lys Tyr Ser 130                 135                 140 Glu Phe Ile Met Asn Ala Leu Lys Thr Val Glu Asp Glu Leu Ser Lys 145                 150                 155                 160 Arg Tyr Arg Val Arg Ile Ile Pro Glu Leu Gln Gly Pro Tyr Tyr Gly 165                 170                 175 Phe Glu Leu Asp Ile Leu Ser Ile Thr Ala 180                 185 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 258 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  5 Met Glu Lys Lys Ile Thr Thr Phe Thr Ile Glu Lys Thr Asp 1               5                 10 Asp Asn Phe Tyr Ala Asn Gly Arg His Gln Cys Met Val Lys Ile Ser 15                  20                  25                  30 Val Leu Lys Gln Glu Tyr Arg Asn Gly Asp Trp Ile Lys Leu Ala Leu 35                  40                  45 Ser Glu Ala Glu Lys Arg Ser Ile Gln Val Ala Ala Leu Ser Asp Ser 50                  55                  60 Leu Ile Tyr Asp Gln Leu Lys Met Pro Ser Gly Trp Thr Thr Thr Asp 65                  70                  75 Ala Arg Asn Lys Phe Asp Leu Gly Leu Leu Asn Gly Val Tyr His Ala 80                  85                  90 Asp Ala Phe Ile Asp Glu Gln Val Thr Asp Arg Ala Gly Asp Cys Cys 95                  100                 105                 110 Thr Asn Glu Asn Tyr Gln Asn Ser Val Lys Ser Val Pro Glu Ile Ile 115                 120                 125 Tyr Arg Tyr Val Ser Ser Asn Arg Thr Ser Thr Glu Tyr Leu Met Ala 130                 135                 140 Lys Met Thr Phe Glu Asp Thr Asp Gly Lys Arg Thr Leu Thr Thr Asn 145                 150                 155 Met Ser Val Gly Asp Glu Val Phe Asp Ser Lys Val Leu Leu Lys Ala 160                 165                 170 Ile Ala Pro Tyr Ala Ile Asn Thr Asn Gln Leu His Glu Asn Ile Asn 175                 180                 185                 190 Thr Leu Phe Asp Lys Thr Glu Glu Pro Thr Lys Ser Asp Thr His His 195                 200                 205 Gln Ile Ile Asn Leu Tyr Arg Trp Thr Leu Pro Tyr His Leu Arg Ile 210                 215                 220 Leu Glu Gly Asn Asp Ser Thr Val Asn Arg Ile Tyr Val Leu Gly Lys 225                 230                 235 Glu Pro Ser Asn Asp Arg Phe Leu Thr Arg Gly Arg Val Phe Lys Arg 240                 245                 250 Gly Thr His Met 255 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 1584 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  6 Met Lys Ala Thr Asp Ile Tyr Ser Asn Ala Phe Asn Phe Gly Ser Tyr 1               5                  10                  15 Ile Asn Thr Gly Val Asp Pro Arg Thr Gly Gln Tyr Ser Ala Asn Ile 20                  25                  30 Asn Ile Ile Thr Leu Arg Pro Asn Asn Val Gly Asn Ser Glu Gln Thr 35                  40                  45 Leu Ser Leu Ser Phe Ser Pro Leu Thr Thr Leu Asn Asn Gly Phe Gly 50                  55                  60 Ile Gly Trp Arg Phe Ser Leu Thr Thr Leu Asp Ile Lys Thr Leu Thr 65                  70                  75                  80 Phe Ser Arg Ala Asn Gly Glu Gln Phe Lys Cys Lys Pro Leu Pro Pro 85                  90                  95 Asn Asn Asn Asp Leu Ser Phe Lys Asp Lys Lys Leu Lys Asp Leu Arg 100                 105                 110 Val Tyr Lys Leu Asp Ser Asn Thr Phe Tyr Val Tyr Asn Lys Asn Gly 115                 120                 125 Ile Ile Glu Ile Leu Lys Arg Ile Gly Ser Ser Asp Ile Ala Lys Thr 130                 135                 140 Val Ala Leu Glu Phe Pro Asp Gly Glu Ala Phe Asp Leu Ile Tyr Asn 145                 150                 155                 160 Ser Arg Phe Ala Leu Ser Glu Ile Lys Tyr Arg Val Thr Gly Lys Thr 165                 170                 175 Tyr Leu Lys Leu Asn Tyr Ser Gly Asn Asn Cys Thr Ser Val Glu Tyr 180                 185                 190 Pro Asp Asp Asn Asn Ile Ser Ala Lys Ile Ala Phe Asp Tyr Arg Asn 195                 200                 205 Asp Tyr Leu Ile Thr Val Thr Val Pro Tyr Asp Ala Ser Gly Pro Ile 210                 215                 220 Asp Ser Ala Arg Phe Lys Met Thr Tyr Gln Thr Leu Lys Gly Val Phe 225                 230                 235                 240 Pro Val Ile Ser Thr Phe Arg Thr Pro Thr Gly Tyr Val Glu Leu Val 245                 250                 255 Ser Tyr Lys Glu Asn Gly His Lys Val Thr Asp Thr Glu Tyr Ile Pro 260                 265                 270 Tyr Ala Ala Ala Leu Thr Ile Gln Pro Gly Asn Gly Gln Pro Ala Val 275                 280                 285 Ser Lys Ser Tyr Glu Tyr Ser Ser Val His Asn Phe Leu Gly Tyr Ser 290                 295                 300 Ser Gly Arg Thr Ser Phe Asp Ser Ser Gln Asp Asn Leu Tyr Leu Val 305                 310                 315                 320 Thr Gly Lys Tyr Thr Tyr Ser Ser Ile Glu Arg Val Leu Asp Gly Gln 325                 330                 335 Ser Val Val Ser Val Ile Glu Arg Val Phe Asn Lys Phe His Leu Met 340                 345                 350 Thr Lys Glu Ala Lys Thr Gln Asp Asn Lys Arg Ile Thr Thr Glu Ile 355                 360                 365 Thr Tyr Asn Glu Asp Leu Ser Lys Ser Phe Ser Glu Gln Pro Glu Asn 370                 375                 380 Leu Gln Gln Pro Ser Arg Val Leu Thr Arg Tyr Thr Asp Ile Gln Thr 385                 390                 395                 400 Asn Thr Ser Arg Glu Glu Thr Val Asn Ile Lys Ser Asp Asp Trp Gly 405                 410                 415 Asn Thr Leu Leu Ile Thr Glu Thr Ser Gly Ile Gln Lys Glu Tyr Val 420                 425                 430 Tyr Tyr Pro Val Asn Gly Glu Gly Asn Ser Cys Pro Ala Asp Pro Leu 435                 440                 445 Gly Phe Ser Arg Phe Leu Lys Ser Val Thr Gln Lys Gly Ser Pro Asp 450                 455                 460 Ala Ala Gln Ser Val Ala Asn Lys Val Ile His Tyr Thr Tyr Gln Lys 465                 470                 475                 480 Phe Pro Thr Phe Thr Gly Ala Tyr Val Lys Glu Tyr Val Ser Lys Val 485                 490                 495 Ser Glu Thr Ile Asp Asn Lys Ile Ala Arg Thr Phe Ser Tyr Val Asn 500                 505                 510 Ser Pro Thr Ser Lys Ser His Gly Ser Leu Ala Lys Ile Thr Ser Val 515                 520                 525 Met Asn Asn Gln Gln Thr Val Thr Thr Phe Lys Tyr Glu Tyr Ser Glu 530                 535                 540 Ser Glu Met Thr Thr Asn Ala Thr Val Thr Gly Phe Asp Gly Ala His 545                 550                 555                 560 Met Glu Ser Lys Asn Val Thr Ser Ile Tyr Thr His Arg Gln Leu Arg 565                 570                 575 Lys Val Asp Val Asn His Val Ile Thr Asp Gln Ser Tyr Asp Leu Leu 580                 585                 590 Gly Arg Ile Thr Gly Gln Ile Ile Asp Pro Gly Thr Ala Arg Glu Ile 595                 600                 605 Lys Arg Asn Tyr Val Tyr Gln Tyr Pro Gly Gly Asp Glu Asn Asp Phe 610                 615                 620 Trp Pro Val Met Ile Glu Val Asp Ser Gln Gly Val Arg Arg Lys Thr 625                 630                 635                 640 His Tyr Asp Gly Met Gly Arg Ile Cys Ser Ile Glu Glu Gln Asp Asp 645                 650                 655 Asp Gly Ala Trp Gly Thr Ser Gly Ile Tyr Gln Gly Thr Tyr Arg Lys 660                 665                 670 Val Leu Ala Arg Gln Tyr Asp Val Leu Gly Gln Leu Ser Lys Glu Ile 675                 680                 685 Ser Asn Asp Trp Leu Trp Asn Leu Ser Ala Asn Pro Leu Val Arg Leu 690                 695                 700 Ala Thr Pro Leu Val Thr Thr Lys Thr Tyr Lys Tyr Asp Gly Trp Gly 705                 710                 715                 720 Asn Leu Tyr Ser Thr Glu Tyr Ser Asp Gly Arg Ile Glu Leu Glu Ile 725                 730                 735 His Asp Pro Ile Thr Arg Thr Ile Thr Gln Gly Val Lys Gly Leu Gly 740                 745                 750 Met Leu Asn Ile Gln Gln Asn Asn Phe Glu Gln Pro Ala Ser Ile Lys 755                 760                 765 Ala Val Tyr Pro Asp Gly Thr Ile Tyr Ser Thr Arg Thr Tyr Arg Tyr 770                 775                 780 Asp Gly Phe Gly Arg Thr Val Thr Glu Thr Asp Ala Glu Gly His Ala 785                 790                 795                 800 Thr Gln Ile Gly Tyr Asp Val Phe Asp Arg Ile Val Lys Lys Thr Leu 805                 810                 815 Pro Asp Gly Thr Ile Leu Glu Ser Ala Tyr Ala Ser Phe Ser His Glu 820                 825                 830 Glu Leu Ile Ser Ala Leu Asn Val Asn Gly Thr Gln Leu Gly Ala Leu 835                 840                 845 Val Tyr Asp Gly Leu Gly Arg Val Ile Ser Asp Thr Val Gly Gly Arg 850                 855                 860 Lys Thr Glu Tyr Leu Tyr Gly Pro Gln Gly Asp Lys Pro Ile Gln Ser 865                 870                 875                 880 Ile Thr Pro Ser His Asn Lys Gln Asn Met Asp Tyr Leu Tyr Tyr Leu 885                 890                 895 Gly Ser Val Met Ser Lys Phe Thr Thr Gly Thr Asp Gln Gln Asn Phe 900                 905                 910 Arg Tyr His Ser Lys Thr Gly Thr Leu Leu Ser Ala Ser Glu Gly Val 915                 920                 925 Ser Gln Thr Asn Tyr Ser Tyr Phe Pro Ser Gly Val Leu Gln Arg Glu 930                 935                 940 Ser Phe Leu Arg Asp Asn Lys Pro Ile Ser Ser Gly Glu Tyr Leu Tyr 945                 950                 955                 960 Thr Met Ser Gly Leu Ile Gln Arg His Lys Asp Ser Phe Gly His Asn 965                 970                 975 His Val Tyr Ser Tyr Asp Ala Gln Gly Arg Leu Val Lys Thr Glu Gln 980                 985                 990 Asp Ala Gln Tyr Ala Thr Phe Glu Tyr Asp Asn Val Gly Arg Leu Ile 995                1000                1005 Thr Thr Thr Thr Lys Asp Thr Thr Ser Leu Ser Gln Leu Val Thr Lys 1010                1015                1020 Ile Glu Tyr Asp Ala Phe Asp Arg Glu Ile Lys Arg Ser Leu Ile Ser 1025                1030                1035                1040 Asp Phe Ser Ile Gln Val Ile Thr Leu Ser Tyr Thr Lys Asn Asn Gln 1045                1050                1055 Ile Ser Gln Arg Ile Thr Ser Ile Asp Gly Val Val Met Lys Asn Glu 1060                1065                1070 Arg Tyr Gln Tyr Asp Asn Asn Gln Arg Leu Ser Gln Tyr Gln Cys Glu 1075                1080                1085 Gly Glu Gln Ser Pro Ile Asp His Thr Gly Arg Val Leu Asn Gln Gln 1090                1095                1100 Ile Tyr His Tyr Asp Gln Trp Gly Asn Ile Lys Arg Leu Asp Asn Thr 1105                1110                1115                1120 Tyr Arg Asp Gly Lys Glu Thr Val Asp Tyr His Phe Ser Gln Ala Asp 1125                1130                1135 Pro Thr Gln Leu Ile Arg Ile Thr Ser Asp Lys Gln Gln Ile Glu Leu 1140                1145                1150 Ser Tyr Asp Ala Asn Gly Asn Leu Thr Arg Asp Glu Lys Gly Gln Thr 1155                1160                1165 Leu Ile Tyr Asp Gln Asn Asn Arg Leu Val Gln Val Lys Asp Arg Leu 1170                1175                1180 Gly Asn Leu Val Cys Ser Tyr Gln Tyr Asp Ala Leu Asn Lys Leu Thr 1185                1190                1195                1200 Ala Gln Val Leu Ala Asn Gly Thr Val Asn Arg Gln His Tyr Ala Ser 1205                1210                1215 Gly Lys Val Thr Asn Ile Gln Leu Gly Asp Glu Ala Ile Thr Trp Leu 1220                1225                1230 Ser Ser Asp Lys Gln Arg Ile Gly His Gln Ser Ala Lys Asn Gly Gln 1235                1240                1245 Ser Val Tyr Tyr Gln Tyr Gly Ile Asp His Asn Ser Thr Val Ile Ala 1250                1255                1260 Ser Gln Asn Glu Asn Glu Leu Met Ala Leu Ser Tyr Thr Pro Tyr Gly 1265                1270                1275                1280 Phe Arg Ser Leu Ile Ser Ser Leu Pro Gly Leu Asn Gly Ala Gln Val 1285                1290                1295 Asp Pro Val Thr Gly Trp Tyr Phe Leu Gly Asn Gly Tyr Arg Val Phe 1300                1305                1310 Asn Pro Val Leu Met Arg Phe His Ser Pro Asp Ser Trp Ser Pro Phe 1315                1320                1325 Gly Arg Gly Gly Ile Asn Pro Tyr Thr Tyr Cys Gln Gly Asp Pro Ile 1330                1335                1340 Asn Arg Ile Asp Leu Asn Gly His Leu Ser Ala Gly Gly Ile Leu Gly 1345                1350                1355                1360 Ile Val Leu Gly Ala Ile Gly Ile Ile Val Gly Ile Val Ser Leu Gly 1365                1370                1375 Ala Gly Ala Ala Ile Ser Ala Gly Leu Ile Ala Ala Gly Gly Ala Leu 1380                1385                1390 Gly Ala Ile Ala Ser Thr Ser Ala Leu Ala Val Thr Ala Thr Val Ile 1395                1400                1405 Gly Leu Ala Ala Asp Ser Ile Gly Ile Ala Ser Ala Ala Leu Ser Glu 1410                1415                1420 Lys Asp Pro Lys Thr Ser Gly Ile Leu Asn Trp Ile Ser Ala Gly Leu 1425                1430                1435                1440 Gly Val Leu Ser Phe Gly Ile Ser Ala Ile Thr Phe Thr Ser Ser Leu 1445                1450                1455 Val Lys Ser Ala Arg Ser Gly Ser Gln Ala Val Ser Ala Gly Val Ile 1460                1465                1470 Gly Ser Val Pro Leu Glu Phe Gly Glu Val Ala Ser Arg Ser Ser Arg 1475                1480                1485 Arg Trp Asp Ile Ala Leu Ser Ser Ile Ser Leu Gly Ala Asn Ala Ala 1490                1495                1500 Ser Leu Ser Thr Gly Ile Ala Ala Ala Ala Val Ala Asp Ser Asn Ala 1505                1510                1515                1520 Asn Ala Ala Asn Ile Leu Gly Trp Val Ser Phe Gly Phe Gly Ala Val 1525                1530                1535 Ser Thr Thr Ser Gly Ile Ile Glu Leu Thr Arg Thr Ala Tyr Ala Val 1540                1545                1550 Asn His Gln Thr Trp Glu Leu Ser Ser Ser Ala Gly Thr Ser Glu Glu 1555                1560                1565 Val Lys Pro Ile Arg Cys Leu Val Ser His Arg Trp Asn Gln Lys Gln 1570                1575                1580 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  7 acacagcagg ttcgtcag                                                   18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  8 ggcagaagca ctcaactc                                                   18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  9 attgatagca cgcggcgacc                                                 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  10 ttgtaacgtg gagccgaact gg                                              22 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 37948 <212> TYPE: DNA <213> ORGANISM: Photorhabdus luminescens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (15171)..(18035) <223> OTHER INFORMATION: orf5 <400> SEQUENCE:  11 tgttgctgga ccgtggagat tatgcctatc gtcagttaga acgagacacg ctcaatgaag     60 ccaagatgtg gtatatgcaa gcactgcatc tgttaggcga taaacctcat ctatcgttca    120 gttcagagtg gagcaaaccg agtttaggcg acgctgccgg aacggaaaga caaaagcaac    180 acgcccaagc aatggccgct ctgcgacaag gtgatgttag tcggcacaac aacccgacag    240 atcttttctt gccacaggtc aatgaagtga tgcaaaacta ttggcaaaaa ttggaacaac    300 ggctgtataa cctgcgtcat aacctcacta ttgacggcca accgctacat ctgcctattt    360 acgctacacc ggcagatcca aaagcattac ttagcgccgc tgtcgctagc tccgaaggtg    420 gggtagctct ctcacagcca tttatgtcac tgtggcgttt cccacacatg ctggaaaacg    480 cgcgtggtat ggtcagtcag ctcactcaat tcggctctac gctacaaaat attatcgaac    540 gtcaggatgc ggaagcttta aacacgctct tgcagaatca agcagcagaa ctgatattga    600 ctcatctcag catacaggac aaaaccatcg cagagctgga tgcggaaaaa atcgtactgg    660 aaaaatccaa agccggggcg caatcacgct ttgacagcta caaaaagtta tacgacgaaa    720 atatcaatgc gggtgaaaac cgggctatag cattgcatgc ctccgttgct ggcctcagca    780 ctgccctgca agcatcacgt ctggcgggcg ctgcgcttga tctggcgccc aacattttcg    840 gtctcgctga tggcggtagc cgttggggag cgattgccga agcgacaggt aatgttatgg    900 aattctccgc cagtgtgatg aacaccgaag cggataaaat cagccagtca gaagcctatc    960 gccgtcgccg tcaggaatgg gaaatccagc gtaatcatgc cgaagcagag ataaaacaga   1020 tcgatgctca acttcaatca ctggcagtac gccgtgaagc cgcggtattg cagaaagcca   1080 gcctaaaaac ccaacaggaa cagactcatg ctcaattgac tttcctgcaa cgtaaattca   1140 gtaatcaagc gttgtactac tggctacgcg gtcggctagc tgctatttac ttccaatttt   1200 acgatttggc cgtagcgcgt tgtctgatgg ctgaaatggc ttatcgttgg gagactaatg   1260 agaccgcggc aagctttatc aaacccggcg cctggcaggg aacccatgcg ggtttactgg   1320 ctggtgaaac cttgatgctg aatctggcgc aaatggaaga tgcccatttg aggtgggatc   1380 aacgcgctct ggaagtggaa cggaccattt cattgacgca acactatgga gcactgccag   1440 aaaaatcgtt taatttagcc acacggattt ctaccctgct agcaggtggt acaactgact   1500 ccattgatga tcatcccgtt acattagaaa acgaccaact tagtgccaaa atctctctgt   1560 caggtctgtc attagataat gactacccag atggcaacgg cgtaggcaac attcgacgca   1620 ttaaacaaat cagtgtcacc ttgccagccc tgttaggacc atatcaggat gtacaagcta   1680 ttctgtccta cggaggaagt gaaatcggat tagctgaaag ctgtaaatca ctggcgatct   1740 ctcatgggat caatgacagt ggtcaattcc agttggattt taacaatggt aagttcctgc   1800 cgtttgaagg gattgcgatt aacgatactg gcacattgac actcagtttc ccccaatgcg   1860 actgtcaaac aagaaaacat gttgcagact ttgagtgata ttattctgca tattcgctat   1920 accatccgcc aataaccacc tcaattaaat accaaaaaca ggctcctaaa cggggcctga   1980 acttttcacg aatatatacc actcacagtc tgctctcttt acctgtctga cgctcgttat   2040 aacagagata tttccttttc tcgtgagtcc catcacctac tataaaatat caaccctctt   2100 ctttttcata atatgcaata tgtaacaaat gcaattattt catttagtta ttgttaacta   2160 gttatattac ttatgatgta attataaatt ttgttattgc atcacaatag ccatttaaat   2220 aaataataac gttgtgaaat agttgatagt taaatggtgt ttttatttag ccgttatttt   2280 caacccaatt tcagaccgct atcagacgtt acctgtgttg cctttgtttt gatagatata   2340 aataacctta tttatatcca cggtactcag accagcataa atgttttatt tacctaacat   2400 ttaaaaggaa taaacatgaa cacactcaaa tccgaatatg aaaacgcgtt agtagcaggt   2460 tttaataatc taaccgatat ttgtcatctc tcttttgacg aacttcgcaa aaaagtgaag   2520 gacaaactct catggtcaca gacccaaagc ttatatcttg aagcacagca agtgcaaaag   2580 gacaatctcc tgcatgaagc ccatattctg aaacgcgcca atcctcattt acaaagtgcg   2640 gtccatcttg ccctgacaac acctcatgct gaccagcaag gttataatag cagatttggc   2700 aatcgcgcca gcaaatatac agccccaggc gcaatttctt ccatgttttc tcctgcggct   2760 tatttagctg aactttatcg tcaggcacgg aatttacatg atgaaaattc tatttatcat   2820 ttggatacac ggcgtccgga tctaaaatca ttggtgctca gccagaaaaa tatggatacg   2880 gagatttcca cactttctct gtctaatgac atgttgctag agggtattaa aactctgttc   2940 aaggacaagc tgctggaggc tctgaagaat attaaatctc tgtccaagga cgagctgctg   3000 gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat   3060 attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc   3120 aaggacgatc tgctggaggc tttgaagaat attaaacctc tgtccaagga cgatctgctg   3180 gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat   3240 attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc   3300 aaggacgatc tacaggaatg tattgaaatt ctattcaatc tggacagcca cactaaagta   3360 atgaaagcgt tatccaattt ccgcgtttct ggcatgatgc catatcacga tgcttatgaa   3420 agcgtgcgta aggttgttca attacaggct ccggtgtttg aacacgttgt tagtacatca   3480 ctagaaacga ctatcgatga actaaaatat caagcttctt tgttggaaat taattcttct   3540 gtctcgccta aattatttac tatcttgact gaagaaatta ctacaatcaa tgcaagaagt   3600 ctctatgagg aaaattttgg taatattaaa ccttctctaa taggaaaacc ggaatatctg   3660 aaaagttatt acaatctgag tgatgaagag tttagcgatt tcattaaaat aagaactata   3720 cttcttccag aagaagaaat agcaattact gatcttgcat cgcgtactac tagtacacaa   3780 cagactatcg aaaatcctga ttatcgtgct ctattgaaaa ttaataagtt tattcgtcta   3840 ttcaaagcta taaacttatc accgacggta ttaagtggaa tcctccgcag catcagcaca   3900 gaattcaata tcaataaaga aatattacaa aaaatctttc gtgttaaata ctatatgcaa   3960 cgttatggta ttgacactga gactgcatta atactatgca aggtaccaat ttcacaatat   4020 atcaatgacg gacatctaag tcagtttgat cgtttattta attcccccaa actgaatggt   4080 caagattttt ccgtcaatgg tactcagaat attgatttaa ccctaagcag taccaacaac   4140 tggaataaaa cagtacttaa acgtgctttt aacctcgatg atatctcatt aaatcgacta   4200 ctaaaaatta ccaatccggt caatactacc gaaatgataa ctaatgatat agagaatctt   4260 tctcatctct ataggacaaa attactggca gatatccatc agttaactat tgatgaactg   4320 