Insecticidal toxins from Photorhabdus luminescens and nucleic acid sequences coding therefor

Abstract

Novel nucleic acid sequences isolated from Photorhabdus luminescens, whose expression results in novel insecticidal toxins, are disclosed herein. The invention also discloses compositions and formulations containing the insecticidal toxins that are capable of controlling insect pests. The invention is further drawn to methods of making the toxins and to methods of using the nucleotide sequences, for example in microorganisms to control insect pests or in transgenic plants to confer insect resistance.

Description

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

SUMMARY OF THE INVENTION

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.

DEFINITIONS

“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 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° 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.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

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 ˜46.4 kDa protein encoded by orf1 of SEQ ID NO:1.

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

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

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

SEQ ID NO:6 is the sequence of the ˜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.

DEPOSITS

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

DETAILED DESCRIPTION OF THE INVENTION

Novel Nucleic Acid Sequences whose Expression Results in Insecticidal 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 ˜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 ˜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 ˜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° C. for 2 weeks. One is also shown to retain its full insecticidal activity after being freeze-dried and stored at 22° C. for 2 weeks. However, the insecticidal toxins of the invention lose their insecticidal activity after incubation for 5 minutes at 100° 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.

Expression of the Nucleotide Sequences in Heterologous Microbial Hosts

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.

Expression of the Nucleotide Sequences in Plant Tissue

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.

Formulation of Insecticidal Compositions

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.

EXAMPLES

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).

A. Isolation Of Nucleotide Sequences Whose Expression Results In Toxins Active Against Lepidopteran Insects

Example 1

Construction of Cosmid Library from Photorhabdus luminescens

Photorhabdus luminescens strain ATCC 29999 is grown in nutrient broth at 25° 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° 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° 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×TE.

The DNA is treated with 0.3 units of Sau3A per mg DNA at 37° 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° C. to inactivate the enzyme. Then 2 units of Calf Intestinal Alkaline Phosphatase are added and incubated for 30 minutes at 37° 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×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° 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° 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° C.

Example 2

Insect Bioassays

Plutella xylostella bioassays are performed by aliquoting of 50 μl 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° 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.

Example 3

Isolation of the Nucleotide Sequence Responsible for Insect Control 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 α E. coli cells. Isolated transformed bacterial colonies are grown in L broth with 50 μg/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 α 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 μg of the 17 kb insert is isolated and treated with 0.3 unit of Sau3A per μg DNA for 4, 6, and 8 minutes at 37° C., heated at 75° 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 DH5α cells and plated on L agar with Xgal/Amp as described in Sambrook et al. and grown overnight at 37° C. White colonies are picked and grown in L broth with 100 μ/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.).

Example 4

Subcloning of the 9.7 kb EcoRIJXbal Fragment From pCIB9359

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 α 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.

Example 5

Bioassay Results for Cosmid Clones pCIB9359 and pCIB9359-7

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.

Example 6

Identification of Active Region of pCIB9359-7 By Subcloning

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

Example 7

Characterization of pCIB9359-7 Insect Control Activity By Titration

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 μl and are transferred to diet cups with 5 insects per cup. The results show the percentage (%) of insect mortality.

μl 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.

Example 8

Stability of pCIB9359-7 Activity

The stability of the toxins is tested after storage for 2 weeks at different temperatures and conditions. 300 ml of Luria broth containing 100 (μg/ml ampicillin is inoculated with E. coli strain 9359-7 and grown overnight at 37° C. Samples are placed in sterile 15 ml screw cap tubes and stored at 22° C. and 4° C. Another sample is centrifuged; the supernatant is removed, freeze dried and stored at 22° 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° C. (2weeks)        +++
4° 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° C., 4° C., and freeze-dried, and are therefore very stable.

Example 9

Size Fraction of pCIB9359-7 Activity

The approximate sizes of the insecticidal toxins are determined. P. luminescens cosmid clones pCIB9359-7 and pUC19 in E. coli host DH5α are grown in media consisting of 50% Terrific broth and 50% Luria broth, supplemented with 50 μg/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° 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 μ 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.

Example 10

Heat Inactivitation of pCIB9359-7 Activity

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° 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 μl 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.

Example 11

Leaf Dip Bioassay of pCIB9359-7

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 μl 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.

B. Isolation Of Nucleic Acid Sequences Whose Expression Results In Toxins Active Against Lepidopteran and Coleopteran Insects

Example 12

Total DNA Isolation from Photorhabdus luminescens

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 μg/ml proteinase K, TE to a final volume of 600 μl. After a 1 hour incubation at 37° C., 100 μl 5M NaCl and 80 μl CTAB/NaCl are added and the culture is incubated at 65° 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 μg 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.

Example 13

PCR Amplification of Probes

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 μM of oligos, 200 μM of dNTPs, 1× Taq DNA Polymerase buffer and 2.5 units of Taq DNA Polymerase are included in the reaction. The reaction conditions are as follows:

94° C.-1 minute

94° C.-30 seconds/60° C.-30 seconds/72° C.-30 seconds (25 cycles)

72° C.-5 minutes

4° C.-indefinite soak

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

Example 14

Probing a Photorhabdus luminescens Library

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° 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× SSC and hybridized with the probes for 12-16 hours at 65° 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.

Example 15

Insect Bioassays

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 μl of an overnight culture of the cosmid library in XL-1 Blue MR cells (Stratagene) is sonicated and then mixed with 500 μl 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 μl 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° C. in complete darkness; the assay with Diamondback Moth is incubated at 72° 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.

Example 16

Sequencing of Cosmid 514

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 DH5α and assigned accession no. B-30077.

Example 17

Subcloning Insecticidal Regions of Cosmid 514

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:

(SEQ ID NO:15)
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 ˜22 kb subclone 051011H2O2, which has activity against WCR and ECB, is designated pNOV1001 and deposited with the NRRL in E. coli DH5α 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.

Example 18

Insecticidal Activity of Subclones

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 DH5α 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.

C. Expression of the Nucleic Acid Sequences of the Invention in 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.

Example 19

Expression of the Nucleotide Sequences in E. coli and Other Gram-Negative 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 L (Pharmacia #27-4946-01) uses a tightly regulated bacteriophage λ 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).

Example 20

Expression of the Nucleotide Sequences in Gram-Positive Bacteria

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

Example 21

Expression of the Nucleotide Sequences in Fungi

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.

D. Formulation of the Insecticidal Toxin

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.

Example 22

Liquid Formulation of Insecticidal Compositions

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°)

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.

Example 23

Solid Formulation of Insecticidal Compositions

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.

E. Expression of the Nucleotide Sequences in Transgenic Plants

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 λgtl1, λgtl0 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.

Example 24

Modification of Coding Sequences and Adjacent Sequences

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.

Example 25

Construction of Plant Expression Cassettes

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.

Example 26

Construction of Plant Transformation Vectors

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.

Example 27

Transformation

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® helium device using a burst pressure of ˜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 μm 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 μmol photons/m 2 /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/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 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.

E. Breeding and Seed Production

Example 28

Breeding

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.

Example 29

Seed Production

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®), methalaxyl (Apron®), and pirimiphos-methyl (Actellic®). 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.