gggttactgt tggaagccat aggtaaagga acaaccaatt tatctgagat tactcctgac   4380 aatctggtta ctctaattaa caaactctat gctgtcacta gctggctacg tacacaaaag   4440 tggagtgtct atcagttgtt tatgatgact actgataaat ataacaaaac cctaaccccg   4500 gaaatacgga atttactgga taccgtctac aatggcttgc aagattttaa taagaagatg   4560 ttgaaagctg aagaagatct agagaaaacc aaaaagaaat tgcagagcgc caaggaaaat   4620 ctggaaaaat tcccggaaaa ccagccacaa ctccaagaag acaggaaaaa agcccagaga   4680 agactgaata aagctgaaga gacccacgaa aaagccgaga aaaacctaga tgaggtcagg   4740 aaaaatctgc caaaagccat atctccttat atcgccgccg ctctgcaatt accatctgaa   4800 catgcggcat attccatact catctgggca gataatctgg aacccggcat aggaaaaatg   4860 acagcggaaa aattatggaa ctggttgcgg aaaaatcccg ttacggctca acctgaattc   4920 caaaaacaag ctgaacctgt ggtccagtat tgccagcgcc tggcacaact agcgttgatt   4980 taccgttcta ccggccttaa cgaaaacacc ttaagtctgt ttgtgacaaa gccgcaacac   5040 tttgttatta aaaccaaagc acccgaaaca actgaaacaa caccagcaca tgacgtatca   5100 acactaatgt cactaacgcg ttttactgac tgggttaact cactaggtga aaacgcctct   5160 tctgtactaa ccgaatttaa aaaaggaaca ttaacagcag aactattggc taaggctatg   5220 aatcttgata aaaatctact ggagcaagcc aagattcagg caaaaactga tttgtccaac   5280 tggccatcta tcgacaacct attgcagtgg attaacatct cacgtcaatt gaacatctct   5340 ccacaaggca tttccacact gactcaagta ttgaccgcag aacctcccgc taactatacc   5400 caatgggaaa acgccgctgc gatattaacc gccgggctgg acacccaaaa gactaacgcc   5460 ctacatgcgt ttctggatga gtctcgcagt gctgcgttaa gcacatacta tatttattct   5520 cataaccaaa aagatcgaga agcaagaaaa catacagtaa ttaaaaaccg tgatgatcta   5580 tatcaatacc tattgatcga taaccaagtt tccgccgaca ttaaaactac agagatcgct   5640 gaagctatcg ctagtatcca actgtatatt aaccgcgcgt tgaaaaatat ggagggagat   5700 actgtcacaa gtgtcaccag ccgctcattc ttcaccaact gggataaata caataaacgc   5760 tacagcactt gggccggtat ggctaaactc ctttactatc cagagaatta catcgatccg   5820 acgctacgta ttgggcagac aaaaatgatg gatacgttgc tgcaatccat cagccaaagc   5880 caattaaata tcgataccgt agaagatgcc tttaaatctt acctaacatc attcgaacag   5940 gtggctaatc tggaaatcct cagcgcctac catgacaaca ttaataatga tcaaggatta   6000 acttacttta tcggacgtag taaaacagaa gtgaatcaat attattggcg cagtgtggat   6060 cacaataaat ccagcgaagg taaattcccc gctaatgcct ggagtgagtg gtacaaaatt   6120 gattgtccaa ttaaccccta caaagatact attcacccgg taattttcca atctcgcctg   6180 tatcttatct ggctggaaca aaaaaaggcg actaaacagg aaggtgataa aaccgcctcg   6240 ggttattatt atgaactgaa attagcgcat atccgttatg acggcacctg gaatacacca   6300 gtcacctttg atgtaaacca aaaaatatcc gatttaaatc tgggaaataa aacacctgga   6360 ctttactgct caagctttca aggcagagat gaattgctgg tgatgtttta taaaaaacaa   6420 gatcaattaa atcaatacac aaacacagta ccaataaaag gactatatat cacttccaat   6480 atgtcttcta aggaaatgac acctgaaaat cacaaaccta acgcttataa acagtttgat   6540 actaatagta ttattggtgt caataatcgc tatgcagaaa gctacgaaat cccttcatca   6600 gtaaatagta ataacggtta tgattgggga gatggctatc tgagtatggt gtatggcgga   6660 aatatttcag ccatcaaact ggagtcctca tcagataagt taaaactctc accaaggtta   6720 agaattattc ataatggact tgtaggccga caacgcaacc aatgcaacct gatgaagaaa   6780 tacggtcagc ttggtgataa atttattatt tatactactc taggtattaa ccccaataat   6840 ttgtcgaata aaaaattcat ttaccctgtt tatcagtata gtgggaacac taccaataat   6900 gagaaaggac gtctgctgtt ttatcgagaa agtactacta actttgtaag agcctggttc   6960 cctaaccttc cctctggctc tcaagaaatg tccacaacca ctggcggtga cattagtggt   7020 aactatggtt atattgataa caaacatagt gacgatgttc catttaaaca atatttctat   7080 atggatgacc acggtggtat tgacactgat gtttcaggga tattatctat taatacgaac   7140 attaatcatt caaaagttaa agtaatagtg aaagccgaag gtatcacaga gcaaactttt   7200 gtagcgagcg aaaacagtaa tgtccccacc aatccgtccc gcttcgaaga aatgaattat   7260 cagtttaaag agcttgaaat agatatctcc acactgacat ttcataataa tgaagcaagt   7320 attgatatca cctttatcgc atttgctgag aaatttgacg ataatagtaa tgatcgtaac   7380 ttaggcgaag aacatttcag tattcgtatt atcaaaaaag cggaaactga taatgccctg   7440 accctgcacc ataatgcaaa cggggcgcaa tatatgcagt ggggaaactc ttgtattcgc   7500 cttaatacgc tatttgcccg tcaattaatt agccgagcca acgcggggat agatactatt   7560 ttgagcatgg acactcagaa tattcaggaa cctaaattag gagaagattc tcctgatgct   7620 atggaaccaa tggacttcaa cggcgccaac agcctctatt tctgggaact gttctactac   7680 accccgatgc tgattgctca acgtttgctg cacgaacaaa acttcgatga ggctaaccgt   7740 tggctgaaat atgtctggaa cccatccggt tatattgtca atggtcaaat gcaacattac   7800 cgctggaatg ttcgcccatt acaagaagac actagttgga acgatgatcc gttggattca   7860 tttgatcctg ataccatagc tcaacatgat ccaatgcact acaaagtcgc cacctttatg   7920 cgcaccctag atctgttgat cgaacgggga gattacgcct atcgccaatt ggagcgggac   7980 acactcgctg aagccaaaat gtggtatatg caggcactgc atctattggg tgataaacct   8040 catctaccac tcagttcagc atggaatgat ccagagctag aagaggccgc agctcttgaa   8100 aaacaacagg cacatgccaa agaaatagca gatttacgac aaggacttcc tacatccaca   8160 gggtctaaag atgaaatcaa aacagatctt ttcctgccgc aagtcaacga agtgatgctg   8220 agctactggc agaaactaga acaacggttg tataacctgc gccataacct ctctattgat   8280 ggtcaacctt tacatttgcc tattttcgca acaccagcag atccaaaagc gctgctcagc   8340 gccgctgtcg ccagttcaca aggtggaagt aatcttccat cagaatttat atcagtgtgg   8400 cgtttccctc atatgctgga aaacgcccgt agtatggtca gtcagctaac ccaattcggc   8460 tccacattgc aaaatattat cgaacgtcaa gatgcggagg cattaaacac gctgttgcaa   8520 aatcaggcgg cagaactgat attgaccaat ctcagcatac aggacaaaac catccaagag   8580 ctggatgctg aaaaaactgt gctagaaaaa aaccgcgccg gaacccagtc gcgttttgat   8640 agctacagca aattctacga tgaagacatc aacgcgggtg aaaaacaggc aatggcgttg   8700 cgtgcttccg tcgctggcat ctctacagcc cttcaagcat cacatctggc gggcgcagca   8760 cttgatctgg cgcccaacat cttcggcttc gctgatggtg gcagccgttg gggggcgatc   8820 gcccaagcca caggtaatgt catggagttc tccgccagtg ttatgaacac cgaagcggat   8880 aaaatcagcc aatctgaagc ctaccgtcgg cgtcgtcagg aatgggaaat tcagcgtaat   8940 aacgccgagg cagagctgaa acaaatcgat gctcaacttg gttcgctggc agtgcgccgt   9000 gaagccgcag tattgcagaa aaccagccta aaaacccaac aagagcagac tcatgcacaa   9060 ctgaccttcc tgcaacataa gttcagtaat caggcgctgt acaactggct gcgtggtcga   9120 ttgtccgcca tttacttcca gttctatgat ttaacggtag ctcgctgttt gatggcggaa   9180 atggcctatc gctgggagac taacgatacc gcatcacgct ttatcaaacc cggcgcctgg   9240 cagggaaccc atgccggttt gctcgcgggt gaaaccttaa tgctgaatct ggcacagatg   9300 gaagatgccc acctgaaaca ggataaacgc gtactggagg tagaacgtac cgtttcgctg   9360 gccgaagtct atgccaaatt accgcaagat aaatttatcc tgactcagga aatagagaag   9420 ttggtgagta aaggttcagg cagggccggc aaggacaata ataagctggc gtttagtacc   9480 aataccaata cctctctaga agcgtccatt tcgttatcta ccttgaacat tagcagcgat   9540 tatcctgatt ctattggtaa aacccgtcgt attaaacaga tcagcgttac cctgccagca   9600 ctgctaggac cctatcagga tgtgcaagca attctgtctt acagcggaaa agcctctgaa   9660 ttggctgaaa gttgcaaatc attagcggtt tctcatggga tgaatgacag cggtcagttc   9720 caactggatt tcaacgatgg caaattcctg ccgttcgaag gaatcaaaat cgatgaaggt   9780 acgctgacat tgagcttccc aaatgcaatt agtaaagaag acaaaaaaga cgaaaaaggc   9840 aaacaacaag ccatgctgga gagtctgaac gacatcattc tgcatattcg ctacaccatt   9900 cgccaataac gattttaatt aagtgctaaa acaggcccct aagcggggcc tgcaaggagt   9960 ctttcatgca aaattcacaa gatttcagta ttacagaact atcattgccc aaaggaggag  10020 gcgctatcac gggaatgggg gaagctttaa cccccaccgg gccggatggg atggccgcgc  10080 tgtctctgcc gttgcctatc tctgccgggc gcggttatgc tccgtcactc gccttaaact  10140 acaacagcgg cgccggtaac agcccatttg gtctgggctg ggattgcaac gttatgacca  10200 tccgccgccg cacccatttt ggcgttccac attatgatga aaccgatacc tttctggggc  10260 cagatggcga ggtactggtg gtagcggatc aatcccgcga cgaatcgaca ttacagggta  10320 tcaacttagg caccgccttt accgttaccg gataccgttc ccgtctggag agtcatttca  10380 gccgattgga atattggcaa cccaaggcaa cacccaagac aactggcaaa acagattttt  10440 ggctgatata tagcccagat ggacaagtac atttactggg taaatcacca caagcccgga  10500 tcagcaaccc gtcagacatc actcaaacag cacaatggtt gctagaagcc tctgtgtcac  10560 cacatggtga acaaatttat tatcaatatc gggccgagga taacaccggt tgcgaagctg  10620 atgaaattac tctccatcca caggccgccg cgcaacgtta tctacacaca gtgtattacg  10680 gcaaccggac agccagcaaa acgttacccg gtctggatgg cagcgcccca ccacaagcag  10740 actggttatt ctatctggta tttgattacg gcgaacgcag taacaacctg agaacgccgc  10800 cagcattttc gactacaggt agctggcttt gtcgccagga ccgtttttcc cgttatgaat  10860 atggttttga gattcgtacc cgccgcttat gccgtcaggt attgatgtat caccacctgc  10920 aagctctgga tagcgagata aaagaacaca acggaccaac gctggtttca cgcctgatac  10980 tcaattatga cgaaagcgca atcgccagca cgctggtatt cgttcgtcga gtaggccacg  11040 agcaagacgg tactgccgtc accctgccgc cattagaatt ggcgtatcag gatttttcac  11100 cgcaacataa cgctcgctgg caatcgatga atgtgctggc aaacttcaat gccattcagc  11160 gctggcaact agttgatcta aaaggcgaag gattccccgg tctgctatat caagataaag  11220 gcgcctggtg gtaccgctcc gcacaacgtt ttggcaaaat tggctcagat gccgtcactt  11280 gggaaaaaat gcaacctttg tcggttatcc cttccttgca aagtaatgcc tcgctggtgg  11340 atatcaatgg agacggccaa cttgactggg ttatcaccgg accgggatta cggggatatc  11400 atagtcagca tccagatggc agttggacac gttttacccc gctcaacgct ctgccagtgg  11460 aatatactca tccacgcgcg caactcgccg atttaatggg agctggactt tctgatttag  11520 tactgatcgg ccctaagagt gtacgtttat atgccaatac ccgcgacggc tttgccaaag  11580 gaaaagatgt agtgcaatcc ggtgatatca cactgccagt accgggcgcc gatccgtgta  11640 agttggtggc atttagtgat gtattgggtt ccggtcaggc acatctggtt gaagtgagcg  11700 cgactaaagt cacctgctgg cctaatctgg ggcacggacg ttttggtcaa ccaattactc  11760 ttccgggatt tagccaacca gaagcgacgt ttaatcctgc tcaagtttat ctggccgatc  11820 tagatggcag cggcccgact gatctgattt atgttcacac agatcgtctg gatatcttcc  11880 tgaataaaag cggcaacggc ttcgccgcac cagtaactct ccccttccca gccggagtgc  11940 gttttgatca tacctgtcag ttacaagtgg ccgatgtaca agggttaggc gtcgccagcc  12000 tgatattaag tgtgccgcat atgactcccc atcactggcg ttgcgatctg accaacacaa  12060 aaccgtggtt actcagtgaa atgaacaaca atatgggggc tcatcacacc ctgcgttacc  12120 gtagttccgc ccagttctgg ctggatgaaa aagccacggc actggatgcc ggacaaatac  12180 cagtttgtta tctacccttc ccggtacaca ccctatggca aacggaaata gaggatgaaa  12240 tcagcggcaa caaattagtc acaatactac gttatgcaca tggcgcctgg gatggacgtg  12300 agcgagaatt tcgcggattt ggttatgttg aacagaaaga cagccatcaa ctggcccaag  12360 gcagtgcgcc agaatgcaca ccacctgcac tgacccaagg caacgcgcct gaactcacat  12420 cacccgcgct gacccaaggc aacgctccag aactcacacc acctgcgatg acccaaagca  12480 acgcgcctga actcacatca cccgcgctga cccaaggcaa cgcgccagaa ttcacatcac  12540 ccgcgctggc ccaaggcaat gcgccagaac tcacaccacc tgcgatgacc aaaaactggt  12600 atgccaccgg aatacccatg atagataaca cattatcgac agagtattgg catggtgatc  12660 accaagcttt tgccggtttt tcaccacgct ttacgacctg gcaagatggt caagatattc  12720 tgctcacacc ggaaaatgat aacagtcagt actggctaaa ccgggcactg aaaggtcaac  12780 tgctacgcag tgaactgtac ggcgaggatg gcagtacaca ggaaaaaatt ccctacacag  12840 tcactgaatt tcgcccacag gtacgtcggt tacagcatac cgatagccga taccttgtgc  12900 tttggtcatc tgtagttgaa agccgcaact atcattacga acgtatcgcc agcgatcctc  12960 aatgcagcca aaagattacg ctatccagcg atctatttgg tcaaccgcta aaacaggttt  13020 cggtacagta tccacgccgc cagcaaccgg caagcagtcc gtatcctgat acgttgcctg  13080 ataagttatt tgctaacagc tatgatgacc agcaacacaa attacggctc acctatcaac  13140 agttcagttg gcatcatctg accgacaata ccattctgat gttaggatta ccggatagta  13200 cccgcagcga tatctttgct tatagcgctg aacatgtccc tactggtggt ctaaatctgg  13260 aaatcctaaa tgataaaaat agtctgattg cggagaataa acctcgtgaa tacctcggcc  13320 agcaaaaaac cgtttatacc gacgggcaaa atgcaacgcc atcgcaaacg ccaacacgac  13380 aagcgctgat tgccttcacc gagacaacag tatttaatca atccacacta tcagcgtttg  13440 atgggagtat ctcatctgct caattgtcaa cgacgctgga acaagccgga taccagcaaa  13500 cagattatct attcccgcgc actggagaag ataaagtctg ggcagctcgt cgtggctata  13560 ctgattacgg cacagccgaa cagttctggc ggccgcaaaa acagagcaac actcaactca  13620 cgggcaaaat cacgctcact tgggatgcaa actattgcgt cgtcacacaa acccgggatg  13680 cggctggact gacaacctca gccagatatg attggcgttt tctgaccccc gttcaactca  13740 cggatatcaa cgacaatcag caccttacca cgctggatgc actgggccga ccaatcacac  13800 tgcgcttttg gggaaccgaa aacggtaaga tgactggtta ttcttcaccg gaaaaaatat  13860 cgttttctcc accatctgat gttgacgccg cgattaagtt aacaacgcca atccctgtag  13920 cacagtgtca ggtctacgca cccgaaagct ggatgcccat attaaagaaa accctcaata  13980 acctggcaga gcaagagcgg aaagagttat ataacacccg aatcatcacc gaagacggac  14040 gcatctgtac cctagctcac cgccgctggg taaaaagcca aagtgcagtc acccagccaa  14100 tcaatctgtc aaacggcagt ccccgtttac cccctcatag cctcacattg actacggatc  14160 gttatgaccg cgatcttaag caacagattc gtcaacaagt agtattcagt gatggctttg  14220 gccgtttact gcaagcatct gtacgacatg aagcaggcga agcctggcaa cgtaaccaag  14280 acggcgctct ggtgacaaaa atggaagata ccaaaacgcg ctgggcggtt acgggacgca  14340 ctgaatatga caataaggga caaccgatac gcacctatca accctatttc ctcaacgact  14400 ggcaatacgt cagtaatgac agtgcccggc ggacagaaga agcctatgca gatacccatg  14460 tctatgatcc cattggtcga gaaatcaagg tcactaccgc aaaaggctgg ttccgtcgaa  14520 ccttgttcac tccttggttt actgtcaatg aagatgaaaa tgacacagct actgaggtga  14580 aggtaaagaa gaaagaatgt aaagaaggta aagaaggtaa agatgtaatt tgatcaatcc  14640 cgcccggttg aagggcggga aacataacat aatatagagg tgaaacgtgt cattcataat  14700 gccgtcagat actcaactta tgagttggtt gatcattggt tttattgcgg cctggggcgg  14760 attagtaagg tacctcattg atatacaaaa caaacaatgt aaatggaatt ggatcaacgt  14820 actctgtcaa ctcattatct cctgttttac cggtatattg ggaggactgc tgagttttga  14880 aagcggcggc agcccctata tgacttttgc gattgccggg ctatttggca ccacgggaag  14940 ttctggattg aactggatct ggcgtcgcct ttttatgcat tatcgcgatg atggaggaaa  15000 gcaataaggc attcccactg ccgcaaaaac catctgtctc cggcagttaa accgggaaat  15060 tacctactac aactattgta agaaaacgaa tatatagaaa aactaacatg cagataaaaa  15120 ctgcgattgc agaacagatg acacacaacg ccccaacaac gaggtaaatc atg aaa     15176 Met Lys 1 aac atc gat cct aaa ctt tat caa aag acc cct gtc gtc aac atc tac    15224 Asn Ile Asp Pro Lys Leu Tyr Gln Lys Thr Pro Val Val Asn Ile Tyr 5                  10                  15 gat aac cga ggt cta acg atc cgt aac atc gac ttt cac cgt acc acc    15272 Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His Arg Thr Thr 20                  25                  30 gca aac ggc gat acc gat atc cgt att act cgc cat caa tat gac tcc    15320 Ala Asn Gly Asp Thr Asp Ile Arg Ile Thr Arg His Gln Tyr Asp Ser 35                  40                  45                  50 ctt ggg cac cta agc caa agc acc gat ccg cgt cta tat gaa gcc aaa    15368 Leu Gly His Leu Ser Gln Ser Thr Asp Pro Arg Leu Tyr Glu Ala Lys 55                  60                  65 caa aaa tct aac ttt ctc tgg cag tat gat ttg acc ggt aat att ttg    15416 Gln Lys Ser Asn Phe Leu Trp Gln Tyr Asp Leu Thr Gly Asn Ile Leu 70                  75                  80 tgt aca gaa agc gtc gat gct ggt cgc act gtc acc ttg aat gat att    15464 Cys Thr Glu Ser Val Asp Ala Gly Arg Thr Val Thr Leu Asn Asp Ile 85                  90                  95 gaa ggc cgt ccg cta ctg aca gta act gca aca ggt gtc ata caa acc    15512 Glu Gly Arg Pro Leu Leu Thr Val Thr Ala Thr Gly Val Ile Gln Thr 100                 105                 110 cga caa tat gaa acg tct tcc cta ccc ggt cgt ctg ttg tct gtt acc    15560 Arg Gln Tyr Glu Thr Ser Ser Leu Pro Gly Arg Leu Leu Ser Val Thr 115                 120                 125                 130 gaa caa ata cca gaa aaa aca tcc cgt atc acc gaa cgc ctg att tgg    15608 Glu Gln Ile Pro Glu Lys Thr Ser Arg Ile Thr Glu Arg Leu Ile Trp 135                 140                 145 gct ggc aat agc gaa gca gag aaa aac cat aat ctt gcc agc cag tgc    15656 Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser Gln Cys 150                 155                 160 gtg cgc cac tat gac acg gcg gga gtc acc cga tta gag agt ttg tca    15704 Val Arg His Tyr Asp Thr Ala Gly Val Thr Arg Leu Glu Ser Leu Ser 165                 170                 175 ctg acc ggt act gtt tta tct caa tcc agc caa cta ttg agc gac act    15752 Leu Thr Gly Thr Val Leu Ser Gln Ser Ser Gln Leu Leu Ser Asp Thr 180                 185                 190 caa gaa gct agc tgg aca ggt gat aat gaa acc gtc tgg caa aac atg    15800 Gln Glu Ala Ser Trp Thr Gly Asp Asn Glu Thr Val Trp Gln Asn Met 195                 200                 205                 210 ctg gct gat gac atc tac aca acc ctg agc gcc ttt gat gcc acc ggc    15848 Leu Ala Asp Asp Ile Tyr Thr Thr Leu Ser Ala Phe Asp Ala Thr Gly 215                 220                 225 gct tta ctc act cag acc gat gcg aaa ggg aac att cag agg cta acc    15896 Ala Leu Leu Thr Gln Thr Asp Ala Lys Gly Asn Ile Gln Arg Leu Thr 230                 235                 240 tat gat gtg gcc ggg cag cta aac ggg agc tgg tta acc tta aaa gac    15944 Tyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp Leu Thr Leu Lys Asp 245                 250                 255 caa ccg gaa caa gtg att atc aga tcc ctg acc tat tcc gcc gcc gga    15992 Gln Pro Glu Gln Val Ile Ile Arg Ser Leu Thr Tyr Ser Ala Ala Gly 260                 265                 270 caa aaa tta cgc gag gaa cac ggc aat ggt gtt atc acc gaa tac agt    16040 Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val Ile Thr Glu Tyr Ser 275                 280                 285                 290 tat gaa ccg gaa acc caa cag ctt atc ggt acc aaa acc cac cgt ccg    16088 Tyr Glu Pro Glu Thr Gln Gln Leu Ile Gly Thr Lys Thr His Arg Pro 295                 300                 305 tca gat gcc aaa gtg ttg caa gat cta cgt tat gag tat gac ccg gta    16136 Ser Asp Ala Lys Val Leu Gln Asp Leu Arg Tyr Glu Tyr Asp Pro Val 310                 315                 320 ggc aat gtc atc agt atc cgt aat gac gca gaa gcc acc cgc ttc tgg    16184 Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu Ala Thr Arg Phe Trp 325                 330                 335 cac aat cag aaa gtg gcg ccg gaa aac act tat acc tac gac tcc ttg    16232 His Asn Gln Lys Val Ala Pro Glu Asn Thr Tyr Thr Tyr Asp Ser Leu 340                 345                 350 tat cag ctt atc agc gca acc ggg cgc gag atg gcg aat ata ggt cag    16280 Tyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln 355                 360                 365                 370 caa agt aac caa ctt ccc tcc ctc acc cta cct tct gat aac aac acc    16328 Gln Ser Asn Gln Leu Pro Ser Leu Thr Leu Pro Ser Asp Asn Asn Thr 375                 380                 385 tac acc aac tat acc cgt act tat act tat gac cgt ggc ggc aat ttg    16376 Tyr Thr Asn Tyr Thr Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu 390                 395                 400 act aaa atc cag cac agt tca ccg gcg acg caa aac aac tac acc aca    16424 Thr Lys Ile Gln His Ser Ser Pro Ala Thr Gln Asn Asn Tyr Thr Thr 405                 410                 415 aac atc acg gtt tct aac cgg agc aat cgc gca gta ctc agc act ctg    16472 Asn Ile Thr Val Ser Asn Arg Ser Asn Arg Ala Val Leu Ser Thr Leu 420                 425                 430 acc gaa gat ccg gcg caa gta gat gct tta ttt gat gca ggc gga cat    16520 Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe Asp Ala Gly Gly His 435                 440                 445                 450 cag aac acg ttg ata tca gga caa aac ctg aac tgg aat aca cgc ggt    16568 Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn Trp Asn Thr Arg Gly 455                 460                 465 gaa cta caa cat gtg aca ttg gtg aaa cgg gac aag ggc gcc aat gat    16616 Glu Leu Gln His Val Thr Leu Val Lys Arg Asp Lys Gly Ala Asn Asp 470                 475                 480 gat cgg gaa tgg tat cgc tat agt agt gac ggg aga agg ata tta aaa    16664 Asp Arg Glu Trp Tyr Arg Tyr Ser Ser Asp Gly Arg Arg Ile Leu Lys 485                 490                 495 atc aat gaa cag cag acc agc agc aac tct caa aca cag aga ata act    16712 Ile Asn Glu Gln Gln Thr Ser Ser Asn Ser Gln Thr Gln Arg Ile Thr 500                 505                 510 tat ttg ccg agc tta gaa ctt cgt cta aca caa aac agc acg atc aca    16760 Tyr Leu Pro Ser Leu Glu Leu Arg Leu Thr Gln Asn Ser Thr Ile Thr 515                 520                 525                 530 acc gaa gat ttg caa gtt atc aca gta gga gaa gcg ggt cgg gca cag    16808 Thr Glu Asp Leu Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln 535                 540                 545 gta cga gta tta cat tgg gat agc ggt caa ccg gaa gat atc gac aat    16856 Val Arg Val Leu His Trp Asp Ser Gly Gln Pro Glu Asp Ile Asp Asn 550                 555                 560 aat cag cta cgt tat agc tac gat aat ctt atc ggt tcc agt caa ctt    16904 Asn Gln Leu Arg Tyr Ser Tyr Asp Asn Leu Ile Gly Ser Ser Gln Leu 565                 570                 575 gaa tta gac agc aaa gga gaa att att agt gag gaa gag tac tat ccc    16952 Glu Leu Asp Ser Lys Gly Glu Ile Ile Ser Glu Glu Glu Tyr Tyr Pro 580                 585                 590 tat ggc ggc acg gca tta tgg gca aca agg aag cgg aca gaa gcc agt    17000 Tyr Gly Gly Thr Ala Leu Trp Ala Thr Arg Lys Arg Thr Glu Ala Ser 595                 600                 605                 610 tat aaa acc atc cgt tat tca ggt aaa gag cgg gat gcc acc gga cta    17048 Tyr Lys Thr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr Gly Leu 615                 620                 625 tat tat tac ggt tac cga tat tat cag cct tgg gta gga cga tgg tta    17096 Tyr Tyr Tyr Gly Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu 630                 635                 640 agt gcc gat ccg gca gga aca gta gat ggg ttg aat tta tat cgg atg    17144 Ser Ala Asp Pro Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met 645                 650                 655 gta agg aat aat ccg gtt act ctg ctt gat cct gat gga tta atg cca    17192 Val Arg Asn Asn Pro Val Thr Leu Leu Asp Pro Asp Gly Leu Met Pro 660                 665                 670 aca att gca gaa cgc ata gca gca ctg caa aaa aat aaa gta gca gat    17240 Thr Ile Ala Glu Arg Ile Ala Ala Leu Gln Lys Asn Lys Val Ala Asp 675                 680                 685                 690 tca gcg cct tcg cca aca aat gcc aca aac gta gcg ata aac atc cgc    17288 Ser Ala Pro Ser Pro Thr Asn Ala Thr Asn Val Ala Ile Asn Ile Arg 695                 700                 705 ccg ccc gta gca cca aaa cct acc tta ccc aaa gca tca acg agt agc    17336 Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys Ala Ser Thr Ser Ser 710                 715                 720 caa tca act aca tac ccc atc aaa tct gca agc ata aaa cca acg acg    17384 Gln Ser Thr Thr Tyr Pro Ile Lys Ser Ala Ser Ile Lys Pro Thr Thr 725                 730                 735 tcg gga tca tcc att act gct cca ctg agt cca gta gga aat aaa tct    17432 Ser Gly Ser Ser Ile Thr Ala Pro Leu Ser Pro Val Gly Asn Lys Ser 740                 745                 750 act cct gaa ata tct ctt cca gaa agc act caa agc aat tct tca agc    17480 Thr Pro Glu Ile Ser Leu Pro Glu Ser Thr Gln Ser Asn Ser Ser Ser 755                 760                 765                 770 gct att tca aca aat cta cag aaa aag tca ttt act tta tat aga gcg    17528 Ala Ile Ser Thr Asn Leu Gln Lys Lys Ser Phe Thr Leu Tyr Arg Ala 775                 780                 785 gat aat aga tcc ttt gaa gac atg cag agt aaa ttc cct gaa gga ttt    17576 Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys Phe Pro Glu Gly Phe 790                 795                 800 aaa gcc tgg act cct cta gat act aag atg gca agg cag ttt gct agt    17624 Lys Ala Trp Thr Pro Leu Asp Thr Lys Met Ala Arg Gln Phe Ala Ser 805                 810                 815 gtc ttt att ggt cag aaa gat act tct aat tta cct aaa gaa aca gtc    17672 Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu Pro Lys Glu Thr Val 820                 825                 830 aag aat ata aac aca tgg gga aca aaa cca aaa tta aat gat ctc tca    17720 Lys Asn Ile Asn Thr Trp Gly Thr Lys Pro Lys Leu Asn Asp Leu Ser 835                 840                 845                 850 act tac ata aaa tat acc aag gac aaa tct aca gta tgg gtc tct act    17768 Thr Tyr Ile Lys Tyr Thr Lys Asp Lys Ser Thr Val Trp Val Ser Thr 855                 860                 865 gca att aat act gaa gca ggt gga caa agt tca ggg gct cca ctc cat    17816 Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser Gly Ala Pro Leu His 870                 875                 880 gaa att aat atg gat ctt tat gag ttt acc att gac gga caa aag cta    17864 Glu Ile Asn Met Asp Leu Tyr Glu Phe Thr Ile Asp Gly Gln Lys Leu 885                 890                 895 aat cca cta cca agg gga aga tct aaa gac agg gtg cct tca cta tta    17912 Asn Pro Leu Pro Arg Gly Arg Ser Lys Asp Arg Val Pro Ser Leu Leu 900                 905                 910 ctt gac aca cca gaa ata gaa aca gca tcc ata att gca ctt aat cat    17960 Leu Asp Thr Pro Glu Ile Glu Thr Ala Ser Ile Ile Ala Leu Asn His 915                 920                 925                 930 gga ccg gta aat gat gca gaa gtt tca ttc cta aca aca att ccg ctt    18008 Gly Pro Val Asn Asp Ala Glu Val Ser Phe Leu Thr Thr Ile Pro Leu 935                 940                 945 aaa aat gta aaa cct tat aag aga taa cgaaaaatta atattcttta          18055 Lys Asn Val Lys Pro Tyr Lys Arg 950                 955 tctactttta atagccctct tgaacttaca ctcaaggggg ggaaaccaaa taagaaacca  18115 tctttaataa caagccatga aagaatattt atttcatggc ttgattactt ttaacattca  18175 atattaaata attaaaacaa tatctaacca attaaaataa caatacctta tttatcatat  18235 taaaatatca aatcagaaat taatgaattt aagggttctt tatatttatt tctgagagca  18295 taggcacaat accttaccga tggcgctgga cgtgattcaa aatccagaaa tgctatattt  18355 tcatcaatat gggcagaata gcgcatttca ttgggagtca ttaaacttat cgcgacaccc  18415 gcttttacca gatccaatct attagtaaaa tcagggaccg tcaataacgc taaattttgg  18475 tattcaggga gataattcaa tggcataaaa ttattgcatt gttttaaaaa agcactatta  18535 tgctgaacaa aaggaaaact agatattatt tcatcagcgt gactttctgg ttctaaaata  18595 tcatgggata cagcaagaga cagcatttga taagcaccat ctatcctgat gatatcatca  18655 ttatctggat aacattcagt cgtcacataa actgttatat cccctttcat taaggaagaa  18715 aataccgcat cttgccttat taaatcatca attagaaaat tgttgattat acaaatatcg  18775 cgataatgat aacgttgcac cgctcttttt acgaccgtag atattttatt aacatattct  18835 ccacttgtgc caataaccag tttgtctctg tttgataatt tataatttct acgacaattc  18895 caattattct caactttcag gatcctttca taacacggca gcaactcttg atatagtgcc  18955 tttccctctt ctgtgagctt ggtttttccc ggtagtcgct caaacaattg acaccccaca  19015 cgctgttcca gttgatatac gagcctgcta agtggagaag gggtaataca aagcgtatcc  19075 gccgctaacg tgaatgactc tttcttagct gattccataa aatactttag ttgctttgaa  19135 caaaatatca tcacataccc tcttgttttc attccagaaa tagaatatta accatagaac  19195 atgacaacga tgtttctact ttgcattctt ttacattagg acatgcgtta atggacattg  19255 aatttcacta catcaattgt taatatttat ttaatacttg cacaataatt ataaaataaa  19315 tataacttag ttaattattt cttgatattg atcatggtaa gttttcctca atacctacag  19375 aagtagatat tattttatct tccagtaatc tatcgtttgg cgacggaggt cgattcttcc  19435 attgggatat tcaacccatt cgccgccttt cttattaatt acagtgattt ttggcatttt  19495 ggtttcatcc aacttaggtt tataggtgat tttccattta gcacccggtg ttaacttcaa  19555 cctaaaggga tacataccaa cttcaccttg taagaatatt ctgtttggtc taccttcaac  19615 gactttcaaa atggggtaaa taaccgggct aaaatcaatc gtatccaatg catcaatttc  19675 gctgatattt gtccgggctg catcattgat aaatgcgatt aaatcggttg ctgaatacgg  19735 aatagcatct ttcactagat gacggacatc ggtataactc actgacacaa aggctcggtc  19795 aatcttccac ttacatcgac cgccaccatt aaaaggtagt tttgcctgaa agtaaccggt  19855 tttcggatca gcttttacat ccagacgtaa tccgttataa gttggtacct taaaaggcga  19915 catattggaa tctaaacgat atttaaggca atcttttgag atatacacag cggatacatg  19975 cggctgtgtg tatttaggtg cgactccttc tacagtaatc cactgattct ctttgggagg  20035 agagagcggc tcatttgggt cagcacagcc tgatattaaa atcacggata agacagataa  20095 gtatttcttg atatttatca tggtaagttt tcctcaactc ctacagcgtt atctgcatgt  20155 gtgtccaatt ccagatcttc ctgtttatct atttagaaat aaataagcta cgctgatagc  20215 attacttcat atttccatac atgaatcgaa aatcgacttc ttgagtgccg ttatcaattt  20275 tgccgcccgg atattcaacc cactcgccgc ctttcttatt agtcaccgtg accttcgcca  20335 ttttggtttc atccagctta ggcttaaaaa taattttcca tttagctcct ggagttaacg  20395 tgagttgaaa aggacgcatt tttaatactt caccttgtaa gaatattctg ttcgggcgac  20455 cttcaacgac tttcaaaaca gggtaaataa ccgggctaaa atcaatcgta ttcaatgtcg  20515 agattttgct aatattcatc tggactatgc cattgataga tgcgattaaa ccggttgctg  20575 aatacggaat agcatctttc accagatggc tgacatcagt ataactcacc gatacaaagg  20635 cccggttaat tttccattta catcgtcccc ctccattaaa aggtagtttt gcttgaaaat  20695 aaccggtttg tggatcggcc ttcactttca gacgaagccc attataggtc ggcactttaa  20755 aaggcgacat attggaatcc agacgatact caaggcaatc ctttgatatg tattctgcgg  20815 atacatgtgg ttcggtatat ttcggcgcta ccccttctac cgtgatccat tgattttctt  20875 taggagggga aagcggctca tttgggtcag cacagcctga tattaaaatc actgacaaga  20935 caaataagta ttttttaaca tttatcatgg taagttttcc tcaattccta cagcattatc  20995 cgcataaata tcctgtcaag aatagcgttc attgatttcg tcaccaaaga aacaagatag  21055 taaaaatcct attaccacag ataaaaaaca ccgcttatgc cgtgagtaat agtgagttga  21115 gcgacaggga tacagcagtg catccccatc aattagtccc tttgaataaa gggaacagaa  21175 tttgaaattt ccgtcatacc gtccatatta cggaacttag attatgatta ttaaatcacc  21235 accaaatggc aagaaaaatt ttcatttttt aatttacgaa gaatgaattt gtaagaaagt  21295 gttacaaact taatagaaat taatttactg ttaatctaat gaaggatgaa attataaaaa  21355 taacccattt ctcagggaca acaatccaca atatatagaa ccactggtcc tcacttaatt  21415 tcctgtcagg agtagaaata tcctgatgac tcagtcgatg acatacagca atgtcattgg  21475 tattgagact accgactgtt taataaattt cttttgtctt taatggcgag atacaagtga  21535 ttcactattt aagcactatc gataaataag attccaaaat agcgccatat cttacaccac  21595 tcataattct atgtataaca attggttaaa taggatcatg tgtaacagga ttatgaaacg  21655 ttatttatat caaatctatc aattatttta tatatagttt cacagtcaca ctcgctatct  21715 ggtaccttca taaccaactg ccctccctgc gctaccttct gataacaaca gctacactaa  21775 ctatacccgc gcctataatt atgaccgtgt gaaaattcag cgtagttcac cggccacgca  21835 aaataactac acgaaatatt gctccccaga gaaacaccgt tcgaggttgt ttcaatgaaa  21895 catcaaggta gagacaccta tgtattatta caagatatta aaccctctgc gattactcat  21955 aggaatgtac gtaatactta tacaggcaac ttcacgtcat ccagagaaaa ttaagttgta  22015 caaaatagac atcaactaat atagtaatag aaaatcccct gaaaatagat tcaggggatt  22075 taataaatta accaaaaatc ataataaaaa tttatttcat tattttagga taaatattta  22135 attagcctaa taatgaatta ttacttaaag taattcctaa acaatcaaat cggaaattaa  22195 taaattcaat ggttcttgat atttatgcct gagagtataa gcacaatatt tcactgaggg  22255 tgtcggatgc gatttaaaat tcaaaaaggt aatgccttta tgaaggtcag cagaacagag  22315 cacttcattc ggtgtcatta aacttatcgc gatacctgat ttcactaagt ccaaccgatt  22375 agtaaaatca ggcacagtta gcaaagttaa attctggtat tcaggtagac aattcagtga  22435 aataaaattg ccgcactgtt taaagaaagc actattatgc tgagccaaag gcaatgtata  22495 tataatatta tcagcgttac tttccgatcc taaaatatca ttagctacag caaggcgcaa  22555 agtctgataa gttccatcca ctctgataat atcatcgttg tcaggataat gttcagtcgt  22615 cacatagacg gttatatctt ccttcattaa agaagaaaat attgtctctt ttcttgccga  22675 atcatcaacc agaaaattgt tttttatgta aatatcacga taatgataac gttgtaccgc  22735 tctttttatc actgccgaaa ttttattaat atattctcca cttgtcccga tgaccagttt  22795 gccggtgttt gctaattttc cgcttctacg ataatgccaa ttatcctcaa ctcgctgaat  22855 tctctcataa cacggcaata gctcctgata taatgccttc ccctcttcag tgagtttggt  22915 ccttcccggt agtcgttcaa atagttgaca gcccacacgt tgttccagtt gatatatgat  22975 cctacttaga ggagagggag taatacaaag cgtatccgcg gctaaagtaa atgactcttt  23035 tttcgctgac tccataaaat atttcaagct ctttgaacaa aatagcatca tatatccttc  23095 ttattttaat tcattgttcc atccgaaata gaatggaatg ttaacaagaa aacattacaa  23155 ctacttttct tctttgcatt atttaacatc aaagtatgca ttaactgaga ttgagtttta  23215 tcatctttat tcttaacagt tatcaaacaa ttttcattat tattgcaaaa taaatacaac  23275 cccttcttat gttacaataa tgattataaa gaaatttcac atattatcat taagtaataa  23335 tgggcacaat taaccattta attaaacatt tcaattggtt gacaaagact cattatgttc  23395 aacatgtaat gagcgcaatt ttaacattaa ataaattaca tagttcatat tcattatcac  23455 tgagatcagc ttttttcgta tagtacatca tgtgaacaat accgtgccat ttcctgccaa  23515 atcttattaa aaagtcagtt gcaaattttg catctgcttt ttttgcaaca gctatttaaa  23575 gaaaacagtg agatagtgat tatccgagag atcaagatat gtctgctctt tacgcacaaa  23635 ctgcaaacca tttctatgca tatctcagct atttctcaaa acctgtattt aatcatctct  23695 tattccgatg gaacggaatc attctctgat tgattcatga tgtaaagaca atatggatgt  23755 ttcatttact tt atg att tta aaa gga ata aat atg aat tcg cct gta aaa  23806 Met Ile Leu Lys Gly Ile Asn Met Asn Ser Pro Val Lys 960                 965 gag ata cct gat gta tta aaa atc cag tgt ggt ttt cag tgt ctg aca    23854 Glu Ile Pro Asp Val Leu Lys Ile Gln Cys Gly Phe Gln Cys Leu Thr 970                 975                 980 gat att agc cac agc tct ttt aac gaa ttt cac cag caa gta tcc gaa    23902 Asp Ile Ser His Ser Ser Phe Asn Glu Phe His Gln Gln Val Ser Glu 985                 990                 995                1000 cac ctc tcc tgg tcc gaa gca cac gac tta tat cat gat gca caa cag    23950 His Leu Ser Trp Ser Glu Ala His Asp Leu Tyr His Asp Ala Gln Gln 1005                1010                1015 gcc caa aag gat aat cgg ctg tat gaa gcg cgt att ctt aaa cgc acg    23998 Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Thr 1020                1025                1030 aat cct caa tta caa aat gct gta cat ctt gcc atc gta gcg cct aat    24046 Asn Pro Gln Leu Gln Asn Ala Val His Leu Ala Ile Val Ala Pro Asn 1035                1040                1045 gct gaa ctg ata ggc tat aac aac caa ttt agc ggc agg gcc agt caa    24094 Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln 1050                1055                1060 tat gtc gcg ccg ggt acc gtt tcc tcc atg ttc tcc ccc gcc gct tat    24142 Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr 1065               1070                1075                1080 ttg act gag ctt tat cgt gaa gca cgc aat tta cac gcc agc gat tcc    24190 Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser 1085                1090                1095 gtt tat cgc ctg gat act cgc cgc cca gat ctc aaa tca atg gcg ctc    24238 Val Tyr Arg Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu 1100                1105                1110 agt caa caa aat atg gat acg gaa ctt tcc act ctc tct tta tcc aat    24286 Ser Gln Gln Asn Met Asp Thr Glu Leu Ser Thr Leu Ser Leu Ser Asn 1115                1120                1125 gag cta tta ttg gaa agc att aaa act gag tct aag ctg gat aat tat    24334 Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Asp Asn Tyr 1130                1135                1140 act caa gtg atg gaa atg ctc tcc gct ttc cgt cct tcc ggc gcg acg    24382 Thr Gln Val Met Glu Met Leu Ser Ala Phe Arg Pro Ser Gly Ala Thr 1145               1150                1155                1160 cct tat cac gat gct tac gaa aat gtg cgt aaa gtt atc cag cta caa    24430 Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Lys Val Ile Gln Leu Gln 1165                1170                1175 gat cct ggg ctt gag caa tta aat gct tca cca gcc att gcc ggg ctg    24478 Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu 1180                1185                1190 atg cat caa gct tcc cta tta ggt att aac gct tca atc tca cct gag    24526 Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu 1195                1200                1205 ttg ttt aat att ctg acg gag gag att act gaa ggt aat gct gag gaa    24574 Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu 1210                1215                1220 ctt tat aag aaa aat ttt ggt aat atc gaa ccg gct tca ctg gct atg    24622 Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met 1225               1230                1235                1240 ccg gaa tac ctt aga cgt tat tac aat tta agt gat gaa gaa ctc agc    24670 Pro Glu Tyr Leu Arg Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser 1245                1250                1255 cag ttt att ggt aaa gcc agc aat ttc ggc caa caa gaa tat agt aat    24718 Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn 1260                1265                1270 aac caa ctc att act ccg ata gtc aac agc aat gat ggc aca gtc aag    24766 Asn Gln Leu Ile Thr Pro Ile Val Asn Ser Asn Asp Gly Thr Val Lys 1275                1280                1285 gta tat cga att acc cgc gaa tat aca aca aat gcc aat caa gta gac    24814 Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Asn Gln Val Asp 1290                1295                1300 gtg gag ctg ttt ccc tac ggt gga gaa aat tat cag tta aat tac aaa    24862 Val Glu Leu Phe Pro Tyr Gly Gly Glu Asn Tyr Gln Leu Asn Tyr Lys 1305               1310                1315                1320 ttc aaa gat tct cgt cag gat gtc tcc tat tta tcc atc aaa tta aat    24910 Phe Lys Asp Ser Arg Gln Asp Val Ser Tyr Leu Ser Ile Lys Leu Asn 1325                1330                1335 gac aaa aga gaa ctt atc cga att gaa gga gcg cct cag gtc aac atc    24958 Asp Lys Arg Glu Leu Ile Arg Ile Glu Gly Ala Pro Gln Val Asn Ile 1340                1345                1350 gaa tat tca gaa cat atc aca tta agt aca act gat atc agt caa cct    25006 Glu Tyr Ser Glu His Ile Thr Leu Ser Thr Thr Asp Ile Ser Gln Pro 1355                1360                1365 ttt gaa atc ggc cta aca cga gta tat cct tct agt tct tgg gca tat    25054 Phe Glu Ile Gly Leu Thr Arg Val Tyr Pro Ser Ser Ser Trp Ala Tyr 1370                1375                1380 gca gcc gca aaa ttt acc att gag gaa tat aac caa tac tct ttc ctg    25102 Ala Ala Ala Lys Phe Thr Ile Glu Glu Tyr Asn Gln Tyr Ser Phe Leu 1385               1390                1395                1400 tta aaa ctc aat aaa gct att cgt cta tct cgt gcg aca gaa tta tca    25150 Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser 1405                1410                1415 ccc acc att ctg gaa agt att gtg cgt agt gtt aat cag caa ctg gat    25198 Pro Thr Ile Leu Glu Ser Ile Val Arg Ser Val Asn Gln Gln Leu Asp 1420                1425                1430 atc aac gca gaa gta tta ggt aaa gtt ttt ctg act aaa tat tat atg    25246 Ile Asn Ala Glu Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met 1435                1440                1445 caa cgt tat gct att aat gct gaa act gcc cta ata cta tgc aat gca    25294 Gln Arg Tyr Ala Ile Asn Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala 1450                1455                1460 ctt att tca caa cgt tca tat gat aat caa cct agc caa ttt gat cgc    25342 Leu Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg 1465               1470                1475                1480 ctg ttt aat acg cca tta ctg aac ggc caa tat ttt tct acc gga gat    25390 Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp 1485                1490                1495 gaa gag att gat tta aat cca ggt agt act ggc gat tgg cgt aaa tcc    25438 Glu Glu Ile Asp Leu Asn Pro Gly Ser Thr Gly Asp Trp Arg Lys Ser 1500                1505                1510 gtg ctt aaa cgt gca ttt aat atc gat gat att tcc ctc tac cgc ctg    25486 Val Leu Lys Arg Ala Phe Asn Ile Asp Asp Ile Ser Leu Tyr Arg Leu 1515                1520                1525 ctt aaa att acc aac cat aat aat caa gat gga aag att aaa aat aac    25534 Leu Lys Ile Thr Asn His Asn Asn Gln Asp Gly Lys Ile Lys Asn Asn 1530                1535                1540 tta aat aat ctt tct gat tta tat att ggg aaa tta ctg gca gaa att    25582 Leu Asn Asn Leu Ser Asp Leu Tyr Ile Gly Lys Leu Leu Ala Glu Ile 1545               1550                1555                1560 cat caa tta acc att gat gaa ttg gat tta ttg ctg gtt gcc gtg ggt    25630 His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Val Ala Val Gly 1565                1570                1575 gaa gga gaa act aat tta tcc gct atc agt gat aaa caa ctg gcg gca    25678 Glu Gly Glu Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Ala 1580                1585                1590 ctg atc aga aaa ctc aat acc att acc gtc tgg cta cag aca cag aag    25726 Leu Ile Arg Lys Leu Asn Thr Ile Thr Val Trp Leu Gln Thr Gln Lys 1595                1600                1605 tgg agt gcg ttc caa tta ttt gtt atg