22 1 9717 DNA Photorhabdus luminescens CDS (412)..(1665) orf1 ~46.4 kDa 1gaattcatat gctatgaaat aaacagttgg cgcaataatt aaagctatta tttttatttt 60gtttttatac aatgatatgc tttattaaac agaataatga gttaatgata aataaatcct 120cgggatttat catgatatta tggccgaatg tgatgtgaac aattatttta taattagatt 180aataatataa tggtattaaa ataacaatat atttattcat gggtatttat catcggtttt 240attacatggg gaataatcta taaattagtt ttacataatt cacaaatagc gattccatta 300accaggaata ttaaaaatac ttatttatga ttatggtgat atatcttcat tagcctactt 360ttataactag aaaaattgac attttcaatc catgtataaa tggtaaccaa t atg cag 417 Met Gln 1aga gct caa cga gtt gtt att aca ggt atg ggt gcc gta aca ccg att 465Arg Ala Gln Arg Val Val Ile Thr Gly Met Gly Ala Val Thr Pro Ile 5 10 15ggt gaa gat gtt gaa tca tgt tgg caa agt att att gaa aaa caa cat 513Gly Glu Asp Val Glu Ser Cys Trp Gln Ser Ile Ile Glu Lys Gln His 20 25 30cga ttt cac aga att gaa ttt cct gac tca ttc att aat tcg cgt ttc 561Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe Ile Asn Ser Arg Phe 35 40 45 50ttt tct ttc ctt gca cca aac cca tcc cgc tat cag tta tta cca aaa 609Phe Ser Phe Leu Ala Pro Asn Pro Ser Arg Tyr Gln Leu Leu Pro Lys 55 60 65aag ttg act cat aca ctt tct gac tgc gga aaa gca gca ttg aag gcg 657Lys Leu Thr His Thr Leu Ser Asp Cys Gly Lys Ala Ala Leu Lys Ala 70 75 80act tat caa gct ttt acc caa gca ttc ggc gtg aat ata tca cct gtt 705Thr Tyr Gln Ala Phe Thr Gln Ala Phe Gly Val Asn Ile Ser Pro Val 85 90 95gaa tat tac gat aaa tac gaa tgt ggc gta att ctt ggc agt ggt tgg 753Glu Tyr Tyr Asp Lys Tyr Glu Cys Gly Val Ile Leu Gly Ser Gly Trp 100 105 110gga gct att gat aat gcc gga gat cat gct tgc caa tat aag caa gca 801Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cys Gln Tyr Lys Gln Ala115 120 125 130aaa tta gct cat cct atg agt aat ctt att acc atg cca agc tcc atg 849Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr Met Pro Ser Ser Met 135 140 145acg gct gca tgt tcg att atg tat gga cta cgt ggt tat caa aat acc 897Thr Ala Ala Cys Ser Ile Met Tyr Gly Leu Arg Gly Tyr Gln Asn Thr 150 155 160gtt atg gct gcc tgc gca acg ggc aca atg gcg ata ggc gat gcc ttt 945Val Met Ala Ala Cys Ala Thr Gly Thr Met Ala Ile Gly Asp Ala Phe 165 170 175gaa att att cgc tca ggg cgg gca aaa tgt atg att gcc gga gcc gct 993Glu Ile Ile Arg Ser Gly Arg Ala Lys Cys Met Ile Ala Gly Ala Ala 180 185 190gaa tca ctc acg cgg gaa tgt aat att tgg agt att gat gta ctg aat 1041Glu Ser Leu Thr Arg Glu Cys Asn Ile Trp Ser Ile Asp Val Leu Asn195 200 205 210gca tta tcg aaa gaa caa gcg gac cca aat ctt gca tgt tgt cca ttt 1089Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu Ala Cys Cys Pro Phe 215 220 225agc ctt gat cgc tct gga ttt gta tta gcc gaa gga gcg gcg gta gtt 1137Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu Gly Ala Ala Val Val 230 235 240tgt ctg gaa aat tat gat tca gcc atc gcg cgt ggt gca acg att tta 1185Cys Leu Glu Asn Tyr Asp Ser Ala Ile Ala Arg Gly Ala Thr Ile Leu 245 250 255gcg gaa att aaa ggt tac gcc caa tat tca gat gcc gtt aat tta acc 1233Ala Glu Ile Lys Gly Tyr Ala Gln Tyr Ser Asp Ala Val Asn Leu Thr 260 265 270cgg cca aca gaa gat att gaa cct aaa ata tta gcg ata act aaa gcc 1281Arg Pro Thr Glu Asp Ile Glu Pro Lys Ile Leu Ala Ile Thr Lys Ala275 280 285 290att gag cag gca cag att tcg ccg aaa gat att gac tac att aat gct 1329Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile Asp Tyr Ile Asn Ala 295 300 305cat ggt act tct aca ccg tta aat gat ctt tat gaa act cag gca att 1377His Gly Thr Ser Thr Pro Leu Asn Asp Leu Tyr Glu Thr Gln Ala Ile 310 315 320aaa gca gca ctg ggc caa tat gct tat cag gta cct ata tca agc aca 1425Lys Ala Ala Leu Gly Gln Tyr Ala Tyr Gln Val Pro Ile Ser Ser Thr 325 330 335aaa tct tat acc ggc cac ctt att gct gcc gcc ggt agt ttt gaa acg 1473Lys Ser Tyr Thr Gly His Leu Ile Ala Ala Ala Gly Ser Phe Glu Thr 340 345 350att gta tgt gtg aaa gca tta gct gaa aat tgc ttg cca gca aca ttg 1521Ile Val Cys Val Lys Ala Leu Ala Glu Asn Cys Leu Pro Ala Thr Leu355 360 365 370aat tta cac cgg gcc gat cca gat tgc gat ctc aat tat ttg cct aat 1569Asn Leu His Arg Ala Asp Pro Asp Cys Asp Leu Asn Tyr Leu Pro Asn 375 380 385caa cat tgc tac acc gct caa cca gag gtg aca ctc aat att agc gca 1617Gln His Cys Tyr Thr Ala Gln Pro Glu Val Thr Leu Asn Ile Ser Ala 390 395 400ggt ttc ggc ggg cat aac gct gcg ttg gtt atc gct aag gta agg taa 1665Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile Ala Lys Val Arg 405 410 415ctgatatgtt 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 425ggg gat ttt aac ccc atc cat tat tcg gcg aaa agc gag tct ttg cgc 1766Gly Asp Phe Asn Pro Ile His Tyr Ser Ala Lys Ser Glu Ser Leu Arg430 435 440 445aat ata cag caa cac ccg gtg cag gga atg ttg agt ttg ctc tat gta 1814Asn Ile Gln Gln His Pro Val Gln Gly Met Leu Ser Leu Leu Tyr Val 450 455 460cgg caa cag ttt tct caa tta act tcc gct ttt aca acg gga ata ttg 1862Arg Gln Gln Phe Ser Gln Leu Thr Ser Ala Phe Thr Thr Gly Ile Leu 465 470 475aac att gat gcc tct ttc cgc cag tat gtt tat acc gca tta ccc cat 1910Asn Ile Asp Ala Ser Phe Arg Gln Tyr Val Tyr Thr Ala Leu Pro His 480 485 490caa ctg agg att aat act aaa aac aaa acg ttt aaa tta gaa aat ccc 1958Gln Leu Arg Ile Asn Thr Lys Asn Lys Thr Phe Lys Leu Glu Asn Pro 495 500 505agt aaa gaa aac acg ttg ttc ggc aat acc agc gta gag aat aca atg 2006Ser Lys Glu Asn Thr Leu Phe Gly Asn Thr Ser Val Glu Asn Thr Met510 515 520 525gag tca att gaa gat tgg atc gtt cag gat aat tgt caa aaa cta acg 2054Glu Ser Ile Glu Asp Trp Ile Val Gln Asp Asn Cys Gln Lys Leu Thr 530 535 540ata aca ggg gag gaa gtt tgt gaa aag tat gct gtc ttt aga tac tat 2102Ile Thr Gly Glu Glu Val Cys Glu Lys Tyr Ala Val Phe Arg Tyr Tyr 545 550 555ttc cca agt gtc act tct att gga tgg ttc ctg gat gcg ctt gct