act tcc acc agc tat aac aaa    25774 Trp Ser Ala Phe Gln Leu Phe Val Met Thr Ser Thr Ser Tyr Asn Lys 1610                1615                1620 acg ctg acg cct gaa att aag aat ctg ctg gat acc gtc tac cac ggt    25822 Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly 1625               1630                1635                1640 tta caa ggc ttt gat aaa gac aag gca aat tta ctg cat gtt atg gcg    25870 Leu Gln Gly Phe Asp Lys Asp Lys Ala Asn Leu Leu His Val Met Ala 1645                1650                1655 ccc tat att gcg gcc acc tta caa tta tca tcg gaa aat gtc gcc cat    25918 Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His 1660                1665                1670 tct gtg ctg ctt tgg gca gac aag tta aag ccc ggc gac ggc gca atg    25966 Ser Val Leu Leu Trp Ala Asp Lys Leu Lys Pro Gly Asp Gly Ala Met 1675                1680                1685 aca gcc gaa aaa ttc tgg gac tgg ttg aat act caa tat acg cca gat    26014 Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Gln Tyr Thr Pro Asp 1690                1695                1700 tca tcg gaa gta tta gca aca cag gaa cat att gtt cag tat tgt cag    26062 Ser Ser Glu Val Leu Ala Thr Gln Glu His Ile Val Gln Tyr Cys Gln 1705               1710                1715                1720 gcg ttg gcg caa tta gaa atg gtt tac cat tcc acc ggt atc aat gaa    26110 Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu 1725                1730                1735 aac gcc ttc cgc ctg ttt gtg aca aaa cca gag atg ttt ggc tcg tca    26158 Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ser Ser 1740                1745                1750 act gag gca gta cct gcg cat gat gca ctt tca ctg atc atg ctg acg    26206 Thr Glu Ala Val Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr 1755                1760                1765 cgt ttt gca gat tgg gtt aat gcg tta ggc gaa aaa gcc tct tcc gta    26254 Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val 1770                1775                1780 cta gcg gca ttt gaa gct aac agt tta acg gca gaa caa ttg gct gat    26302 Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp 1785               1790                1795                1800 gcc atg aat ctt gat gct aat ttg cta ttg caa gcc agt act caa gca    26350 Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Thr Gln Ala 1805                1810                1815 caa aac cat caa cat ctt ccc cca gtg acg caa aaa aat gct ttc tcc    26398 Gln Asn His Gln His Leu Pro Pro Val Thr Gln Lys Asn Ala Phe Ser 1820                1825                1830 tgt tgg aca tct atc gac act atc ctg caa tgg gtt aat gtt gca caa    26446 Cys Trp Thr Ser Ile Asp Thr Ile Leu Gln Trp Val Asn Val Ala Gln 1835                1840                1845 caa ttg aat gtc gcc cca cag gga gtt tcc gct ttg gtc ggg ctg gat    26494 Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp 1850                1855                1860 tat att caa tta aat caa aaa atc ccc acc tat gcc cag tgg gaa agt    26542 Tyr Ile Gln Leu Asn Gln Lys Ile Pro Thr Tyr Ala Gln Trp Glu Ser 1865               1870                1875                1880 gct ggg gaa ata ttg act gcc gga ttg aat tca caa cag gct gat ata    26590 Ala Gly Glu Ile Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asp Ile 1885                1890                1895 tta cac gct ttt ttg gac gaa tct cgc agt gcc gca tta agc acc tac    26638 Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr 1900                1905                1910 tat atc cgt caa gtc gcc aag cca gcg gca gcc ata aaa agc cgt gat    26686 Tyr Ile Arg Gln Val Ala Lys Pro Ala Ala Ala Ile Lys Ser Arg Asp 1915                1920                1925 gac ttg tac caa tac tta cta att gat aat cag gtt tcc gct gca atc    26734 Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile 1930                1935                1940 aaa act acc cgg att gcc gaa gcc att gcc agc att caa ctg tac gtc    26782 Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val 1945               1950                1955                1960 aac cgc acg ctg gaa aat gta gaa gaa aat gcc cat tca ggg gtt atc    26830 Asn Arg Thr Leu Glu Asn Val Glu Glu Asn Ala His Ser Gly Val Ile 1965                1970                1975 agc cgt cag ttc ttt atc gac tgg gac aaa tat aac aaa cgc tac agc    26878 Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser 1980                1985                1990 acc tgg gcg ggt gtt tct caa tta gtt tac tac ccg gaa aac tat att    26926 Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile 1995                2000                2005 gat ccc acc atg cgt atc gga caa acc aaa atg atg gac gca tta ttg    26974 Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu 2010                2015                2020 caa tcc gtc agc caa agc caa tta aat gcc gat act gtc gaa gac gcc    27022 Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala 2025               2030                2035                2040 ttt atg tct tat ctg aca tcg ttt gag caa gtg gct aat ctt aaa gtt    27070 Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val 2045                2050                2055 att agc gcg tat cac gat aat att aac aac gat caa ggg ctg acc tat    27118 Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr 2060                2065                2070 ttt atc ggc ctc agt gaa act gat acc ggt gaa tac tat tgg cgc agt    27166 Phe Ile Gly Leu Ser Glu Thr Asp Thr Gly Glu Tyr Tyr Trp Arg Ser 2075                2080                2085 gtc gat cac agt aaa ttc agc gac ggt aaa ttc gcc gct aat gcc tgg    27214 Val Asp His Ser Lys Phe Ser Asp Gly Lys Phe Ala Ala Asn Ala Trp 2090                2095                2100 agt gaa tgg cac aaa att gat tgt cca att aat cct tac cga agc act    27262 Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Arg Ser Thr 2105               2110                2115                2120 atc cgt cct gtg atg tac aaa tcc cgc ttg tat ctg ctc tgg ttg gaa    27310 Ile Arg Pro Val Met Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu 2125                2130                2135 caa aag gag atc act aaa caa aca gga aat agc aaa gat ggc tat caa    27358 Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln 2140                2145                2150 acc gag aca gat tat cgt tat gag cta aaa ttg gcg cat atc cgt tat    27406 Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr 2155                2160                2165 gac ggt acc tgg aat acg cca atc act ttt gat gtc aat gaa aaa ata    27454 Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Glu Lys Ile 2170                2175                2180 tcc aag cta gaa ctg gca aaa aat aaa gcg cct ggg ctc tat tgt gct    27502 Ser Lys Leu Glu Leu Ala Lys Asn Lys Ala Pro Gly Leu Tyr Cys Ala 2185               2190                2195                2200 ggt tat caa ggt gaa gat acg ttg ctg gtt atg ttt tat aac caa caa    27550 Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln 2205                2210                2215 gat aca ctc gat agt tat aaa acc gct tca atg caa ggg cta tat atc    27598 Asp Thr Leu Asp Ser Tyr Lys Thr Ala Ser Met Gln Gly Leu Tyr Ile 2220                2225                2230 ttt gcc gat atg gaa tat aaa gat atg acc gat gga caa tac aaa tct    27646 Phe Ala Asp Met Glu Tyr Lys Asp Met Thr Asp Gly Gln Tyr Lys Ser 2235                2240                2245 tat cgg gac aac agc tat aaa caa ttc gat act aat agt gtc aga aga    27694 Tyr Arg Asp Asn Ser Tyr Lys Gln Phe Asp Thr Asn Ser Val Arg Arg 2250                2255                2260 gtg aat aac cgc tat gca gag gat tat gaa att ccc tca tcg gta aat    27742 Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Asn 2265               2270                2275                2280 agc cgt aaa ggc tat gat tgg gga gat tat tat ctc agt atg gta tat    27790 Ser Arg Lys Gly Tyr Asp Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr 2285                2290                2295 aac gga gat att cca act att agt tac aaa gcc aca tca agt gat tta    27838 Asn Gly Asp Ile Pro Thr Ile Ser Tyr Lys Ala Thr Ser Ser Asp Leu 2300                2305                2310 aaa atc tat atc tcg cca aaa tta aga att att cat aat gga tat gaa    27886 Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu 2315                2320                2325 ggg cag caa cgc aat caa tgc aat cta atg aat aaa tat ggc aaa cta    27934 Gly Gln Gln Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu 2330                2335                2340 ggt gat aaa ttt att gtt tat act agc ttg gga gtt aat cca aat aat    27982 Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn 2345               2350                2355                2360 tcg tca aat aag ctg atg ttt tac ccc gtt tat caa tat aac gga aat    28030 Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Asn Gly Asn 2365                2370                2375 gtc agt ggg ctt agt caa ggg aga tta cta ttc cac cgt gac acc aat    28078 Val Ser Gly Leu Ser Gln Gly Arg Leu Leu Phe His Arg Asp Thr Asn 2380                2385                2390 tat tca tct aaa gta gaa gct tgg att cct gga gca gga cgt tct cta    28126 Tyr Ser Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Gly Arg Ser Leu 2395                2400                2405 acc aat ccg aat gct gcc att ggt gat gat tat gct aca gac tcg tta    28174 Thr Asn Pro Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu 2410                2415                2420 aac aaa ccg aat gat ctt aag caa tac gtc tat atg act gac agt aaa    28222 Asn Lys Pro Asn Asp Leu Lys Gln Tyr Val Tyr Met Thr Asp Ser Lys 2425               2430                2435                2440 ggt act gct acc gat gtc tca gga cca gta gat atc aat act gca att    28270 Gly Thr Ala Thr Asp Val Ser Gly Pro Val Asp Ile Asn Thr Ala Ile 2445                2450                2455 tcc ccg gca aaa gtt cag gta aca gta aaa gcc ggt agc aaa gaa caa    28318 Ser Pro Ala Lys Val Gln Val Thr Val Lys Ala Gly Ser Lys Glu Gln 2460                2465                2470 acg ttt acc gcg gat aaa aat gtc tcc att cag cca tcc cct agc ttt    28366 Thr Phe Thr Ala Asp Lys Asn Val Ser Ile Gln Pro Ser Pro Ser Phe 2475                2480                2485 gat gaa atg aat tat caa ttt aat gct ctc gaa ata gat ggc tca agt    28414 Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Ser 2490                2495                2500 ctg aat ttt act aac aat tca gcc agt att gat att acc ttt acc gca    28462 Leu Asn Phe Thr Asn Asn Ser Ala Ser Ile Asp Ile Thr Phe Thr Ala 2505               2510                2515                2520 ttt gca gag gat gga cgt aaa ctg ggt tat gaa agt ttc agt att cct    28510 Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro 2525                2530                2535 att acc cgc aag gtg agt act gat aat tcc ctg acc ctg cgc cat aat    28558 Ile Thr Arg Lys Val Ser Thr Asp Asn Ser Leu Thr Leu Arg His Asn 2540                2545                2550 gaa aat ggt gcg caa tat atg caa tgg gga gtc tat cgc att cgt ctt    28606 Glu Asn Gly Ala Gln Tyr Met Gln Trp Gly Val Tyr Arg Ile Arg Leu 2555                2560                2565 aat act tta ttt gct cgc caa tta gtt gcg cga gcc act acc ggt att    28654 Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile 2570                2575                2580 gat acg att ctg agt atg gaa act cag aat att cag gaa cca cag tta    28702 Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu 2585               2590                2595                2600 ggc aaa ggt ttc tac gct acg ttc gtg ata cct ccg tat aac cca tca    28750 Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Pro Ser 2605                2610                2615 act cat ggt gat gaa cgt tgg ttt aag ctt tat atc aaa cat gtt gtt    28798 Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val 2620                2625                2630 gat aat aat tca cat att atc tat tca ggt cag cta aaa gat aca aat    28846 Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Lys Asp Thr Asn 2635                2640                2645 ata agc acc acg tta ttt atc cct ctt gat gat gtt cca ttg aac caa    28894 Ile Ser Thr Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln 2650                2655                2660 gat tac agc gcc aag gtt tac atg acc ttc aag aaa tca cca tca gat    28942 Asp Tyr Ser Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp 2665               2670                2675                2680 ggt acc tgg tgg ggc cct cac ttt gtt aga gat gat aaa gga ata gta    28990 Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val 2685                2690                2695 aca ata aac cct aaa tcc att ttg acc cac ttt gag agc gtc aat gtc    29038 Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val 2700                2705                2710 ctg aat aat att agt agc gaa cca atg gat ttc agc ggc gct aac agc    29086 Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser 2715                2720                2725 ctc tat ttt tgg gaa ctg ttc tac tat acc ccg atg ctg gtt gcc caa    29134 Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln 2730                2735                2740 cgt ttg ttg cat gag caa aac ttt gat gaa gcg aac cgc tgg ctg aaa    29182 Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys 2745               2750                2755                2760 tat gtc tgg agc cca tcc ggg tat att gtt cac ggc cag att cag aat    29230 Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn 2765                2770                2775 tat caa tgg aac gtc cgc ccg tta ttg gaa gat acc agt tgg aac agt    29278 Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser 2780                2785                2790 gat cct ttg gat tcc gtc gat cct gac gcg gta gcg cag cac gat ccg    29326 Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro 2795                2800                2805 atg cac tat aaa gtt tca acc ttt atg cgc acc ctt gat ctg ttg atc    29374 Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile 2810                2815                2820 gcg cgc ggc gac cat gct tac cgc caa ttg gag cgc gat acg ctt aac    29422 Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn 2825               2830                2835                2840 gaa gcg aag atg tgg tat atg caa gcg ctg cat ctg tta ggc gat aaa    29470 Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys 2845                2850                2855 cct tat ctg ccg ctg agt acc aca tgg aat gat cca cga ctg gac aaa    29518 Pro Tyr Leu Pro Leu Ser Thr Thr Trp Asn Asp Pro Arg Leu Asp Lys 2860                2865                2870 gcc gcg gat att act acc caa agt gct cat tcc agc tca ata gtc gct    29566 Ala Ala Asp Ile Thr Thr Gln Ser Ala His Ser Ser Ser Ile Val Ala 2875                2880                2885 ttg cgg cag agt aca ccg gcg ctt tta tca ttg cgc agc gcc aat acc    29614 Leu Arg Gln Ser Thr Pro Ala Leu Leu Ser Leu Arg Ser Ala Asn Thr 2890                2895                2900 ctg acc gat ctc ttc ctg ccg caa atc aat gaa gtg atg atg aat tac    29662 Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr 2905               2910                2915                2920 tgg caa aca tta gct cag aga gta tac aac ctg cgc cac aac ctc tct    29710 Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser 2925                2930                2935 atc gac ggt cag ccg tta tat ctg cca atc tat gcc aca ccg gcg gac    29758 Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp 2940                2945                2950 ccg aaa gcg tta ctc agc gcc gct gtt gcc act tct caa ggt gga ggc    29806 Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly 2955                2960                2965 aag ctg ccg gag tca ttt atg tcc ctg tgg cgt ttc ccg cac atg ctg    29854 Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu 2970                2975                2980 gaa aat gct cgc agc atg gtt agc cag ctc acc caa ttc ggc tcc acg    29902 Glu Asn Ala Arg Ser Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr 2985               2990                2995                3000 tta caa aat att atc gaa cgt cag gac gca gaa gcg ctc aat gcg tta    29950 Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu 3005                3010                3015 tta caa aat cag gcc gca gag ctg ata ttg act aac ctg agt att caa    29998 Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln 3020                3025                3030 gac aaa acc att gaa gaa ctg gat gcc gag aaa acc gtg ctg gaa aaa    30046 Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys 3035                3040                3045 tcc aaa gcg gga gca caa tcg cgc ttt gat agc tat agc aaa ctg cat    30094 Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Ser Lys Leu His 3050                3055                3060 gat gaa aac atc aac gcc ggt gaa aac caa gct atg acg cta cga gcg    30142 Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala 3065               3070                3075                3080 tcc gca gcc ggg ctt acc acg gcg gtt cag gca tcc cgt ctg gcc ggc    30190 Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly 3085                3090                3095 gca gcg gct gat ctg gtg cct aac atc ttc ggc ttc gcc ggt ggt ggt    30238 Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly 3100                3105                3110 agc cgt tgg ggg gct atc gct gag gcg acc ggc tat gta atg gaa ttt    30286 Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe 3115                3120                3125 tcc gct aat gtt atg aat acc gaa gcg gat aaa att agc caa tct gaa    30334 Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu 3130                3135                3140 acc tac cgt cgt cgc cgt cag gag tgg gaa att cag cgt aat aat gcc    30382 Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala 3145               3150                3155                3160 gaa gcg gag ctg aaa caa ctc gat gcc caa ctt aaa tcg ctg gca gta    30430 Glu Ala Glu Leu Lys Gln Leu Asp Ala Gln Leu Lys Ser Leu Ala Val 3165                3170                3175 cgc cgt gaa gcc gcc gta ttg caa aaa acc agc ctg aaa acc caa caa    30478 Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln 3180                3185                3190 gag cag acc caa gcc caa ttg gcc ttc ctg caa cgt aag ttc agc aat    30526 Glu Gln Thr Gln Ala Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn 3195                3200                3205 caa gcg ttg tac aac tgg cta cgt ggc cga ctg gca gca att tac ttc    30574 Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe 3210                3215                3220 caa ttc tac gac ttg gct atc gcg cgt tgt tta atg gca gag cag gct    30622 Gln Phe Tyr Asp Leu Ala Ile Ala Arg Cys Leu Met Ala Glu Gln Ala 3225               3230                3235                3240 tac cgt tgg gaa att agc gat gac tct gct cgc ttt att aaa ccg ggc    30670 Tyr Arg Trp Glu Ile Ser Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly 3245                3250                3255 gcc tgg caa gga acc tat gca ggt ctg ctg gca ggt gaa acc ttg atg    30718 Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met 3260                3265                3270 cta agt ttg gca caa atg gaa gac gcc cat tta aga cgc gat aaa cgc    30766 Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Arg Arg Asp Lys Arg 3275                3280                3285 gca tta gag gtc gaa cgt aca gta tcg ctg gcc gaa att tat gct ggt    30814 Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Ile Tyr Ala Gly 3290                3295                3300 tta ccg caa gat aaa ggc cca ttc tcc ctg acg caa gaa atc gag aag    30862 Leu Pro Gln Asp Lys Gly Pro Phe Ser Leu Thr Gln Glu Ile Glu Lys 3305               3310                3315                3320 ctg gtg aat gca ggt tca ggc agc gcc ggc agt ggt aat aat aat ttg    30910 Leu Val Asn Ala Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu 3325                3330                3335 gca ttt ggc gcc ggc acg gac act aaa act tct ttg cag gca tcc att    30958 Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Ile 3340                3345                3350 tca tta gct gat tta aaa att cgt gag gat tac ccg gaa tct att ggc    31006 Ser Leu Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Glu Ser Ile Gly 3355                3360                3365 aaa atc cga cgc atc aaa cag atc agc gtt acc ctg ccg gcg cta ttg    31054 Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu 3370                3375                3380 gga cct tat cag gat gtg cag gca ata tta tct tac ggc gat aaa gcc    31102 Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala 3385               3390                3395                3400 gga tta gcg aac ggc tgt gca gcg ctg gcc gtt tcc cac ggt acg aat    31150 Gly Leu Ala Asn Gly Cys Ala Ala Leu Ala Val Ser His Gly Thr Asn 3405                3410                3415 gac agc ggt caa ttc cag ctc gat ttc aac gat ggc aaa ttc ctg ccg    31198 Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro 3420                3425                3430 ttt gaa ggt atc gcc att gat caa ggt acg cta aca ctg agt ttt cct    31246 Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro 3435                3440                3445 aat gca tca acg cca gcc aaa ggt aaa caa gcc act atg tta aaa acc    31294 Asn Ala Ser Thr Pro Ala Lys Gly Lys Gln Ala Thr Met Leu Lys Thr 3450                3455                3460 ctg aac gat atc att ttg cat att cgc tac acc att aag taa            31336 Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys 3465               3470                3475 ccatcccaac acagaactaa gacaggcccc gaatcggggt ctggtaagga gtttct atg  31395 Met cag aat tca cag aca ttc agc atg acc gag ctg tca tta cct aag ggc    31443 Gln Asn Ser Gln Thr Phe Ser Met Thr Glu Leu Ser Leu Pro Lys Gly 3480               3485                3490                3495 ggc ggc gcc att acc ggt atg ggt gaa gca tta acg ccg gcc ggg ccg    31491 Gly Gly Ala Ile Thr Gly Met Gly Glu Ala Leu Thr Pro Ala Gly Pro 3500                3505                3510 gat ggt atg gca gcc tta tcg ctg cca ttg ccc att tct gcc gga cgt    31539 Asp Gly Met Ala Ala Leu Ser Leu Pro Leu Pro Ile Ser Ala Gly Arg 3515                3520                3525 ggt tat gcc ccc tcg ctc acg ctg aac tac aac agc gga acc ggt aac    31587 Gly Tyr Ala Pro Ser Leu Thr Leu Asn Tyr Asn Ser Gly Thr Gly Asn 3530                3535                3540 agc ccg ttc ggt ctc ggt tgg gac tgt aac gtc atg aca att cgt cgt    31635 Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val Met Thr Ile Arg Arg 3545                3550                3555 cgc acc agt acc ggc gtg ccg aat tat gat gaa acc gat act ttt ctg    31683 Arg Thr Ser Thr Gly Val Pro Asn Tyr Asp Glu Thr Asp Thr Phe Leu 3560               3565                3570                3575 ggg ccg gaa ggt gaa gtg ttg gtc gta gca tta aat gag gca ggt caa    31731 Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu Asn Glu Ala Gly Gln 3580                3585                3590 gct gat atc cgc agt gaa tcc tca tta cag ggc atc aat ttg ggg atg    31779 Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly Ile Asn Leu Gly Met 3595                3600                3605 acc ttc acc gtt acc ggt tat cgc tcc cgt ttg gaa agc cac ttt agc    31827 Thr Phe Thr Val Thr Gly Tyr Arg Ser Arg Leu Glu Ser His Phe Ser 3610                3615                3620 cgg ttg gaa tac tgg caa ccc caa aca aca ggc gca acc gat ttc tgg    31875 Arg Leu Glu Tyr Trp Gln Pro Gln Thr Thr Gly Ala Thr Asp Phe Trp 3625                3630                3635 ctg ata tac agc ccc gac gga caa gcc cat tta ctg ggc aaa aat cct    31923 Leu Ile Tyr Ser Pro Asp Gly Gln Ala His Leu Leu Gly Lys Asn Pro 3640               3645                3650                3655 caa gca cgc atc agc aat cca cta aat gtt aac caa aca gcg caa tgg    31971 Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn Gln Thr Ala Gln Trp 3660                3665                3670 cta ttg gaa gcc tcg gta tca tcc cac ggc gag cag att tat tat cag    32019 Leu Leu Glu Ala Ser Val Ser Ser His Gly Glu Gln Ile Tyr Tyr Gln 3675                3680                3685 tat cga gcc gaa gat gaa act gat tgc gaa act gac gaa ctc aca gcc    32067 Tyr Arg Ala Glu Asp Glu Thr Asp Cys Glu Thr Asp Glu Leu Thr Ala 3690                3695                3700 cac ccg aac aca acc gtc cag cgc tac ctg caa gta gta cat tac ggt    32115 His Pro Asn Thr Thr Val Gln Arg Tyr Leu Gln Val Val His Tyr Gly 3705                3710                3715 aat cta acc gcc agc gaa gta ttt ccc acg cta aat gga gat gat cca    32163 Asn Leu Thr Ala Ser Glu Val Phe Pro Thr Leu Asn Gly Asp Asp Pro 3720               3725                3730                3735 ctc aaa tct ggc tgg ttg ttc tgt tta gta ttt gat tac ggt gag cgc    32211 Leu Lys Ser Gly Trp Leu Phe Cys Leu Val Phe Asp Tyr Gly Glu Arg 3740                3745                3750 aaa aac agc tta tct gaa atg ccg cca ttt aaa gcc aca agt aac tgg    32259 Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys Ala Thr Ser Asn Trp 3755                3760                3765 ctt tgc cgc aaa gac cgt ttt tcc cgt tat gaa tac ggt ttt gca ttg    32307 Leu Cys Arg Lys Asp Arg Phe Ser Arg Tyr Glu Tyr Gly Phe Ala Leu 3770                3775                3780 cgc acc cgg cgc tta tgt cgc caa ata ctg atg ttt cac cgt ctg caa    32355 Arg Thr Arg Arg Leu Cys Arg Gln Ile Leu Met Phe His Arg Leu Gln 3785                3790                3795 acc ctg tct ggt cag gca aaa ggc gac gat gaa ccc gca tta gtt tca    32403 Thr Leu Ser Gly Gln Ala Lys Gly Asp Asp Glu Pro Ala Leu Val Ser 3800               3805                3810                3815 cgt ctg ata ctg gat tat gac gaa aac gcg gtg gtc agt acg ctc gtt    32451 Arg Leu Ile Leu Asp Tyr Asp Glu Asn Ala Val Val Ser Thr Leu Val 3820                3825                3830 tct gtc cgc cga gtg gga cat gag caa gat ggc aca acg gcg gtc gcc    32499 Ser Val Arg Arg Val Gly His Glu Gln Asp Gly Thr Thr Ala Val Ala 3835                3840                3845 ctg ccg cca ttg gaa ctg gct tat cag cct ttt gaa cca gaa caa aaa    32547 Leu Pro Pro Leu Glu Leu Ala Tyr Gln Pro Phe Glu Pro Glu Gln Lys 3850                3855                3860 gca ctc tgg cga cca atg gat gta ctg gcg aat ttc aac acc atc caa    32595 Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn Phe Asn Thr Ile Gln 3865                3870                3875 cgc tgg caa ctg ctt gat ctg caa ggc gaa ggc gta ccc ggt att ctg    32643 Arg Trp Gln Leu Leu Asp Leu Gln Gly Glu Gly Val Pro Gly Ile Leu 3880               3885                3890                3895 tat cag gat aaa aat ggc tgg tgg tat cga tct gct caa cgt cag aca    32691 Tyr Gln Asp Lys Asn Gly Trp Trp Tyr Arg Ser Ala Gln Arg Gln Thr 3900                3905                3910 ggg gaa gag atg aat gcg gtc acc tgg ggc aaa atg caa ctc ctt cct    32739 Gly Glu Glu Met Asn Ala Val Thr Trp Gly Lys Met Gln Leu Leu Pro 3915                3920                3925 atc acg ccc gct att cag gat aac gcc tca ctg atg gat att aat ggt    32787 Ile Thr Pro Ala Ile Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly 3930                3935                3940 gat ggg caa ctg gat tgg gtt atc acc ggt ccg ggg cta agg ggt tat    32835 Asp Gly Gln Leu Asp Trp Val Ile Thr Gly Pro Gly Leu Arg Gly Tyr 3945                3950                3955 cac agc cag cat cca gat ggc agt tgg aca cgt ttt acg ccg ttg cac    32883 His Ser Gln His Pro Asp Gly Ser Trp Thr Arg Phe Thr Pro Leu His 3960               3965                3970                3975 gcc tta ccg ata gaa tat acc cat ccc cgc gcc caa ctt gcg gat tta    32931 Ala Leu Pro Ile Glu Tyr Thr His Pro Arg Ala Gln Leu Ala Asp Leu 3980                3985                3990 atg ggg gcc ggg ctg tcc gat tta gtg ctg att ggt ccc aaa agc gtg    32979 Met Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val 3995                4000                4005 cgt ttg tat gcc aat aac cgt gat ggt ttt acc gaa gga cgg gat gtg    33027 Arg Leu Tyr Ala Asn Asn Arg Asp Gly Phe Thr Glu Gly Arg Asp Val 4010                4015                4020 gtg caa tcc ggt ggt atc acc ctg ccg tta ccg ggc gcc gat gcg cgt    33075 Val Gln Ser Gly Gly Ile Thr Leu Pro Leu Pro Gly Ala Asp Ala Arg 4025                4030                4035 aag tta gtg gcc ttt agc gac gta ctc ggt tca ggc caa gca cat ttg    33123 Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser Gly Gln Ala His Leu 4040               4045                4050                4055 gtt gaa gtt agt gcg acg aaa gtc acc tgc tgg cca aat ctg gga cat    33171 Val Glu Val Ser Ala Thr Lys Val Thr Cys Trp Pro Asn Leu Gly His 4060                4065                4070 ggc cgt ttt ggt cag cca atc aca ttg ccg gga ttt agc caa tcc gcc    33219 Gly Arg Phe Gly Gln Pro Ile Thr Leu Pro Gly Phe Ser Gln Ser Ala 4075                4080                4085 gcc aat ttt aat cct gat cga gtt cat ctg gcc gat ctg gac ggt agt    33267 Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala Asp Leu Asp Gly Ser 4090                4095                4100 ggt cct gcc gat ctg att tat gtt cat gct gac cat ctg gat att ttc    33315 Gly Pro Ala Asp Leu Ile Tyr Val His Ala Asp His Leu Asp Ile Phe 4105                4110                4115 agc aat gaa agt ggt aac ggt ttt gca caa cca ttc aca ctc cgt ttt    33363 Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro Phe Thr Leu Arg Phe 4120               4125                4130                4135 cct gac ggc ctg cgt ttt gat gat act tgc cag cta caa gtg gct gat    33411 Pro Asp Gly Leu Arg Phe Asp Asp Thr Cys Gln Leu Gln Val Ala Asp 4140                4145                4150 gta cag gga tta ggg gtt gtc agc ctg atc ctg agc gta ccg cat atg    33459 Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu Ser Val Pro His Met 4155                4160                4165 gcg cca cac cat tgg cgc tgc gat ctg acc aac gcg aaa ccg tgg tta    33507 Ala Pro His His Trp Arg Cys Asp Leu Thr Asn Ala Lys Pro Trp Leu 4170                4175                4180 ctc agt gaa atg aac aac aac atg gga gcc cat cac acc ctg cat tac    33555 Leu Ser Glu Met Asn Asn Asn Met Gly Ala His His Thr Leu His Tyr 4185                4190                4195 cgt agc tcc gtc cag ttt tgg ctg gat gaa aaa gcc gca gcc tta gct    33603 Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys Ala Ala Ala Leu Ala 4200               4205                4210                4215 acc gga caa aca ccg gtc tgt tac ctg ccc ttc ccg gtc cat acc ctg    33651 Thr Gly Gln Thr Pro Val Cys Tyr Leu Pro Phe Pro Val His Thr Leu 4220                4225                4230 tgg caa aca gaa acc gag gat gaa atc agc ggc aat aaa tta gtg acc    33699 Trp Gln Thr Glu Thr Glu Asp Glu Ile Ser Gly Asn Lys Leu Val Thr 4235                4240                4245 act tta cgt tac gct cac ggc gcc tgg gat gga cgt gag cgg gaa ttt    33747 Thr Leu Arg Tyr Ala His Gly Ala Trp Asp Gly Arg Glu Arg Glu Phe 4250                4255                4260 cgc ggc ttt ggc tat gtt gag cag aca gac agc cat caa ctg gct caa    33795 Arg Gly Phe Gly Tyr Val Glu Gln Thr Asp Ser His Gln Leu Ala Gln 4265                4270                4275 ggc aat gcg ccg gaa cgt aca tca ccg gca ctt acc aaa aac tgg tat    33843 Gly Asn Ala Pro Glu Arg Thr Ser Pro Ala Leu Thr Lys Asn Trp Tyr 4280               4285                4290                4295 gcc acc gga atc cct gag gta gac aat acg cta tct gcc ggg tat tgg    33891 Ala Thr Gly Ile Pro Glu Val Asp Asn Thr Leu Ser Ala Gly Tyr Trp 4300                4305                4310 cgc ggt gat acg cag gct ttc act ggt ttt acg cca cac ttt act ctc    33939 Arg Gly Asp Thr Gln Ala Phe Thr Gly Phe Thr Pro His Phe Thr Leu 4315                4320                4325 tgg aaa gag ggc aaa gat gtt cca ctg aca ccg gaa gat gac cac aat    33987 Trp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro Glu Asp Asp His Asn 4330                4335                4340 ctg tac tgg tta aac cgg gca cta aaa ggt caa cca ctg cgt agt gaa    34035 Leu Tyr Trp Leu Asn Arg Ala Leu Lys Gly Gln Pro Leu Arg Ser Glu 4345                4350                4355 ctc tac ggg cta gat ggc agc gca cag cag aag atc ccc tat aca gtg    34083 Leu Tyr Gly Leu Asp Gly Ser Ala Gln Gln Lys Ile Pro Tyr Thr Val 4360               4365                4370                4375 act gaa tcc cgc cca caa gtg cgc caa tta caa gat aac act acc ctt    34131 Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln Asp Asn Thr Thr Leu 4380                4385                4390 tcc ccg gtg ctc tgg gcc tca gtg gtg gaa agt cgt agt tat cac tat    34179 Ser Pro Val Leu Trp Ala Ser Val Val Glu Ser Arg Ser Tyr His Tyr 4395                4400                4405 gaa cgt atc atc agc gat ccc caa tgc aat cag gat atc act ctg tcc    34227 Glu Arg Ile Ile Ser Asp Pro Gln Cys Asn Gln Asp Ile Thr Leu Ser 4410                4415                4420 agt gac cta ttc ggg caa ccg ctg aaa cag gtt tca gtg caa tat ccc    34275 Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val Ser Val Gln Tyr Pro 4425                4430                4435 cgc cgc aat aaa cca aca acc aat ccg tat ccc gat aca cta cca gat    34323 Arg Arg Asn Lys Pro Thr Thr Asn Pro Tyr Pro Asp Thr Leu Pro Asp 4440               4445                4450                4455 act ctg ttt gcc agc agt tat gac gac caa caa caa cta ttg cgg tta    34371 Thr Leu Phe Ala Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg Leu 4460                4465                4470 acc tac cag caa tcc agt tgg cat cat cta att gct aat gaa ctc aga    34419 Thr Tyr Gln Gln Ser Ser Trp His His Leu Ile Ala Asn Glu Leu Arg 4475                4480                4485 gtg tta gga tta ccg gat ggt aca cgc agt gat gct ttc act tac gat    34467 Val Leu Gly Leu Pro Asp Gly Thr Arg Ser Asp Ala Phe Thr Tyr Asp 4490                4495                4500 gct aaa cac gtg cct gtt gat ggt tta aat ctg gaa gct cta tgt gct    34515 Ala Lys His Val Pro Val Asp Gly Leu Asn Leu Glu Ala Leu Cys Ala 4505                4510                4515 gaa aat agc ctg att gcc gat gat aaa cct cgc gaa tac ctc aac cag    34563 Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg Glu Tyr Leu Asn Gln 4520               4525                4530                4535 caa cga acg ttc tat acc gat ggg aaa acc gat gga aaa aat cca acg    34611 Gln Arg Thr Phe Tyr Thr Asp Gly Lys Thr Asp Gly Lys Asn Pro Thr 4540                4545                4550 cca ctg aaa aca ccg aca cga cag gct tta atc gcc ttt acc gaa acg    34659 Pro Leu Lys Thr Pro Thr Arg Gln Ala Leu Ile Ala Phe Thr Glu Thr 4555                4560                4565 gcg gta tta acg gaa tct ctg tta tcc gca ttt gat ggc ggt atc acg    34707 Ala Val Leu Thr Glu Ser Leu Leu Ser Ala Phe Asp Gly Gly Ile Thr 4570                4575                4580 cca gat gaa tta ccc ggc ctt ctg aca caa gca gga tac caa caa gaa    34755 Pro Asp Glu Leu Pro Gly Leu Leu Thr Gln Ala Gly Tyr Gln Gln Glu 4585                4590                4595 cct tat ctg ttc cca ctc agt ggc gaa aac caa gtc tgg gta gca cgc    34803 Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln Val Trp Val Ala Arg 4600               4605                4610                4615 aaa ggc tat acc gat tac gga act gag gta caa ttt tgg cgt cct gtc    34851 Lys Gly Tyr Thr Asp Tyr Gly Thr Glu Val Gln Phe Trp Arg Pro Val 4620                4625                4630 gca caa cgt aac acc cag tta acc ggg aaa acg act cta aaa tgg gat    34899 Ala Gln Arg Asn Thr Gln Leu Thr Gly Lys Thr Thr Leu Lys Trp Asp 4635                4640                4645 acc cac tac tgt gtc atc act caa acc caa gac gcg gct ggt ttg act    34947 Thr His Tyr Cys Val Ile Thr Gln Thr Gln Asp Ala Ala Gly Leu Thr 4650                4655                4660 gtc tca gcc aat tat gac tgg cgt ttt ctc aca cct atg caa ctg act    34995 Val Ser Ala Asn Tyr Asp Trp Arg Phe Leu Thr Pro Met Gln Leu Thr 4665                4670                4675 gat atc aac gat aat gtg cat atc ata acc ttg gat gcg cta gga cgc    35043 Asp Ile Asn Asp Asn Val His Ile Ile Thr Leu Asp Ala Leu Gly Arg 4680               4685                4690                4695 cct gtc act caa cgt ttc tgg gga atc gaa aat ggt gtg gca aca ggt    35091 Pro Val Thr Gln Arg Phe Trp Gly Ile Glu Asn Gly Val Ala Thr Gly 4700                4705                4710 tac tct tca cca gaa gca aaa cca ttc act cca cca gtc gat gtc aat    35139 Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro Pro Val Asp Val Asn 4715                4720                4725 gct gcc att gct ctg acc gga cca ctc cct gtc gcg cag tgt ctg gtc    35187 Ala Ala Ile Ala Leu Thr Gly Pro Leu Pro Val Ala Gln Cys Leu Val 4730                4735                4740 tat gcg ccg gac agt tgg atg ccg cta ttc ggt cag gaa acc ttc aac    35235 Tyr Ala Pro Asp Ser Trp Met Pro Leu Phe Gly Gln Glu Thr Phe Asn 4745                4750                4755 aca tta acg cag gaa gag caa aag aca ctg cgt gat tta cgg att atc    35283 Thr Leu Thr Gln Glu Glu Gln Lys Thr Leu Arg Asp Leu Arg Ile Ile 4760               4765                4770                4775 aca gaa gat tgg cgt att tgc gca ctg gct cgc cgc cgt tgg cta caa    35331 Thr Glu Asp Trp Arg Ile Cys Ala Leu Ala Arg Arg Arg Trp Leu Gln 4780                4785                4790 agt caa aaa gcc ggc aca cca ttg gtt aag ctg tta acc aac agc atc    35379 Ser Gln Lys Ala Gly Thr Pro Leu Val Lys Leu Leu Thr Asn Ser Ile 4795                4800                4805 ggt tta cct ccc cac aac ctc atg ctg gct acg gac cgt tat gac cgt    35427 Gly Leu Pro Pro His Asn Leu Met Leu Ala Thr Asp Arg Tyr Asp Arg 4810                4815                4820 gat tct gaa cag caa att cgt caa caa gtc gca ttc agt gat ggt ttt    35475 Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala Phe Ser Asp Gly Phe 4825                4830                4835 ggc cgt ttg ttg caa gcg gct gtg cgg cat gag gca ggc gaa gcc tgg    35523 Gly Arg Leu Leu Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala Trp 4840               4845                4850                4855 caa cgt aac caa gac ggt tct ctg gtg aca aaa atg gaa gat acc aaa    35571 Gln Arg Asn Gln Asp Gly Ser Leu Val Thr Lys Met Glu Asp Thr Lys 4860                4865                4870 acg cgc tgg gcg att acg gga cgc act gaa tat gac aat aag ggg cag    35619 Thr Arg Trp Ala Ile Thr Gly Arg Thr Glu Tyr Asp Asn Lys Gly Gln 4875                4880                4885 gcg ata cga act tat cag ccc tat ttc ctc aat gac tgg cga tat gtg    35667 Ala Ile Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val 4890                4895                4900 agt gat gac agc gcc aga aaa gag gcc tat gcc gat act cat atc tat    35715 Ser Asp Asp Ser Ala Arg Lys Glu Ala Tyr Ala Asp Thr His Ile Tyr 4905                4910                4915 gat ccg att ggg cgg gaa atc caa gtt atc acg gca aaa ggc tgg ctg    35763 Asp Pro Ile Gly Arg Glu Ile Gln Val Ile Thr Ala Lys Gly Trp Leu 4920               4925                4930                4935 cgg cag aac caa tat ttc ccg tgg ttt acc gtg agt gaa gat gaa aat    35811 Arg Gln Asn Gln Tyr Phe Pro Trp Phe Thr Val Ser Glu Asp Glu Asn 4940                4945                4950 gat ttg tcc gct gac gcg ctc gtg taa ttgaatcaag attcgctcgt          35858 Asp Leu Ser Ala Asp Ala Leu Val 4955                4960 ttaatgttaa