ttt 2150Phe Pro Ser Val Thr Ser Ile Gly Trp Phe Leu Asp Ala Leu Ala Phe 560 565 570cat ctt att att aat tcg aca gga ttt ctt aat ttt gag cac tac cat 2198His Leu Ile Ile Asn Ser Thr Gly Phe Leu Asn Phe Glu His Tyr His 575 580 585ttt aac caa tta cag gat tat ctg agt caa tct ttt act ttg cat act 2246Phe Asn Gln Leu Gln Asp Tyr Leu Ser Gln Ser Phe Thr Leu His Thr590 595 600 605ggg caa gcg att aaa atc agg aag gag att gtt aat agt aca gta tta 2294Gly Gln Ala Ile Lys Ile Arg Lys Glu Ile Val Asn Ser Thr Val Leu 610 615 620tta tct tca ccg gat atc tgt gtt gaa tta aat cct cct tta ttg att 2342Leu Ser Ser Pro Asp Ile Cys Val Glu Leu Asn Pro Pro Leu Leu Ile 625 630 635aag aat ggc gat aaa gat tat att cgt att ttc tat tat cga tgt tta 2390Lys Asn Gly Asp Lys Asp Tyr Ile Arg Ile Phe Tyr Tyr Arg Cys Leu 640 645 650tat gat aaa aaa cct att ttt gta tca aag act tca att atc tct aag 2438Tyr Asp Lys Lys Pro Ile Phe Val Ser Lys Thr Ser Ile Ile Ser Lys 655 660 665atg aaa taa aaggaaagcg aaatgccaac acaaagtgat attttcactg 2487Met Lys670aaataaagaa tagaatatta atgatgaagg atatagaaga tgaagaaata acaccagagt 2547cctcttttgt ttcgcttgaa tttgatagtc ttgactatgt ggaaatccaa gtttttgtgt 2607tggaagcgta tggtattgtg cttaaagccg aacttttttc aaatcattct atttcaacat 2667taaatgagct cactgactat ttaaaatcaa aattgtaatc tgaattttta cttaattatg 2727ttttttcacc attaacatta agaggttata atg aac gtt tta gaa caa ggt aag 2781 Met Asn Val Leu Glu Gln Gly Lys 675 680gtt gct gct tta tat tca gcc tat tcg gaa aca gaa ggt tct tcg tgg 2829Val Ala Ala Leu Tyr Ser Ala Tyr Ser Glu Thr Glu Gly Ser Ser Trp 685 690 695gtg gga aac ttg tgc tgt ttt tca agt gat cgg gag cat ttg cct att 2877Val Gly Asn Leu Cys Cys Phe Ser Ser Asp Arg Glu His Leu Pro Ile 700 705 710atc gtg aat ggg cgt cgt ttc ttg att gaa ttt gtt att cca gat cat 2925Ile Val Asn Gly Arg Arg Phe Leu Ile Glu Phe Val Ile Pro Asp His 715 720 725tta ctt gat aaa acg gtt aaa ccc aga gta ttc gat ttg gat atc aat 2973Leu Leu Asp Lys Thr Val Lys Pro Arg Val Phe Asp Leu Asp Ile Asn 730 735 740aaa caa ttt tta ctg cgt cgt gac cat cgt gag ata aat att tat ctt 3021Lys Gln Phe Leu Leu Arg Arg Asp His Arg Glu Ile Asn Ile Tyr Leu745 750 755 760tta ggt gaa gga aat ttt atg gat agg acg acg aca gat aaa aat cta 3069Leu Gly Glu Gly Asn Phe Met Asp Arg Thr Thr Thr Asp Lys Asn Leu 765 770 775ttc gag tta aat gag gat ggt tca cta ttt att aag acg tta cgc cat 3117Phe Glu Leu Asn Glu Asp Gly Ser Leu Phe Ile Lys Thr Leu Arg His 780 785 790gct ctt ggt aaa tat gtt gct att aat cct tca act acg caa ttt atc 3165Ala Leu Gly Lys Tyr Val Ala Ile Asn Pro Ser Thr Thr Gln Phe Ile 795 800 805ttc ttt gca caa gga aag tac agt gaa ttt atc atg aat gcc tta aag 3213Phe Phe Ala Gln Gly Lys Tyr Ser Glu Phe Ile Met Asn Ala Leu Lys 810 815 820aca gtt gaa gac gaa tta tca aaa cgt tat cga gtc aga att att cct 3261Thr Val Glu Asp Glu Leu Ser Lys Arg Tyr Arg Val Arg Ile Ile Pro825 830 835 840gaa ttg caa ggg ccg tat tat ggc ttt gaa ctt gat att ctt tct att 3309Glu Leu Gln Gly Pro Tyr Tyr Gly Phe Glu Leu Asp Ile Leu Ser Ile 845 850 855aca gct taa ttcacaatat tatggagagt gtt atg gaa aag aaa ata aca aca 3362Thr Ala Met Glu Lys Lys Ile Thr Thr 860 865ttt acc att gag aaa act gat gac aat ttt tat gct aat ggg cgt cat 3410Phe Thr Ile Glu Lys Thr Asp Asp Asn Phe Tyr Ala Asn Gly Arg His 870 875 880caa tgt atg gta aaa atc tct gta ctt aaa caa gaa tat agg aat ggt 3458Gln Cys Met Val Lys Ile Ser Val Leu Lys Gln Glu Tyr Arg Asn Gly 885 890 895gat tgg ata aaa tta gca ctt agt gag gct gaa aaa aga tcg att cag 3506Asp Trp Ile Lys Leu Ala Leu Ser Glu Ala Glu Lys Arg Ser Ile Gln 900 905 910gtg gcg gca tta agt gat agc ctc ata tat gac caa tta aaa atg cct 3554Val Ala Ala Leu Ser Asp Ser Leu Ile Tyr Asp Gln Leu Lys Met Pro915 920 925 930tca ggt tgg aca acg aca gat gca aga aat aaa ttt gat ctt ggg tta 3602Ser Gly Trp Thr Thr Thr Asp Ala Arg Asn Lys Phe Asp Leu Gly Leu 935 940 945tta aat ggt gtt tat cat gct gat gct ttt att gac gaa cag gta aca 3650Leu Asn Gly Val Tyr His Ala Asp Ala Phe Ile Asp Glu Gln Val Thr 950 955 960gat cgt gcg gga gat tgc tgc aca aat gaa aac tat cag aac agt gtg 3698Asp Arg Ala Gly Asp Cys Cys Thr Asn Glu Asn Tyr Gln Asn Ser Val 965 970 975aaa agt gtt cct gaa att atc tat cgt tat gtc agt agt aat aga aca 3746Lys Ser Val Pro Glu Ile Ile Tyr Arg Tyr Val Ser Ser Asn Arg Thr 980 985 990agc aca gaa tac cta atg gca aaa atg aca ttt gaa gat acg gat ggg 3794Ser Thr Glu Tyr Leu Met Ala Lys Met Thr Phe Glu Asp Thr Asp Gly995 1000 1005 1010aaa cgc aca tta aca acg aat atg tca gtt ggt gat gaa gtt ttt gac 3842Lys Arg Thr Leu Thr Thr Asn Met Ser Val Gly Asp Glu Val Phe Asp 1015 1020 1025agc aag gtt tta tta aaa gcc att gct cct tat gca att aat aca aat 3890Ser Lys Val Leu Leu Lys Ala Ile Ala Pro Tyr Ala Ile Asn Thr Asn 1030 1035 1040caa ttg cat gaa aac atc aat aca ttg ttt gat aaa aca gaa gag ccg 3938Gln Leu His Glu Asn Ile Asn Thr Leu Phe Asp Lys Thr Glu Glu Pro 1045 1050 1055aca aaa tcc gat act cat cat caa ata att aat ctt tat cgc tgg aca 3986Thr Lys Ser Asp Thr His His Gln Ile Ile Asn Leu Tyr Arg Trp Thr 1060 1065 1070ttg cca tat cat ttg agg att ctt gaa ggg aat gac agt act gtt aat 4034Leu Pro Tyr His Leu Arg Ile Leu Glu Gly Asn Asp Ser Thr Val Asn1075 1080 1085 1090aga ata tat gtc ctt ggt aaa gag cca tca aat gat aga ttc ctg aca 4082Arg Ile Tyr Val Leu Gly Lys Glu Pro Ser Asn Asp Arg Phe Leu Thr 1095 1100 1105aga gga agg gta ttt aaa cga gga act cat atg tga atgcacgtga 4128Arg Gly Arg Val Phe Lys Arg Gly Thr His Met 1110 1115taatgtgagt ggaggatgtg ttatggacta tgcttatacc gtaactattc cggacacgca 4188gcttgctgct gaagtgcttc atgtgacagg gtgttcgtgg acgagtggtt attatgatgg 4248atatcatgat gtcacaatca ttgataacta cggttgtcag cataaattta gaatttcttc 4308ggttaatatt ggacgtgcgc