cgagcgaata taatatacct aatagatttc gagttgcagc gcggcggcaa  35918 gtgaacgaat ccccaggagc atagataact atgtgactgg ggtgagtgaa agcagccaac  35978 aaagcagcag cttgaaagat gaagggtata aataagaaac tgcattgtga gttctaaata  36038 gagtagcagc atattttatt gccttttatt tcataggtaa taaaattcaa ttgctgtaaa  36098 aatctgtcat catgagaact aaaaataaca actttctctt ctgcaagaga aatcaataat  36158 tcaattaaaa atgttataga atctgaatca agaccatttg ttggctcatc aaaaatataa  36218 acatccgcat cggtaataaa agctgatgtc aatagaaatt tcttttttat cccaagtgac  36278 atatgtccat actcaatacc agaataatta gatataccaa aaccatttaa atagtaatct  36338 aattgatatt ttaaattact tttcctataa cgctgactta aattaatcac atccattccc  36398 gtgatgaaat tataaaagtt aacattatcc gatagataaa aaccatgctg ttgcaaatta  36458 aatcggctct tttctccctt ttttataaaa ttaaccattc cttttttaac cttatttaca  36518 ccagcaatac ttgaaagaaa agtcgtttta cccgccccat taactcccgc aatacggttt  36578 aatccaaccc gaaaatcaca attgactcct gaaaaaatag tcttaccatt aataacaacc  36638 tctaacccaa taacttcaag cataaataac ccctaaaaat aacgtaaaaa agaaaataac  36698 accaacaata ataattttcg tgtattgcgt tctcaacaga gaaatagaag aaacaataat  36758 agaaagaaaa gcataagata aaaatataat cacaggaaaa gatttaacaa caagaaagca  36818 aaaaataaaa aaacaaagca aataaaaaaa caaagaaata ccataattaa aaaagaatat  36878 tttccgcaca gataaaaagt tggacaaata tgaaagataa tttatttcaa tatatgatag  36938 attataaaat aacaacatgc atatatataa aacaacactg gcatatatta atgatatata  36998 atcagccttg tttgggattt gagaaaaggc actttcacat aatagatata aaagcagaac  37058 agataatgcc ataataagca cagacatttt atttttatta aaacaataac gaagattcat  37118 tatataaggc aatgaaaaaa aacctgatga aaataatttt ttatttctat taattatata  37178 acatggtgtg aaattcaaat ataatatcaa tgctactaat ggaataacta atgtaaaaat  37238 caaatcatat aatattccac tcctgaatga tgccgccaga agaaagaaca cagcaacaat  37298 aaaaaaatgc aaaaaactta attcaaataa gcaaaatcca attacagcaa aagaaactat  37358 caaaaaaaac acagatgaaa ggtaatgcaa ataattaaca ttttcgtaaa aaaaacctat  37418 aaagaagaaa ataactatcg gaaaagcact ataaataaaa aaaacgatac gactaaaaaa  37478 caacgttttt ttacctacca aagaaacgat gattgaattc tcctttgcag aaggaaaaaa  37538 ccttatgtta atcaaataaa ataccatata taccattaaa gatatggcag taaaataaaa  37598 tgattttatg tagccatctg gaataataat attggaagat aaagttatta aaacctcaaa  37658 gataccactg aactttgccg gaagtaataa aagaaaaagg aatataatga catttttatt  37718 cccagacgca aatttcttta tcctaccttt atattccaag gcatcagcga ttattaaatt  37778 catactgcct ctctaaaacc aaaatctaaa taatgtcctt ggtgaatctt tagggaattt  37838 cgtcctggaa tgcaaatata aatagttact gaaaacaata cattgatttt taattaaata  37898 ctggcgatat gaccttaatg atgctacttt attttccagt attcaattcg             37948 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 954 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  12 Met Lys Asn Ile Asp Pro Lys Leu Tyr Gln Lys Thr Pro Val Val Asn 1               5                  10                  15 Ile Tyr Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His Arg 20                  25                  30 Thr Thr Ala Asn Gly Asp Thr Asp Ile Arg Ile Thr Arg His Gln Tyr 35                  40                  45 Asp Ser Leu Gly His Leu Ser Gln Ser Thr Asp Pro Arg Leu Tyr Glu 50                  55                  60 Ala Lys Gln Lys Ser Asn Phe Leu Trp Gln Tyr Asp Leu Thr Gly Asn 65                  70                  75                  80 Ile Leu Cys Thr Glu Ser Val Asp Ala Gly Arg Thr Val Thr Leu Asn 85                  90                  95 Asp Ile Glu Gly Arg Pro Leu Leu Thr Val Thr Ala Thr Gly Val Ile 100                 105                 110 Gln Thr Arg Gln Tyr Glu Thr Ser Ser Leu Pro Gly Arg Leu Leu Ser 115                 120                 125 Val Thr Glu Gln Ile Pro Glu Lys Thr Ser Arg Ile Thr Glu Arg Leu 130                 135                 140 Ile Trp Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser 145                 150                 155                 160 Gln Cys Val Arg His Tyr Asp Thr Ala Gly Val Thr Arg Leu Glu Ser 165                 170                 175 Leu Ser Leu Thr Gly Thr Val Leu Ser Gln Ser Ser Gln Leu Leu Ser 180                 185                 190 Asp Thr Gln Glu Ala Ser Trp Thr Gly Asp Asn Glu Thr Val Trp Gln 195                 200                 205 Asn Met Leu Ala Asp Asp Ile Tyr Thr Thr Leu Ser Ala Phe Asp Ala 210                 215                 220 Thr Gly Ala Leu Leu Thr Gln Thr Asp Ala Lys Gly Asn Ile Gln Arg 225                 230                 235                 240 Leu Thr Tyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp Leu Thr Leu 245                 250                 255 Lys Asp Gln Pro Glu Gln Val Ile Ile Arg Ser Leu Thr Tyr Ser Ala 260                 265                 270 Ala Gly Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val Ile Thr Glu 275                 280                 285 Tyr Ser Tyr Glu Pro Glu Thr Gln Gln Leu Ile Gly Thr Lys Thr His 290                 295                 300 Arg Pro Ser Asp Ala Lys Val Leu Gln Asp Leu Arg Tyr Glu Tyr Asp 305                 310                 315                 320 Pro Val Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu Ala Thr Arg 325                 330                 335 Phe Trp His Asn Gln Lys Val Ala Pro Glu Asn Thr Tyr Thr Tyr Asp 340                 345                 350 Ser Leu Tyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met Ala Asn Ile 355                 360                 365 Gly Gln Gln Ser Asn Gln Leu Pro Ser Leu Thr Leu Pro Ser Asp Asn 370                 375                 380 Asn Thr Tyr Thr Asn Tyr Thr Arg Thr Tyr Thr Tyr Asp Arg Gly Gly 385                 390                 395                 400 Asn Leu Thr Lys Ile Gln His Ser Ser Pro Ala Thr Gln Asn Asn Tyr 405                 410                 415 Thr Thr Asn Ile Thr Val Ser Asn Arg Ser Asn Arg Ala Val Leu Ser 420                 425                 430 Thr Leu Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe Asp Ala Gly 435                 440                 445 Gly His Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn Trp Asn Thr 450                 455                 460 Arg Gly Glu Leu Gln His Val Thr Leu Val Lys Arg Asp Lys Gly Ala 465                 470                 475                 480 Asn Asp Asp Arg Glu Trp Tyr Arg Tyr Ser Ser Asp Gly Arg Arg Ile 485                 490                 495 Leu Lys Ile Asn Glu Gln Gln Thr Ser Ser Asn Ser Gln Thr Gln Arg 500                 505                 510 Ile Thr Tyr Leu Pro Ser Leu Glu Leu Arg Leu Thr Gln Asn Ser Thr 515                 520                 525 Ile Thr Thr Glu Asp Leu Gln Val Ile Thr Val Gly Glu Ala Gly Arg 530                 535                 540 Ala Gln Val Arg Val Leu His Trp Asp Ser Gly Gln Pro Glu Asp Ile 545                 550                 555                 560 Asp Asn Asn Gln Leu Arg Tyr Ser Tyr Asp Asn Leu Ile Gly Ser Ser 565                 570                 575 Gln Leu Glu Leu Asp Ser Lys Gly Glu Ile Ile Ser Glu Glu Glu Tyr 580                 585                 590 Tyr Pro Tyr Gly Gly Thr Ala Leu Trp Ala Thr Arg Lys Arg Thr Glu 595                 600                 605 Ala Ser Tyr Lys Thr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr 610                 615                 620 Gly Leu Tyr Tyr Tyr Gly Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg 625                 630                 635                 640 Trp Leu Ser Ala Asp Pro Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr 645                 650                 655 Arg Met Val Arg Asn Asn Pro Val Thr Leu Leu Asp Pro Asp Gly Leu 660                 665                 670 Met Pro Thr Ile Ala Glu Arg Ile Ala Ala Leu Gln Lys Asn Lys Val 675                 680                 685 Ala Asp Ser Ala Pro Ser Pro Thr Asn Ala Thr Asn Val Ala Ile Asn 690                 695                 700 Ile Arg Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys Ala Ser Thr 705                 710                 715                 720 Ser Ser Gln Ser Thr Thr Tyr Pro Ile Lys Ser Ala Ser Ile Lys Pro 725                 730                 735 Thr Thr Ser Gly Ser Ser Ile Thr Ala Pro Leu Ser Pro Val Gly Asn 740                 745                 750 Lys Ser Thr Pro Glu Ile Ser Leu Pro Glu Ser Thr Gln Ser Asn Ser 755                 760                 765 Ser Ser Ala Ile Ser Thr Asn Leu Gln Lys Lys Ser Phe Thr Leu Tyr 770                 775                 780 Arg Ala Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys Phe Pro Glu 785                 790                 795                 800 Gly Phe Lys Ala Trp Thr Pro Leu Asp Thr Lys Met Ala Arg Gln Phe 805                 810                 815 Ala Ser Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu Pro Lys Glu 820                 825                 830 Thr Val Lys Asn Ile Asn Thr Trp Gly Thr Lys Pro Lys Leu Asn Asp 835                 840                 845 Leu Ser Thr Tyr Ile Lys Tyr Thr Lys Asp Lys Ser Thr Val Trp Val 850                 855                 860 Ser Thr Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser Gly Ala Pro 865                 870                 875                 880 Leu His Glu Ile Asn Met Asp Leu Tyr Glu Phe Thr Ile Asp Gly Gln 885                 890                 895 Lys Leu Asn Pro Leu Pro Arg Gly Arg Ser Lys Asp Arg Val Pro Ser 900                 905                 910 Leu Leu Leu Asp Thr Pro Glu Ile Glu Thr Ala Ser Ile Ile Ala Leu 915                 920                 925 Asn His Gly Pro Val Asn Asp Ala Glu Val Ser Phe Leu Thr Thr Ile 930                 935                 940 Pro Leu Lys Asn Val Lys Pro Tyr Lys Arg 945                 950 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211> LENGTH: 2522 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  13 Met Ile Leu Lys Gly Ile Asn Met Asn Ser Pro Val Lys Glu Ile Pro 1               5                  10                  15 Asp Val Leu Lys Ile Gln Cys Gly Phe Gln Cys Leu Thr Asp Ile Ser 20                  25                  30 His Ser Ser Phe Asn Glu Phe His Gln Gln Val Ser Glu His Leu Ser 35                  40                  45 Trp Ser Glu Ala His Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys 50                  55                  60 Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Thr Asn Pro Gln 65                  70                  75                  80 Leu Gln Asn Ala Val His Leu Ala Ile Val Ala Pro Asn Ala Glu Leu 85                  90                  95 Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala 100                 105                 110 Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu 115                 120                 125 Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Arg 130                 135                 140 Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln 145                 150                 155                 160 Asn Met Asp Thr Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu 165                 170                 175 Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Asp Asn Tyr Thr Gln Val 180                 185                 190 Met Glu Met Leu Ser Ala Phe Arg Pro Ser Gly Ala Thr Pro Tyr His 195                 200                 205 Asp Ala Tyr Glu Asn Val Arg Lys Val Ile Gln Leu Gln Asp Pro Gly 210                 215                 220 Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln 225                 230                 235                 240 Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn 245                 250                 255 Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys 260                 265                 270 Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr 275                 280                 285 Leu Arg Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile 290                 295                 300 Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu 305                 310                 315                 320 Ile Thr Pro Ile Val Asn Ser Asn Asp Gly Thr Val Lys Val Tyr Arg 325                 330                 335 Ile Thr Arg Glu Tyr Thr Thr Asn Ala Asn Gln Val Asp Val Glu Leu 340                 345                 350 Phe Pro Tyr Gly Gly Glu Asn Tyr Gln Leu Asn Tyr Lys Phe Lys Asp 355                 360                 365 Ser Arg Gln Asp Val Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg 370                 375                 380 Glu Leu Ile Arg Ile Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser 385                 390                 395                 400 Glu His Ile Thr Leu Ser Thr Thr Asp Ile Ser Gln Pro Phe Glu Ile 405                 410                 415 Gly Leu Thr Arg Val Tyr Pro Ser Ser Ser Trp Ala Tyr Ala Ala Ala 420                 425                 430 Lys Phe Thr Ile Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu 435                 440                 445 Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile 450                 455                 460 Leu Glu Ser Ile Val Arg Ser Val Asn Gln Gln Leu Asp Ile Asn Ala 465                 470                 475                 480 Glu Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr 485                 490                 495 Ala Ile Asn Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Leu Ile Ser 500                 505                 510 Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn 515                 520                 525 Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile 530                 535                 540 Asp Leu Asn Pro Gly Ser Thr Gly Asp Trp Arg Lys Ser Val Leu Lys 545                 550                 555                 560 Arg Ala Phe Asn Ile Asp Asp Ile Ser Leu Tyr Arg Leu Leu Lys Ile 565                 570                 575 Thr Asn His Asn Asn Gln Asp Gly Lys Ile Lys Asn Asn Leu Asn Asn 580                 585                 590 Leu Ser Asp Leu Tyr Ile Gly Lys Leu Leu Ala Glu Ile His Gln Leu 595                 600                 605 Thr Ile Asp Glu Leu Asp Leu Leu Leu Val Ala Val Gly Glu Gly Glu 610                 615                 620 Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Ala Leu Ile Arg 625                 630                 635                 640 Lys Leu Asn Thr Ile Thr Val Trp Leu Gln Thr Gln Lys Trp Ser Ala 645                 650                 655 Phe Gln Leu Phe Val Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr 660                 665                 670 Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly 675                 680                 685 Phe Asp Lys Asp Lys Ala Asn Leu Leu His Val Met Ala Pro Tyr Ile 690                 695                 700 Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu 705                 710                 715                 720 Leu Trp Ala Asp Lys Leu Lys Pro Gly Asp Gly Ala Met Thr Ala Glu 725                 730                 735 Lys Phe Trp Asp Trp Leu Asn Thr Gln Tyr Thr Pro Asp Ser Ser Glu 740                 745                 750 Val Leu Ala Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala 755                 760                 765 Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe 770                 775                 780 Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ser Ser Thr Glu Ala 785                 790                 795                 800 Val Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala 805                 810                 815 Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala 820                 825                 830 Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn 835                 840                 845 Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Thr Gln Ala Gln Asn His 850                 855                 860 Gln His Leu Pro Pro Val Thr Gln Lys Asn Ala Phe Ser Cys Trp Thr 865                 870                 875                 880 Ser Ile Asp Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn 885                 890                 895 Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln 900                 905                 910 Leu Asn Gln Lys Ile Pro Thr Tyr Ala Gln Trp Glu Ser Ala Gly Glu 915                 920                 925 Ile Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asp Ile Leu His Ala 930                 935                 940 Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg 945                 950                 955                 960 Gln Val Ala Lys Pro Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr 965                 970                 975 Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr 980                 985                 990 Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Thr 995                1000                1005 Leu Glu Asn Val Glu Glu Asn Ala His Ser Gly Val Ile Ser Arg Gln 1010                1015                1020 Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala 1025                1030                1035                1040 Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr 1045                1050                1055 Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val 1060                1065                1070 Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser 1075                1080                1085 Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala 1090                1095                1100 Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly 1105                1110                1115                1120 Leu Ser Glu Thr Asp Thr Gly Glu Tyr Tyr Trp Arg Ser Val Asp His 1125                1130                1135 Ser Lys Phe Ser Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp 1140                1145                1150 His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Arg Ser Thr Ile Arg Pro 1155                1160                1165 Val Met Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu 1170                1175                1180 Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr 1185                1190                1195                1200 Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr 1205                1210                1215 Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Glu Lys Ile Ser Lys Leu 1220                1225                1230 Glu Leu Ala Lys Asn Lys Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln 1235                1240                1245 Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu 1250                1255                1260 Asp Ser Tyr Lys Thr Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp 1265                1270                1275                1280 Met Glu Tyr Lys Asp Met Thr Asp Gly Gln Tyr Lys Ser Tyr Arg Asp 1285                1290                1295 Asn Ser Tyr Lys Gln Phe Asp Thr Asn Ser Val Arg Arg Val Asn Asn 1300                1305                1310 Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Asn Ser Arg Lys 1315                1320                1325 Gly Tyr Asp Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp 1330                1335                1340 Ile Pro Thr Ile Ser Tyr Lys Ala Thr Ser Ser Asp Leu Lys Ile Tyr 1345                1350                1355                1360 Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Gln 1365                1370                1375 Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys 1380                1385                1390 Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn 1395                1400                1405 Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Asn Gly Asn Val Ser Gly 1410                1415                1420 Leu Ser Gln Gly Arg Leu Leu Phe His Arg Asp Thr Asn Tyr Ser Ser 1425                1430                1435                1440 Lys Val Glu Ala Trp Ile Pro Gly Ala Gly Arg Ser Leu Thr Asn Pro 1445                1450                1455 Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro 1460                1465                1470 Asn Asp Leu Lys Gln Tyr Val Tyr Met Thr Asp Ser Lys Gly Thr Ala 1475                1480                1485 Thr Asp Val Ser Gly Pro Val Asp Ile Asn Thr Ala Ile Ser Pro Ala 1490                1495                1500 Lys Val Gln Val Thr Val Lys Ala Gly Ser Lys Glu Gln Thr Phe Thr 1505                1510                1515                1520 Ala Asp Lys Asn Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met 1525                1530                1535 Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Ser Leu Asn Phe 1540                1545                1550 Thr Asn Asn Ser Ala Ser Ile Asp Ile Thr Phe Thr Ala Phe Ala Glu 1555                1560                1565 Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Ile Thr Arg 1570                1575                1580 Lys Val Ser Thr Asp Asn Ser Leu Thr Leu Arg His Asn Glu Asn Gly 1585                1590                1595                1600 Ala Gln Tyr Met Gln Trp Gly Val Tyr Arg Ile Arg Leu Asn Thr Leu 1605                1610                1615 Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile 1620                1625                1630 Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly 1635                1640                1645 Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Pro Ser Thr His Gly 1650                1655                1660 Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn 1665                1670                1675                1680 Ser His Ile Ile Tyr Ser Gly Gln Leu Lys Asp Thr Asn Ile Ser Thr 1685                1690                1695 Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr Ser 1700                1705                1710 Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp 1715                1720                1725 Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn 1730                1735                1740 Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn 1745                1750                1755                1760 Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe 1765                1770                1775 Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu 1780                1785                1790 His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp 1795                1800                1805 Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp 1810                1815                1820 Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu 1825                1830                1835                1840 Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr 1845                1850                1855 Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly 1860                1865                1870 Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys 1875                1880                1885 Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu 1890                1895                1900 Pro Leu Ser Thr Thr Trp Asn Asp Pro Arg Leu Asp Lys Ala Ala Asp 1905                1910                1915                1920 Ile Thr Thr Gln Ser Ala His Ser Ser Ser Ile Val Ala Leu Arg Gln 1925                1930                1935 Ser Thr Pro Ala Leu Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp 1940                1945                1950 Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr 1955                1960                1965 Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly 1970                1975                1980 Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala 1985                1990                1995                2000 Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro 2005                2010                2015 Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala 2020                2025                2030 Arg Ser Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn 2035                2040                2045 Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn 2050                2055                2060 Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr 2065                2070                2075                2080 Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala 2085                2090                2095 Gly Ala Gln Ser Arg Phe Asp Ser Tyr Ser Lys Leu His Asp Glu Asn 2100                2105                2110 Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala 2115                2120                2125 Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala 2130                2135                2140 Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp 2145                2150                2155                2160 Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn 2165                2170                2175 Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg 2180                2185                2190 Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu 2195                2200                2205 Leu Lys Gln Leu Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu 2210                2215                2220 Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr 2225                2230                2235                2240 Gln Ala Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu 2245                2250                2255 Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr 2260                2265                2270 Asp Leu Ala Ile Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp 2275                2280                2285 Glu Ile Ser Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln 2290                2295                2300 Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu 2305                2310                2315                2320 Ala Gln Met Glu Asp Ala His Leu Arg Arg Asp Lys Arg Ala Leu Glu 2325                2330                2335 Val Glu Arg Thr Val Ser Leu Ala Glu Ile Tyr Ala Gly Leu Pro Gln 2340                2345                2350 Asp Lys Gly Pro Phe Ser Leu Thr Gln Glu Ile Glu Lys Leu Val Asn 2355                2360                2365 Ala Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly 2370                2375                2380 Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Ile Ser Leu Ala 2385                2390                2395                2400 Asp Leu Lys Ile Arg Glu Asp Tyr Pro Glu Ser Ile Gly Lys Ile Arg 2405                2410                2415 Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr 2420                2425                2430 Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala 2435                2440                2445 Asn Gly Cys Ala Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly 2450                2455                2460 Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly 2465                2470                2475                2480 Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser 2485                2490                2495 Thr Pro Ala Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp 2500                2505                2510 Ile Ile Leu His Ile Arg Tyr Thr Ile Lys 2515                2520 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 1481 <212> TYPE: PRT <213> ORGANISM: Photorhabdus luminescens <400> SEQUENCE:  14 Met Gln Asn Ser Gln Thr Phe Ser Met Thr Glu Leu Ser Leu Pro Lys 1               5                  10                  15 Gly Gly Gly Ala Ile Thr Gly Met Gly Glu Ala Leu Thr Pro Ala Gly 20                  25                  30 Pro Asp Gly Met Ala Ala Leu Ser Leu Pro Leu Pro Ile Ser Ala Gly 35                  40                  45 Arg Gly Tyr Ala Pro Ser Leu Thr Leu Asn Tyr Asn Ser Gly Thr Gly 50                  55                  60 Asn Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val Met Thr Ile Arg 65                  70                  75                  80 Arg Arg Thr Ser Thr Gly Val Pro Asn Tyr Asp Glu Thr Asp Thr Phe 85                  90                  95 Leu Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu Asn Glu Ala Gly 100                 105                 110 Gln Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly Ile Asn Leu Gly 115                 120                 125 Met Thr Phe Thr Val Thr Gly Tyr Arg Ser Arg Leu Glu Ser His Phe 130                 135                 140 Ser Arg Leu Glu Tyr Trp Gln Pro Gln Thr Thr Gly Ala Thr Asp Phe 145                 150                 155                 160 Trp Leu Ile Tyr Ser Pro Asp Gly Gln Ala His Leu Leu Gly Lys Asn 165                 170                 175 Pro Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn Gln Thr Ala Gln 180                 185                 190 Trp Leu Leu Glu Ala Ser Val Ser Ser His Gly Glu Gln Ile Tyr Tyr 195                 200                 205 Gln Tyr Arg Ala Glu Asp Glu Thr Asp Cys Glu Thr Asp Glu Leu Thr 210                 215                 220 Ala His Pro Asn Thr Thr Val Gln Arg Tyr Leu Gln Val Val His Tyr 225                 230                 235                 240 Gly Asn Leu Thr Ala Ser Glu Val Phe Pro Thr Leu Asn Gly Asp Asp 245                 250                 255 Pro Leu Lys Ser Gly Trp Leu Phe Cys Leu Val Phe Asp Tyr Gly Glu 260                 265                 270 Arg Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys Ala Thr Ser Asn 275                 280                 285 Trp Leu Cys Arg Lys Asp Arg Phe Ser Arg Tyr Glu Tyr Gly Phe Ala 290                 295                 300 Leu Arg Thr Arg Arg Leu Cys Arg Gln Ile Leu Met Phe His Arg Leu 305                 310                 315                 320 Gln Thr Leu Ser Gly Gln Ala Lys Gly Asp Asp Glu Pro Ala Leu Val 325                 330                 335 Ser Arg Leu Ile Leu Asp Tyr Asp Glu Asn Ala Val Val Ser Thr Leu 340                 345                 350 Val Ser Val Arg Arg Val Gly His Glu Gln Asp Gly Thr Thr Ala Val 355                 360                 365 Ala Leu Pro Pro Leu Glu Leu Ala Tyr Gln Pro Phe Glu Pro Glu Gln 370                 375                 380 Lys Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn Phe Asn Thr Ile 385                 390                 395                 400 Gln Arg Trp Gln Leu Leu Asp Leu Gln Gly Glu Gly Val Pro Gly Ile 405                 410                 415 Leu Tyr Gln Asp Lys Asn Gly Trp Trp Tyr Arg Ser Ala Gln Arg Gln 420                 425                 430 Thr Gly Glu Glu Met Asn Ala Val Thr Trp Gly Lys Met Gln Leu Leu 435                 440                 445 Pro Ile Thr Pro Ala Ile Gln Asp Asn Ala Ser Leu Met Asp Ile Asn 450                 455                 460 Gly Asp Gly Gln Leu Asp Trp Val Ile Thr Gly Pro Gly Leu Arg Gly 465                 470                 475                 480 Tyr His Ser Gln His Pro Asp Gly Ser Trp Thr Arg Phe Thr Pro Leu 485                 490                 495 His Ala Leu Pro Ile Glu Tyr Thr His Pro Arg Ala Gln Leu Ala Asp 500                 505                 510 Leu Met Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser 515                 520                 525 Val Arg Leu Tyr Ala Asn Asn Arg Asp Gly Phe Thr Glu Gly Arg Asp 530                 535                 540 Val Val Gln Ser Gly Gly Ile Thr Leu Pro Leu Pro Gly Ala Asp Ala 545                 550                 555                 560 Arg Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser Gly Gln Ala His 565                 570                 575 Leu Val Glu Val Ser Ala Thr Lys Val Thr Cys Trp Pro Asn Leu Gly 580                 585                 590 His Gly Arg Phe Gly Gln Pro Ile Thr Leu Pro Gly Phe Ser Gln Ser 595                 600                 605 Ala Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala Asp Leu Asp Gly 610                 615                 620 Ser Gly Pro Ala Asp Leu Ile Tyr Val His Ala Asp His Leu Asp Ile 625                 630                 635                 640 Phe Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro Phe Thr Leu Arg 645                 650                 655 Phe Pro Asp Gly Leu Arg Phe Asp Asp Thr Cys Gln Leu Gln Val Ala 660                 665                 670 Asp Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu Ser Val Pro His 675                 680                 685 Met Ala Pro His His Trp Arg Cys Asp Leu Thr Asn Ala Lys Pro Trp 690                 695                 700 Leu Leu Ser Glu Met Asn Asn Asn Met Gly Ala His His Thr Leu His 705                 710                 715                 720 Tyr Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys Ala Ala Ala Leu 725                 730                 735 Ala Thr Gly Gln Thr Pro Val Cys Tyr Leu Pro Phe Pro Val His Thr 740                 745                 750 Leu Trp Gln Thr Glu Thr Glu Asp Glu Ile Ser Gly Asn Lys Leu Val 755                 760                 765 Thr Thr Leu Arg Tyr Ala His Gly Ala Trp Asp Gly Arg Glu Arg Glu 770                 775                 780 Phe Arg Gly Phe Gly Tyr Val Glu Gln Thr Asp Ser His Gln Leu Ala 785                 790                 795                 800 Gln Gly Asn Ala Pro Glu Arg Thr Ser Pro Ala Leu Thr Lys Asn Trp 805                 810                 815 Tyr Ala Thr Gly Ile Pro Glu Val Asp Asn Thr Leu Ser Ala Gly Tyr 820                 825                 830 Trp Arg Gly Asp Thr Gln Ala Phe Thr Gly Phe Thr Pro His Phe Thr 835                 840                 845 Leu Trp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro Glu Asp Asp His 850                 855                 860 Asn Leu Tyr Trp Leu Asn Arg Ala Leu Lys Gly Gln Pro Leu Arg Ser 865                 870                 875                 880 Glu Leu Tyr Gly Leu Asp Gly Ser Ala Gln Gln Lys Ile Pro Tyr Thr 885                 890                 895 Val Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln Asp Asn Thr Thr 900                 905                 910 Leu Ser Pro Val Leu Trp Ala Ser Val Val Glu Ser Arg Ser Tyr His 915                 920                 925 Tyr Glu Arg Ile Ile Ser Asp Pro Gln Cys Asn Gln Asp Ile Thr Leu 930                 935                 940 Ser Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val Ser Val Gln Tyr 945                 950                 955                 960 Pro Arg Arg Asn Lys Pro Thr Thr Asn Pro Tyr Pro Asp Thr Leu Pro 965                 970                 975 Asp Thr Leu Phe Ala Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg 980                 985                 990 Leu Thr Tyr Gln Gln Ser Ser Trp His His Leu Ile Ala Asn Glu Leu 995                1000                1005 Arg Val Leu Gly Leu Pro Asp Gly Thr Arg Ser Asp Ala Phe Thr Tyr 1010                1015                1020 Asp Ala Lys His Val Pro Val Asp Gly Leu Asn Leu Glu Ala Leu Cys 1025                1030                1035                1040 Ala Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg Glu Tyr Leu Asn 1045                1050                1055 Gln Gln Arg Thr Phe Tyr Thr Asp Gly Lys Thr Asp Gly Lys Asn Pro 1060                1065                1070 Thr Pro Leu Lys Thr Pro Thr Arg Gln Ala Leu Ile Ala Phe Thr Glu 1075                1080                1085 Thr Ala Val Leu Thr Glu Ser Leu Leu Ser Ala Phe Asp Gly Gly Ile 1090                1095                1100 Thr Pro Asp Glu Leu Pro Gly Leu Leu Thr Gln Ala Gly Tyr Gln Gln 1105                1110                1115                1120 Glu Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln Val Trp Val Ala 1125                1130                1135 Arg Lys Gly Tyr Thr Asp Tyr Gly Thr Glu Val Gln Phe Trp Arg Pro 1140                1145                1150 Val Ala Gln Arg Asn Thr Gln Leu Thr Gly Lys Thr Thr Leu Lys Trp 1155                1160                1165 Asp Thr His Tyr Cys Val Ile Thr Gln Thr Gln Asp Ala Ala Gly Leu 1170                1175                1180 Thr Val Ser Ala Asn Tyr Asp Trp Arg Phe Leu Thr Pro Met Gln Leu 1185                1190                1195                1200 Thr Asp Ile Asn Asp Asn Val His Ile Ile Thr Leu Asp Ala Leu Gly 1205                1210                1215 Arg Pro Val Thr Gln Arg Phe Trp Gly Ile Glu Asn Gly Val Ala Thr 1220                1225                1230 Gly Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro Pro Val Asp Val 1235                1240                1245 Asn Ala Ala Ile Ala Leu Thr Gly Pro Leu Pro Val Ala Gln Cys Leu 1250                1255                1260 Val Tyr Ala Pro Asp Ser Trp Met Pro Leu Phe Gly Gln Glu Thr Phe 1265                1270                1275                1280 Asn Thr Leu Thr Gln Glu Glu Gln Lys Thr Leu Arg Asp Leu Arg Ile 1285                1290                1295 Ile Thr Glu Asp Trp Arg Ile Cys Ala Leu Ala Arg Arg Arg Trp Leu 1300                1305                1310 Gln Ser Gln Lys Ala Gly Thr Pro Leu Val Lys Leu Leu Thr Asn Ser 1315                1320                1325 Ile Gly Leu Pro Pro His Asn Leu Met Leu Ala Thr Asp Arg Tyr Asp 1330                1335                1340 Arg Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala Phe Ser Asp Gly 1345                1350                1355                1360 Phe Gly Arg Leu Leu Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala 1365                1370                1375 Trp Gln Arg Asn Gln Asp Gly Ser Leu Val Thr Lys Met Glu Asp Thr 1380                1385                1390 Lys Thr Arg Trp Ala Ile Thr Gly Arg Thr Glu Tyr Asp Asn Lys Gly 1395                1400                1405 Gln Ala Ile Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr 1410                1415                1420 Val Ser Asp Asp Ser Ala Arg Lys Glu Ala Tyr Ala Asp Thr His Ile 1425                1430                1435                1440 Tyr Asp Pro Ile Gly Arg Glu Ile Gln Val Ile Thr Ala Lys Gly Trp 1445                1450                1455 Leu Arg Gln Asn Gln Tyr Phe Pro Trp Phe Thr Val Ser Glu Asp Glu 1460                1465                1470 Asn Asp Leu Ser Ala Asp Ala Leu Val 1475                1480 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  15 cgggatccga tgattttaaa agg                                             23 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  16 gcgccattga tttgag                                                     16 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 17 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  17 cattagaggt cgaacgtac                                                  19 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  18 gagcgagctc ttacttaatg gtgtag                                          26 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  19 cagcgagctc catgcagaat tcacagac                                        28 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  20 ggcaatggca gcgataag                                                   18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  21 cattaacgca ggaagagc                                                   18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence oligonucleotide <400> SEQUENCE:  22 gacctcgagt tacacgagcg cgtcag                                          26