taagcatagc gagaataagt tgattttcct tagtaaaaaa 4368cctttgttta tgctggtaaa cgcatgtgcg tttgccagca attaatatat tccattattg 4428aaataggaat atagccatat ctgtaattat acataaacga atttttactc gaatataatt 4488ttaattgatc aaacaggaaa tttaaa atg aaa gct acc gat ata tat tcc aat 4541 Met Lys Ala Thr Asp Ile Tyr Ser Asn 1120 1125gct ttt aat ttc ggt tct tat att aat act ggt gtc gat ccc aga aca 4589Ala Phe Asn Phe Gly Ser Tyr Ile Asn Thr Gly Val Asp Pro Arg Thr 1130 1135 1140ggt caa tat agt gca aat att aat att atc acg tta aga cct aat aat 4637Gly Gln Tyr Ser Ala Asn Ile Asn Ile Ile Thr Leu Arg Pro Asn Asn 1145 1150 1155gtg ggt aat tcg gaa caa aca ttg agc cta tca ttc tcg cca tta aca 4685Val Gly Asn Ser Glu Gln Thr Leu Ser Leu Ser Phe Ser Pro Leu Thr1160 1165 1170 1175acg tta aac aat ggc ttt ggt att ggc tgg cgc ttt tca tta aca aca 4733Thr Leu Asn Asn Gly Phe Gly Ile Gly Trp Arg Phe Ser Leu Thr Thr 1180 1185 1190tta gat ata aaa aca ctt aca ttt agc cga gca aat ggg gag caa ttt 4781Leu Asp Ile Lys Thr Leu Thr Phe Ser Arg Ala Asn Gly Glu Gln Phe 1195 1200 1205aaa tgt aag cca ttg ccg cct aat aat aat gat ctt agt ttt aaa gat 4829Lys Cys Lys Pro Leu Pro Pro Asn Asn Asn Asp Leu Ser Phe Lys Asp 1210 1215 1220aaa aaa cta aaa gat ttg cgc gta tat aag ctc gat agc aat act ttt 4877Lys Lys Leu Lys Asp Leu Arg Val Tyr Lys Leu Asp Ser Asn Thr Phe 1225 1230 1235tat gtt tat aac aaa aac ggc att ata gag ata ctt aaa cga att ggg 4925Tyr Val Tyr Asn Lys Asn Gly Ile Ile Glu Ile Leu Lys Arg Ile Gly1240 1245 1250 1255tcg agt gat att gca aaa aca gtt gca ctt gaa ttt cct gat ggt gaa 4973Ser Ser Asp Ile Ala Lys Thr Val Ala Leu Glu Phe Pro Asp Gly Glu 1260 1265 1270gca ttt gat tta att tat aat tca aga ttt gca ttg tcc gaa ata aaa 5021Ala Phe Asp Leu Ile Tyr Asn Ser Arg Phe Ala Leu Ser Glu Ile Lys 1275 1280 1285tac cgt gtg aca ggt aaa act tat ctt aaa ctc aat tac tct gga aat 5069Tyr Arg Val Thr Gly Lys Thr Tyr Leu Lys Leu Asn Tyr Ser Gly Asn 1290 1295 1300aac tgt aca tca gtg gaa tac cct gat gat aat aat att tct gcg aaa 5117Asn Cys Thr Ser Val Glu Tyr Pro Asp Asp Asn Asn Ile Ser Ala Lys 1305 1310 1315ata gca ttc gat tat cgt aac gat tac ctt att acg gtg act gta cct 5165Ile Ala Phe Asp Tyr Arg Asn Asp Tyr Leu Ile Thr Val Thr Val Pro1320 1325 1330 1335tac gat gct tct ggt cct att gat tct gcc cga ttt aag atg acc tat 5213Tyr Asp Ala Ser Gly Pro Ile Asp Ser Ala Arg Phe Lys Met Thr Tyr 1340 1345 1350cag aca tta aaa ggc gta ttt cca gtt atc agc acc ttc cgt aca cca 5261Gln Thr Leu Lys Gly Val Phe Pro Val Ile Ser Thr Phe Arg Thr Pro 1355 1360 1365acc ggt tat gtt gag ctg gtg agt tat aaa gag aat ggg cat aaa gtg 5309Thr Gly Tyr Val Glu Leu Val Ser Tyr Lys Glu Asn Gly His Lys Val 1370 1375 1380acg gac acg gaa tat att cct tat gcg gct gca ctc act att caa ccc 5357Thr Asp Thr Glu Tyr Ile Pro Tyr Ala Ala Ala Leu Thr Ile Gln Pro 1385 1390 1395ggc aat gga caa cct gcg gtc agc aaa tcc tat gaa tat agt tca gta 5405Gly Asn Gly Gln Pro Ala Val Ser Lys Ser Tyr Glu Tyr Ser Ser Val1400 1405 1410 1415cat aac ttc ttg ggc tat tct tct ggc cgg aca agc ttt gat tcc agt 5453His Asn Phe Leu Gly Tyr Ser Ser Gly Arg Thr Ser Phe Asp Ser Ser 1420 1425 1430caa gat aat ttg tat ttg gtc aca ggg aaa tac act tat tca tcc att 5501Gln Asp Asn Leu Tyr Leu Val Thr Gly Lys Tyr Thr Tyr Ser Ser Ile 1435 1440 1445gaa cgg gtt tta gat ggt caa agt gtg gtt tca gta ata gaa cga gta 5549Glu Arg Val Leu Asp Gly Gln Ser Val Val Ser Val Ile Glu Arg Val 1450 1455 1460ttt aat aaa ttc cat tta atg acc aaa gaa gca aaa aca caa gat aat 5597Phe Asn Lys Phe His Leu Met Thr Lys Glu Ala Lys Thr Gln Asp Asn 1465 1470 1475aag aga att aca aca gaa att act tac aat gag gat cta tca aaa agt 5645Lys Arg Ile Thr Thr Glu Ile Thr Tyr Asn Glu Asp Leu Ser Lys Ser1480 1485 1490 1495ttc tca gag caa cca gaa aat tta caa caa cct tct cgc gtg tta acc 5693Phe Ser Glu Gln Pro Glu Asn Leu Gln Gln Pro Ser Arg Val Leu Thr 1500 1505 1510cgt tat acg gat ata caa aca aat act tca cga gaa gag act gtc aat 5741Arg Tyr Thr Asp Ile Gln Thr Asn Thr Ser Arg Glu Glu Thr Val Asn 1515 1520 1525att aaa agt gat gat tgg gga aat act cta ctt att act gag acc agt 5789Ile Lys Ser Asp Asp Trp Gly Asn Thr Leu Leu Ile Thr Glu Thr Ser 1530 1535 1540ggg ata cag aaa gaa tac gtt tat tat ccg gtc aat ggc gaa ggt aat 5837Gly Ile Gln Lys Glu Tyr Val Tyr Tyr Pro Val Asn Gly Glu Gly Asn 1545 1550 1555agt tgc cct gcc gat ccc ttg ggt ttt tct cgg ttc tta aaa tca gtt 5885Ser Cys Pro Ala Asp Pro Leu Gly Phe Ser Arg Phe Leu Lys Ser Val1560 1565 1570 1575acg caa aaa gga tcg cct gat gct gct caa agt gtc gca aat aaa gtg 5933Thr Gln Lys Gly Ser Pro Asp Ala Ala Gln Ser Val Ala Asn Lys Val 1580 1585 1590att cat tat aca tat caa aaa ttt cct act ttt acc ggc gct tat gtt 5981Ile His Tyr Thr Tyr Gln Lys Phe Pro Thr Phe Thr Gly Ala Tyr Val 1595 1600 1605aag gaa tat gtc agt aaa gtc tca gag acg ata gac aat aaa ata gcg 6029Lys Glu Tyr Val Ser Lys Val Ser Glu Thr Ile Asp Asn Lys Ile Ala 1610 1615 1620aga acc ttt agc tat gtt aac tca ccg acg agt aaa tct cat ggt tcg 6077Arg Thr Phe Ser Tyr Val Asn Ser Pro Thr Ser Lys Ser His Gly Ser 1625 1630 1635tta gca aaa ata acg tca gtg atg aat aac cag caa acg gtc acc aca 6125Leu Ala Lys Ile Thr Ser Val Met Asn Asn Gln Gln Thr Val Thr Thr1640 1645 1650 1655ttt aaa tat gaa tat tca gaa agt gag atg acc aca aat gct acg gtg 6173Phe Lys Tyr Glu Tyr Ser Glu Ser Glu Met Thr Thr Asn Ala Thr Val 1660 1665 1670acc ggt ttt gat ggc gca cat atg gaa tcg aaa aat gtg acg tct att 6221Thr Gly Phe Asp Gly Ala His Met Glu Ser Lys Asn Val Thr Ser Ile 1675 1680 1685tat acc cat cgg caa ctt cgt aaa gtt gat gta aac cac gtg att acc 6269Tyr Thr His Arg Gln Leu Arg Lys Val Asp Val Asn His Val Ile Thr 1690 1695 1700gat cag tct tat gat ctt ttg ggt cgc att aca ggg caa att att gat 6317Asp Gln Ser Tyr Asp Leu Leu Gly Arg Ile Thr Gly Gln Ile Ile Asp 1705 1710 1715ccc ggc acg gca aga gaa att aaa cgt aat tac gtt tat caa tat ccc 6365Pro Gly Thr Ala Arg Glu Ile Lys Arg Asn Tyr Val Tyr Gln Tyr Pro1720 1725 1730 1735ggc ggt gac gaa aat gat ttt tgg ccg gtg atg ata gaa gtt gat tct 6413Gly Gly Asp Glu Asn Asp Phe Trp Pro Val Met Ile Glu Val Asp Ser 1740 1745 1750caa ggc gtc aga cgt aaa acc cat tac gat gga atg gga cgt att tgt 6461Gln Gly Val Arg Arg Lys Thr His Tyr Asp Gly Met Gly Arg Ile Cys 1755 1760 1765tcg att gaa gaa caa gat gat gat ggc gcc tgg ggc aca tcg ggg att 6509Ser Ile Glu Glu Gln Asp Asp Asp Gly Ala Trp Gly Thr Ser Gly Ile 1770 1775 1780tat caa ggc aca tat cga aaa gtt ctt gcc aga caa tat gat gtt ttg 6557Tyr Gln Gly Thr Tyr Arg Lys Val Leu Ala Arg Gln Tyr Asp Val Leu 1785 1790 1795ggg cag ttg agc aag gaa att tca aat gat tgg tta tgg aat tta tct 6605Gly Gln Leu Ser Lys Glu Ile Ser Asn Asp Trp Leu Trp Asn Leu Ser1800 1805 1810 1815gcc aat cct ttg gtt cgt ctt gct acc ccg ttg gtt aca acg aaa acc 6653Ala Asn Pro Leu Val Arg Leu Ala Thr Pro Leu Val Thr Thr Lys Thr 1820 1825 1830tat aaa tat gat ggt tgg gga aat ctt tac agc acg gaa tac agt gat 6701Tyr Lys Tyr Asp Gly Trp Gly Asn Leu Tyr Ser Thr Glu Tyr Ser Asp 1835 1840 1845ggt cgg ata gag ctg gaa atc cat gat cct att acg agg aca att act 6749Gly Arg Ile Glu Leu Glu Ile His Asp Pro Ile Thr Arg Thr Ile Thr 1850 1855 1860caa ggg gtc aaa gga tta ggg atg tta aat att cag caa aat aat ttt 6797Gln Gly Val Lys Gly Leu Gly Met Leu Asn Ile Gln Gln Asn Asn Phe 1865 1870 1875gag caa ccg gct tcg atc aaa gct gtg tat cct gat ggt acg ata tat 6845Glu Gln Pro Ala Ser Ile Lys Ala Val Tyr Pro Asp Gly Thr Ile Tyr1880 1885 1890 1895agc acc cgt act tat cgt tat gat gga ttt ggt cgt aca gtg acg gaa 6893Ser Thr Arg Thr Tyr Arg Tyr Asp Gly Phe Gly Arg Thr Val Thr Glu 1900 1905 1910aca gat gca gaa ggt cat gct acc caa att gga tat gat gtg ttt gat 6941Thr Asp Ala Glu Gly His Ala Thr Gln Ile Gly Tyr Asp Val Phe Asp 1915 1920 1925cgt ata gtg aaa aaa acg ttg cca gac gga aca ata tta gaa tcc gct 6989Arg Ile Val Lys Lys Thr Leu Pro Asp Gly Thr Ile Leu Glu Ser Ala 1930 1935 1940tat gca agc ttt agc cat gaa gaa tta att tcg gca ctg aac gtg aat 7037Tyr Ala Ser Phe Ser His Glu Glu Leu Ile Ser Ala Leu Asn Val Asn 1945 1950 1955ggc aca cag ttg ggg gca tta gtt tat gat ggt ctt ggg cgg gta ata 7085Gly Thr Gln Leu Gly Ala Leu Val Tyr Asp Gly Leu Gly Arg Val Ile1960 1965 1970 1975agt gat acg gtg ggt ggt cgc aaa acg gaa tat tta tat ggg cct caa 7133Ser Asp Thr Val Gly Gly Arg Lys Thr Glu Tyr Leu Tyr Gly Pro Gln 1980 1985 1990ggt gac aaa ccg att cag tca att act cct tcg cat aat aag caa aat 7181Gly Asp Lys Pro Ile Gln Ser Ile Thr Pro Ser His Asn Lys Gln Asn 1995 2000 2005atg gat tac ctc tac tat ctt ggt agt gtg atg tcc aaa ttt acc acg 7229Met Asp Tyr Leu Tyr Tyr Leu Gly Ser Val Met Ser Lys Phe Thr Thr 2010 2015 2020ggg aca gac caa caa aac ttt cgt tat cat tcg aaa acg gga aca tta 7277Gly Thr Asp Gln Gln Asn Phe Arg Tyr His Ser Lys Thr Gly Thr Leu 2025 2030 2035tta tct gcg tca gaa ggc gta tct cag act aat tac agt tat ttc cca 7325Leu Ser Ala Ser Glu Gly Val Ser Gln Thr Asn Tyr Ser Tyr Phe Pro2040 2045 2050 2055tcg ggt gta tta cag cga gaa tca ttt tta cgg gat aat aaa ccg att 7373Ser Gly Val Leu Gln Arg Glu Ser Phe Leu Arg Asp Asn Lys Pro Ile 2060 2065 2070tca tcg ggc gag tac ctt tat acg atg tcc ggt ttg att caa cgt cat 7421Ser Ser Gly Glu Tyr Leu Tyr Thr Met Ser Gly Leu Ile Gln Arg His 2075 2080 2085aaa gat agt ttt ggt cat aat cat gtt tat agt tac gat gct cag gga 7469Lys Asp Ser Phe Gly His Asn His Val Tyr Ser Tyr Asp Ala Gln Gly 2090 2095 2100aga ttg gtc aaa aca gaa cag gat gca caa tac gct aca ttt gaa tat 7517Arg Leu Val Lys Thr Glu Gln Asp Ala Gln Tyr Ala Thr Phe Glu Tyr 2105 2110 2115gac aat gtt ggg cga ttg ata aca acg acg acc aaa gac acg acg tca 7565Asp Asn Val Gly Arg Leu Ile Thr Thr Thr Thr Lys Asp Thr Thr Ser2120 2125 2130 2135tta tcc caa tta gtg aca aaa atc gaa tat gat gct ttt gat cga gaa 7613Leu Ser Gln Leu Val Thr Lys Ile Glu Tyr Asp Ala Phe Asp Arg Glu 2140 2145 2150ata aaa cgc tcg cta att agt gac ttc tca ata caa gtt att acc tta 7661Ile Lys Arg Ser Leu Ile Ser Asp Phe Ser Ile Gln Val Ile Thr Leu 2155 2160 2165agc tat acg aag aat aat caa atc agt caa cgt atc acc tcc atc gat 7709Ser Tyr Thr Lys Asn Asn Gln Ile Ser Gln Arg Ile Thr Ser Ile Asp 2170 2175 2180ggg gtg gtt atg aaa aat gaa cgt tat caa tat gat aat aat caa cgc 7757Gly Val Val Met Lys Asn Glu Arg Tyr Gln Tyr Asp Asn Asn Gln Arg 2185 2190 2195tta agc caa tac caa tgt gag gga gaa caa tct ccg att gat cat acg 7805Leu Ser Gln Tyr Gln Cys Glu Gly Glu Gln Ser Pro Ile Asp His Thr2200 2205 2210 2215ggt cgt gta tta aat cag cag att tac cat tat gac caa tgg gga aat 7853Gly Arg Val Leu Asn Gln Gln Ile Tyr His Tyr Asp Gln Trp Gly Asn 2220 2225 2230att aag cgg ctc gat aat aca tat cga gat ggt aag gaa acg gtg gat 7901Ile Lys Arg Leu Asp Asn Thr Tyr Arg Asp Gly Lys Glu Thr Val Asp 2235 2240 2245tat cat ttc agt caa gcc gat cca act caa ctt att cgt att acc agc 7949Tyr His Phe Ser Gln Ala Asp Pro Thr Gln Leu Ile Arg Ile Thr Ser 2250 2255 2260gac aaa cag cag ata gag tta agt tat gat gct aat ggc aac cta aca 7997Asp Lys Gln Gln Ile Glu Leu Ser Tyr Asp Ala Asn Gly Asn Leu Thr 2265 2270 2275cgt gac gaa aaa ggg caa acg ctc att tac gat cag aat aat cgc ttg 8045Arg Asp Glu Lys Gly Gln Thr Leu Ile Tyr Asp Gln Asn Asn Arg Leu2280 2285 2290 2295gta cag gtc aaa gac cgg ttg ggc aat ctg gtg tgc agc tac cag tat 8093Val Gln Val Lys Asp Arg Leu Gly Asn Leu Val Cys Ser Tyr Gln Tyr 2300 2305 2310gat gca ttg aac aaa tta acc gca cag gtt ttg gcg aat ggt acc gtt 8141Asp Ala Leu