## Claims

1. An isolated nucleic acid molecule comprising sequence that encodes at least one toxin that is active against lepidopteran and coleopteran insects, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEO ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

2. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:12.

3. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises nucleotides 15,171-18,035 of SEQ ID NO:11.

4. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:14.

5. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises nucleotides 31,393-35,838 of SEQ ID NO:11.

6. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:13.

7. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises nicleotides 23,768-31,336 of SEQ ID NO:11.

8. An isolated nucleic acid molecule acording to claim 1, wherein said nucleotide sequence comprises the approximately 38 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-30077.

9. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises the approximately 22.2 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-30078.

10. an isolated nuleic aciid molecule according to claim 1, wherin the toxin has insecticidal activity against insects selected from the group consisting of Plutella Xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

11. A chimeric gene comprising a heterologous promoter sequence opeatively linked to the nucleic acid molecule of claim 1.

12. A recombinant vector comprising the chimeric gene of claim 11.

13. A transgenic host cell comprising the chimeric gene of claim 11.

14. A transgenic host cell according to claim 13, which is a transgenic plant cell.

15. A transgenic plant comprising the transgenic plant cell of claim 14.

16. A transgenic plant acoording to claim 15, which is maize.

17. Seed from the transgenic plant of claim 15.

18. Seed from the transgenic plant of claim 16.

19. A method of producing an insect-resistant plant, comprising introducing a nuleic acid molecule according to claim 1 into said plant, wherein said nucleic acid molecule is expressible in said plant in an effective amount to control lepidopteran and coleopteran insects.

20. The method of claim 19, wherein the insects are selected from the group consisiting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).