Asn Lys Leu Thr Ala Gln Val Leu Ala Asn Gly Thr Val 2315 2320 2325aat cga cag cat tat gct tcc ggt aaa gtg acg aat att caa ttg ggt 8189Asn Arg Gln His Tyr Ala Ser Gly Lys Val Thr Asn Ile Gln Leu Gly 2330 2335 2340gat gaa gcg att act tgg ttg agc agt gat aag caa cga att gga cat 8237Asp Glu Ala Ile Thr Trp Leu Ser Ser Asp Lys Gln Arg Ile Gly His 2345 2350 2355caa agc gcc aag aat ggt caa tca gtc tac tat caa tat ggt att gac 8285Gln Ser Ala Lys Asn Gly Gln Ser Val Tyr Tyr Gln Tyr Gly Ile Asp2360 2365 2370 2375cat aac agt acg gtt atc gcc agt cag aac gaa aac gag ttg atg gct 8333His Asn Ser Thr Val Ile Ala Ser Gln Asn Glu Asn Glu Leu Met Ala 2380 2385 2390tta tcc tat aca cct tat ggc ttt agg agt tta att tcc tca tta ccg 8381Leu Ser Tyr Thr Pro Tyr Gly Phe Arg Ser Leu Ile Ser Ser Leu Pro 2395 2400 2405ggt ttg aat ggc gca cag gtt gat cca gta aca ggc tgg tac ttc tta 8429Gly Leu Asn Gly Ala Gln Val Asp Pro Val Thr Gly Trp Tyr Phe Leu 2410 2415 2420ggt aac gga tat cgt gtt ttc aac ccg gtt ctc atg agg ttt cac agc 8477Gly Asn Gly Tyr Arg Val Phe Asn Pro Val Leu Met Arg Phe His Ser 2425 2430 2435ccc gat agt tgg agt cct ttt ggt cgg gga ggg att aac cct tat acc 8525Pro Asp Ser Trp Ser Pro Phe Gly Arg Gly Gly Ile Asn Pro Tyr Thr2440 2445 2450 2455tat tgc caa ggc gat ccc ata aac cgg att gat ctg aac ggt cat ctt 8573Tyr Cys Gln Gly Asp Pro Ile Asn Arg Ile Asp Leu Asn Gly His Leu 2460 2465 2470agt gcc ggc ggg ata tta ggc att gtg cta ggg gca att ggc atc att 8621Ser Ala Gly Gly Ile Leu Gly Ile Val Leu Gly Ala Ile Gly Ile Ile 2475 2480 2485gtc ggg att gta tca ctg gga gcc gga gcg gcg att agc gcg ggt ctc 8669Val Gly Ile Val Ser Leu Gly Ala Gly Ala Ala Ile Ser Ala Gly Leu 2490 2495 2500att gct gcg ggg ggc gct ttg ggg gcg att gct tct acc agc gcg ctt 8717Ile Ala Ala Gly Gly Ala Leu Gly Ala Ile Ala Ser Thr Ser Ala Leu 2505 2510 2515gca gtt act gcg act gtc att gga ttg gct gcc gat tcg ata ggg att 8765Ala Val Thr Ala Thr Val Ile Gly Leu Ala Ala Asp Ser Ile Gly Ile2520 2525 2530 2535gcg tca gca gca tta tcg gaa aaa gat ccg aaa aca tct ggg ata tta 8813Ala Ser Ala Ala Leu Ser Glu Lys Asp Pro Lys Thr Ser Gly Ile Leu 2540 2545 2550aat tgg att agt gcg gga ttg ggg gtt tta agc ttt ggt atc agc gca 8861Asn Trp Ile Ser Ala Gly Leu Gly Val Leu Ser Phe Gly Ile Ser Ala 2555 2560 2565ata acc ttt acc tct tcg ctg gta aaa tcg gca cgg agt ggt tct cag 8909Ile Thr Phe Thr Ser Ser Leu Val Lys Ser Ala Arg Ser Gly Ser Gln 2570 2575 2580gca gtc agc gcg ggt gtt atc ggg tca gtg cct ctt gaa ttt ggt gaa 8957Ala Val Ser Ala Gly Val Ile Gly Ser Val Pro Leu Glu Phe Gly Glu 2585 2590 2595gtt gct agc cgt tcc agc aga cga tgg gat att gcg tta tct tcg ata 9005Val Ala Ser Arg Ser Ser Arg Arg Trp Asp Ile Ala Leu Ser Ser Ile2600 2605 2610 2615tcg ttg ggc gca aat gcg gcg tct ctc tct acg ggg ata gcg gcg gcg 9053Ser Leu Gly Ala Asn Ala Ala Ser Leu Ser Thr Gly Ile Ala Ala Ala 2620 2625 2630gcg gtt gca gac agt aat gcg aat gca gct aat att ctg gga tgg gta 9101Ala Val Ala Asp Ser Asn Ala Asn Ala Ala Asn Ile Leu Gly Trp Val 2635 2640 2645tcc ttt ggt ttt ggt gca gta tcg aca acc tca gga ata att gag ctt 9149Ser Phe Gly Phe Gly Ala Val Ser Thr Thr Ser Gly Ile Ile Glu Leu 2650 2655 2660acg cgt aca gct tat gca gtg aat cat cag act tgg gaa ctg agt tca 9197Thr Arg Thr Ala Tyr Ala Val Asn His Gln Thr Trp Glu Leu Ser Ser 2665 2670 2675tca gca ggt act tcg gag gaa gtg aag cct ata cgt tgt ctc gtt tca 9245Ser Ala Gly Thr Ser Glu Glu Val Lys Pro Ile Arg Cys Leu Val Ser2680 2685 2690 2695cac cgc tgg aat cag aag cag tga atgttaaccc tcctcgggca gttgagttaa 9299His Arg Trp Asn Gln Lys Gln 2700tcaaacgttt cgaaatagta ccgggaacta tttagccaat cgtccattga aacccgtaat 9359gtgttgcgac gtcgtttgac aatataaaga ttctgcgaac cgattggtta agtctcacga 9419aaaataacta ttaggcgaca tttgcgtcgc cttttttaag gaactttatc aggttacatt 9479tataagaagc tattttgttt tcgacggatg ttggtttctc tgagataaaa aatagaggga 9539aatgatgtca agggtgataa tggttaattg taaaatatgt gatattattc gcatttatat 9599gtcaatgtaa ttcctcttat tatttaattt tattgcattt gctacgcgaa atcgccttat 9659aattttattt ttaataaatt attatttcat cattaaacta aaataaatta tttctaga 9717 2 417 PRT Photorhabdus luminescens 2Met Gln Arg Ala Gln Arg Val Val Ile Thr Gly Met Gly Ala Val Thr 1 5 10 15Pro Ile Gly Glu Asp Val Glu Ser Cys Trp Gln Ser Ile Ile Glu Lys 20 25 30Gln His Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe Ile Asn Ser 35 40 45Arg Phe Phe Ser Phe Leu Ala Pro Asn Pro Ser Arg Tyr Gln Leu Leu 50 55 60Pro Lys Lys Leu Thr His Thr Leu Ser Asp Cys Gly Lys Ala Ala Leu 65 70 75 80Lys Ala Thr Tyr Gln Ala Phe Thr Gln Ala Phe Gly Val Asn Ile Ser 85 90 95Pro Val Glu Tyr Tyr Asp Lys Tyr Glu Cys Gly Val Ile Leu Gly Ser 100 105 110Gly Trp Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cys Gln Tyr Lys 115 120 125Gln Ala Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr Met Pro Ser 130 135 140Ser Met Thr Ala Ala Cys Ser Ile Met Tyr Gly Leu Arg Gly Tyr Gln145 150 155 160Asn Thr Val Met Ala Ala Cys Ala Thr Gly Thr Met Ala Ile Gly Asp 165 170 175Ala Phe Glu Ile Ile Arg Ser Gly Arg Ala Lys Cys Met Ile Ala Gly 180 185 190Ala Ala Glu Ser Leu Thr Arg Glu Cys Asn Ile Trp Ser Ile Asp Val 195 200 205Leu Asn Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu Ala Cys Cys 210 215 220Pro Phe Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu Gly Ala Ala225 230 235 240Val Val Cys Leu Glu Asn Tyr Asp Ser Ala Ile Ala Arg Gly Ala Thr 245 250 255Ile Leu Ala Glu Ile Lys Gly Tyr Ala Gln Tyr Ser Asp Ala Val Asn 260 265 270Leu Thr Arg Pro Thr Glu Asp Ile Glu Pro Lys Ile Leu Ala Ile Thr 275 280 285Lys Ala Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile Asp Tyr Ile 290 295 300Asn Ala His Gly Thr Ser Thr Pro Leu Asn Asp Leu Tyr Glu Thr Gln305 310 315 320Ala Ile Lys Ala Ala Leu Gly Gln Tyr Ala Tyr Gln Val Pro Ile Ser 325 330 335Ser Thr Lys Ser Tyr Thr Gly His Leu Ile Ala Ala Ala Gly Ser Phe 340 345 350Glu Thr Ile Val Cys Val Lys Ala Leu Ala Glu Asn Cys Leu Pro Ala 355 360 365Thr Leu Asn Leu His Arg Ala Asp Pro Asp Cys Asp Leu Asn Tyr Leu 370 375 380Pro Asn Gln His Cys Tyr Thr Ala Gln Pro Glu Val Thr Leu Asn Ile385 390 395 400Ser Ala Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile Ala Lys Val 405 410 415Arg 3 253 PRT Photorhabdus luminescens 3Met Glu Asp Ile Glu His Trp Ser Asn Phe Ser Gly Asp Phe Asn Pro 1 5 10 15Ile His Tyr Ser Ala Lys Ser Glu Ser Leu Arg Asn Ile Gln Gln His 20 25 30Pro Val Gln Gly Met Leu Ser Leu Leu Tyr Val Arg Gln Gln Phe Ser 35 40 45Gln Leu Thr Ser Ala Phe Thr Thr Gly Ile Leu Asn Ile Asp Ala Ser 50 55 60Phe Arg Gln Tyr Val Tyr Thr Ala Leu Pro His Gln Leu Arg Ile Asn 65 70 75 80Thr Lys Asn Lys Thr Phe Lys Leu Glu Asn Pro Ser Lys Glu Asn Thr 85 90 95Leu Phe Gly Asn Thr Ser Val Glu Asn Thr Met Glu Ser Ile Glu Asp 100 105 110Trp Ile Val Gln Asp Asn Cys Gln Lys Leu Thr Ile Thr Gly Glu Glu 115 120 125Val Cys Glu Lys Tyr Ala Val Phe Arg Tyr Tyr Phe Pro Ser Val Thr 130 135 140Ser Ile Gly Trp Phe Leu Asp Ala Leu Ala Phe His Leu Ile Ile Asn145 150 155 160Ser Thr Gly Phe Leu Asn Phe Glu His Tyr His Phe Asn Gln Leu Gln 165 170 175Asp Tyr Leu Ser Gln Ser Phe Thr Leu His Thr Gly Gln Ala Ile Lys 180 185 190Ile Arg Lys Glu Ile Val Asn Ser Thr Val Leu Leu Ser Ser Pro Asp 195 200 205Ile Cys Val Glu Leu Asn Pro Pro Leu Leu Ile Lys Asn Gly Asp Lys 210 215 220Asp Tyr Ile Arg Ile Phe Tyr Tyr Arg Cys Leu Tyr Asp Lys Lys Pro225 230 235 240Ile Phe Val Ser Lys Thr Ser Ile Ile Ser Lys Met Lys 245 250 4 186 PRT Photorhabdus luminescens 4Met Asn Val Leu Glu Gln Gly Lys Val Ala Ala Leu Tyr Ser Ala Tyr 1 5 10 15Ser Glu Thr Glu Gly Ser Ser Trp Val Gly Asn Leu Cys Cys Phe Ser 20 25 30Ser Asp Arg Glu His Leu Pro Ile Ile Val Asn Gly Arg Arg Phe Leu 35 40 45Ile Glu Phe Val Ile Pro Asp His Leu Leu Asp Lys Thr Val Lys Pro 50 55 60Arg Val Phe Asp Leu Asp Ile Asn Lys Gln Phe Leu Leu Arg Arg Asp 65 70 75 80His Arg Glu Ile Asn Ile Tyr Leu Leu Gly Glu Gly Asn Phe Met Asp 85 90 95Arg Thr Thr Thr Asp Lys Asn Leu Phe Glu Leu Asn Glu Asp Gly Ser 100 105 110Leu Phe Ile Lys Thr Leu Arg His Ala Leu Gly Lys Tyr Val Ala Ile 115 120 125Asn Pro Ser Thr Thr Gln Phe Ile Phe Phe Ala Gln Gly Lys Tyr Ser 130 135 140Glu Phe Ile Met Asn Ala Leu Lys Thr Val Glu Asp Glu Leu Ser Lys145 150 155 160Arg Tyr Arg Val Arg Ile Ile Pro Glu Leu Gln Gly Pro Tyr Tyr Gly 165 170 175Phe Glu Leu Asp Ile Leu Ser Ile Thr Ala 180 185 5 258 PRT Photorhabdus luminescens 5Met Glu Lys Lys Ile Thr Thr Phe Thr Ile Glu Lys Thr Asp 1 5 10Asp Asn Phe Tyr Ala Asn Gly Arg His Gln Cys Met Val Lys Ile Ser 15 20 25 30Val Leu Lys Gln Glu Tyr Arg Asn Gly Asp Trp Ile Lys Leu Ala Leu 35 40 45Ser Glu Ala Glu Lys Arg Ser Ile Gln Val Ala Ala Leu Ser Asp Ser 50 55 60Leu Ile Tyr Asp Gln Leu Lys Met Pro Ser Gly Trp Thr Thr Thr Asp 65 70 75Ala Arg Asn Lys Phe Asp Leu Gly Leu Leu Asn Gly Val Tyr His Ala 80 85 90Asp Ala Phe Ile Asp Glu Gln Val Thr Asp Arg Ala Gly Asp Cys Cys95 100 105 110Thr Asn Glu Asn Tyr Gln Asn Ser Val Lys Ser Val Pro Glu Ile Ile 115 120 125Tyr Arg Tyr Val Ser Ser Asn Arg Thr Ser Thr Glu Tyr Leu Met Ala 130 135 140Lys Met Thr Phe Glu Asp Thr Asp Gly Lys Arg Thr Leu Thr Thr Asn 145 150 155Met Ser Val Gly Asp Glu Val Phe Asp Ser Lys Val Leu Leu Lys Ala 160 165 170Ile Ala Pro Tyr Ala Ile Asn Thr Asn Gln Leu His Glu Asn Ile Asn175 180 185 190Thr Leu Phe Asp Lys Thr Glu Glu Pro Thr Lys Ser Asp Thr His His 195 200 205Gln Ile Ile Asn Leu Tyr Arg Trp Thr Leu Pro Tyr His Leu Arg Ile 210 215 220Leu Glu Gly Asn Asp Ser Thr Val Asn Arg Ile Tyr Val Leu Gly Lys 225 230 235Glu Pro Ser Asn Asp Arg Phe Leu Thr Arg Gly Arg Val Phe Lys Arg 240 245 250Gly Thr His Met255 6 1584 PRT Photorhabdus luminescens 6Met Lys Ala Thr Asp Ile Tyr Ser Asn Ala Phe Asn Phe Gly Ser Tyr 1 5 10 15Ile Asn Thr Gly Val Asp Pro Arg Thr Gly Gln Tyr Ser Ala Asn Ile 20 25 30Asn Ile Ile Thr Leu Arg Pro Asn Asn Val Gly Asn Ser Glu Gln Thr 35 40 45Leu Ser Leu Ser Phe Ser Pro Leu Thr Thr Leu Asn Asn Gly Phe Gly 50 55 60Ile Gly Trp Arg Phe Ser Leu Thr Thr Leu Asp Ile Lys Thr Leu Thr 65 70 75 80Phe Ser Arg Ala Asn Gly Glu Gln Phe Lys Cys Lys Pro Leu Pro Pro 85 90 95Asn Asn Asn Asp Leu Ser Phe Lys Asp Lys Lys Leu Lys Asp Leu Arg 100 105 110Val Tyr Lys Leu Asp Ser Asn Thr Phe Tyr Val Tyr Asn Lys Asn Gly 115 120 125Ile Ile Glu Ile Leu Lys Arg Ile Gly Ser Ser Asp Ile Ala Lys Thr 130 135 140Val Ala Leu Glu Phe Pro Asp Gly Glu Ala Phe Asp Leu Ile Tyr Asn145 150 155 160Ser Arg Phe Ala Leu Ser Glu Ile Lys Tyr Arg Val Thr Gly Lys Thr 165 170 175Tyr Leu Lys Leu Asn Tyr Ser Gly Asn Asn Cys Thr Ser Val Glu Tyr 180 185 190Pro Asp Asp Asn Asn Ile Ser Ala Lys Ile Ala Phe Asp Tyr Arg Asn 195 200 205Asp Tyr Leu Ile Thr Val Thr Val Pro Tyr Asp Ala Ser Gly Pro Ile 210 215 220Asp Ser Ala Arg Phe Lys Met Thr Tyr Gln Thr Leu Lys Gly Val Phe225 230 235 240Pro Val Ile Ser Thr Phe Arg Thr Pro Thr Gly Tyr Val Glu Leu Val 245 250 255Ser Tyr Lys Glu Asn Gly His Lys Val Thr Asp Thr Glu Tyr Ile Pro 260 265 270Tyr Ala Ala Ala Leu Thr Ile Gln Pro Gly Asn Gly Gln Pro Ala Val 275 280 285Ser Lys Ser Tyr Glu Tyr Ser Ser Val His Asn Phe Leu Gly Tyr Ser 290 295 300Ser Gly Arg Thr Ser Phe Asp Ser Ser Gln Asp Asn Leu Tyr Leu Val305 310 315 320Thr Gly Lys Tyr Thr Tyr Ser Ser Ile Glu Arg Val Leu Asp Gly Gln 325 330 335Ser Val Val Ser Val Ile Glu Arg Val Phe Asn Lys Phe His Leu Met 340 345 350Thr Lys Glu Ala Lys Thr Gln Asp Asn Lys Arg Ile Thr Thr Glu Ile 355 360 365Thr Tyr Asn Glu Asp Leu Ser Lys Ser Phe Ser Glu Gln Pro Glu Asn 370 375 380Leu Gln Gln Pro Ser Arg Val Leu Thr Arg Tyr Thr Asp Ile Gln Thr385 390 395 400Asn Thr Ser Arg Glu Glu Thr Val Asn Ile Lys Ser Asp Asp Trp Gly 405 410 415Asn Thr Leu Leu Ile Thr Glu Thr Ser Gly Ile Gln Lys Glu Tyr Val 420 425 430Tyr Tyr Pro Val Asn Gly Glu Gly Asn Ser Cys Pro Ala Asp Pro Leu 435 440 445Gly Phe Ser Arg Phe Leu Lys Ser Val Thr Gln Lys Gly Ser Pro Asp 450 455 460Ala Ala Gln Ser Val Ala Asn Lys Val Ile His Tyr Thr Tyr Gln Lys465 470 475 480Phe Pro Thr Phe Thr Gly Ala Tyr Val Lys Glu Tyr Val Ser Lys Val 485 490 495Ser Glu Thr Ile Asp Asn Lys Ile Ala Arg Thr Phe Ser Tyr Val Asn 500 505 510Ser Pro Thr Ser Lys Ser His Gly Ser Leu Ala Lys Ile Thr Ser Val 515 520 525Met Asn Asn Gln Gln Thr Val Thr Thr Phe Lys Tyr Glu Tyr Ser Glu 530 535 540Ser Glu Met Thr Thr Asn Ala Thr Val Thr Gly Phe Asp Gly Ala His545 550 555 560Met Glu Ser Lys Asn Val Thr Ser Ile Tyr Thr His Arg Gln Leu Arg 565 570 575Lys Val Asp Val Asn His Val Ile Thr Asp Gln Ser Tyr Asp Leu Leu 580 585 590Gly Arg Ile Thr Gly Gln Ile Ile Asp Pro Gly Thr Ala Arg Glu Ile 595 600 605Lys Arg Asn Tyr Val Tyr Gln Tyr Pro Gly Gly Asp Glu Asn Asp Phe 610 615 620Trp Pro Val Met Ile Glu Val Asp Ser Gln Gly Val Arg Arg Lys Thr625 630 635 640His Tyr Asp Gly Met Gly Arg Ile Cys Ser Ile Glu Glu Gln Asp Asp 645 650 655Asp Gly Ala Trp Gly Thr Ser Gly Ile Tyr Gln Gly Thr Tyr Arg Lys 660 665 670Val Leu Ala Arg Gln Tyr Asp Val Leu Gly Gln Leu Ser Lys Glu Ile 675 680 685Ser Asn Asp Trp Leu Trp Asn Leu Ser Ala Asn Pro Leu Val Arg Leu 690 695 700Ala Thr Pro Leu Val Thr Thr Lys Thr Tyr Lys Tyr Asp Gly Trp Gly705 710 715 720Asn Leu Tyr Ser Thr Glu Tyr Ser Asp Gly Arg Ile Glu Leu Glu Ile 725 730 735His Asp Pro Ile Thr Arg Thr Ile Thr Gln Gly Val Lys Gly Leu Gly 740 745 750Met Leu Asn Ile Gln Gln Asn Asn Phe Glu Gln Pro Ala Ser Ile Lys 755 760 765Ala Val Tyr Pro Asp Gly Thr Ile Tyr Ser Thr Arg Thr Tyr Arg Tyr 770 775 780Asp Gly Phe Gly Arg Thr Val Thr Glu Thr Asp Ala Glu Gly His Ala785 790 795 800Thr Gln Ile Gly Tyr Asp Val Phe Asp Arg Ile Val Lys Lys Thr Leu 805 810 815Pro Asp Gly Thr Ile Leu Glu Ser Ala Tyr Ala Ser Phe Ser His Glu 820 825 830Glu Leu Ile Ser Ala Leu Asn Val Asn Gly Thr Gln Leu Gly Ala Leu 835 840 845Val Tyr Asp Gly Leu Gly Arg Val Ile Ser Asp Thr Val Gly Gly Arg 850 855 860Lys Thr Glu Tyr Leu Tyr Gly Pro Gln Gly Asp Lys Pro Ile Gln Ser865 870 875 880Ile Thr Pro Ser His Asn Lys Gln Asn Met Asp Tyr Leu Tyr Tyr Leu 885 890 895Gly Ser Val Met Ser Lys Phe Thr Thr Gly Thr Asp Gln Gln Asn Phe 900 905 910Arg Tyr His Ser Lys Thr Gly Thr Leu Leu Ser Ala Ser Glu Gly Val 915 920 925Ser Gln Thr Asn Tyr Ser Tyr Phe Pro Ser Gly Val Leu Gln Arg Glu 930 935 940Ser Phe Leu Arg Asp Asn Lys Pro Ile Ser Ser Gly Glu Tyr Leu Tyr945 950 955 960Thr Met Ser Gly Leu Ile Gln Arg His Lys Asp Ser Phe Gly His Asn 965 970 975His Val Tyr Ser Tyr Asp Ala Gln Gly Arg Leu Val Lys Thr Glu Gln 980 985 990Asp Ala Gln Tyr Ala Thr Phe Glu Tyr Asp Asn Val Gly Arg Leu Ile 995 1000 1005Thr Thr Thr Thr Lys Asp Thr Thr Ser Leu Ser Gln Leu Val Thr Lys 1010 1015 1020Ile Glu Tyr Asp Ala Phe Asp Arg Glu Ile Lys Arg Ser Leu Ile Ser1025 1030 1035 1040Asp Phe Ser Ile Gln Val Ile Thr Leu Ser Tyr Thr Lys Asn Asn Gln 1045 1050 1055Ile Ser Gln Arg Ile Thr Ser Ile Asp Gly Val Val Met Lys Asn Glu 1060 1065 1070Arg Tyr Gln Tyr Asp Asn Asn Gln Arg Leu Ser Gln Tyr Gln Cys Glu 1075 1080 1085Gly Glu Gln Ser Pro Ile Asp His Thr Gly Arg Val Leu Asn Gln Gln 1090 1095 1100Ile Tyr His Tyr Asp Gln Trp Gly Asn Ile Lys Arg Leu Asp Asn Thr1105 1110 1115 1120Tyr Arg Asp Gly Lys Glu Thr Val Asp Tyr His Phe Ser Gln Ala Asp 1125 1130 1135Pro Thr Gln Leu Ile Arg Ile Thr Ser Asp Lys Gln Gln Ile Glu Leu 1140 1145 1150Ser Tyr Asp Ala Asn Gly Asn Leu Thr Arg Asp Glu Lys Gly Gln Thr 1155 1160 1165Leu Ile Tyr Asp Gln Asn Asn Arg Leu Val Gln Val Lys Asp Arg Leu 1170 1175 1180Gly Asn Leu Val Cys Ser Tyr Gln Tyr Asp Ala Leu Asn Lys Leu Thr1185 1190 1195 1200Ala Gln Val Leu Ala Asn Gly Thr Val Asn Arg Gln His Tyr Ala Ser 1205 1210 1215Gly Lys Val Thr Asn Ile Gln Leu Gly Asp Glu Ala Ile Thr Trp Leu 1220 1225 1230Ser Ser Asp Lys Gln Arg Ile Gly His Gln Ser Ala Lys Asn Gly Gln 1235 1240 1245Ser Val Tyr Tyr Gln Tyr Gly Ile Asp His Asn Ser Thr Val Ile Ala 1250 1255 1260Ser Gln Asn Glu Asn Glu Leu Met Ala Leu Ser Tyr Thr Pro Tyr Gly1265 1270 1275 1280Phe Arg Ser Leu Ile Ser Ser Leu Pro Gly Leu Asn Gly Ala Gln Val 1285 1290 1295Asp Pro Val Thr Gly Trp Tyr Phe Leu Gly Asn Gly Tyr Arg Val Phe 1300 1305 1310Asn Pro Val Leu Met Arg Phe His Ser Pro Asp Ser Trp Ser Pro Phe 1315 1320 1325Gly Arg Gly Gly Ile Asn Pro Tyr Thr Tyr Cys Gln Gly Asp Pro Ile 1330 1335 1340Asn Arg Ile Asp Leu Asn Gly His Leu Ser Ala Gly Gly Ile Leu Gly1345 1350 1355 1360Ile Val Leu Gly Ala Ile Gly Ile Ile Val Gly Ile Val Ser Leu Gly 1365 1370 1375Ala Gly Ala Ala Ile Ser Ala Gly Leu Ile Ala Ala Gly Gly Ala Leu 1380 1385 1390Gly Ala Ile Ala Ser Thr Ser Ala Leu Ala Val Thr Ala Thr Val Ile 1395 1400 1405Gly Leu Ala Ala Asp Ser Ile Gly Ile Ala Ser Ala Ala Leu Ser Glu 1410 1415 1420Lys Asp Pro Lys Thr Ser Gly Ile Leu Asn Trp Ile Ser Ala Gly Leu1425 1430 1435 1440Gly Val Leu Ser Phe Gly Ile Ser Ala Ile Thr Phe Thr Ser Ser Leu 1445 1450 1455Val Lys Ser Ala Arg Ser Gly Ser Gln Ala Val Ser Ala Gly Val Ile 1460 1465 1470Gly Ser Val Pro Leu Glu Phe Gly Glu Val Ala Ser Arg Ser Ser Arg 1475 1480 1485Arg Trp Asp Ile Ala Leu Ser Ser Ile Ser Leu Gly Ala Asn Ala Ala 1490 1495 1500Ser Leu Ser Thr Gly Ile Ala Ala Ala Ala Val Ala Asp Ser Asn Ala1505 1510 1515 1520Asn Ala Ala Asn Ile Leu Gly Trp Val Ser Phe Gly Phe Gly Ala Val 1525 1530 1535Ser Thr Thr Ser Gly Ile Ile Glu Leu Thr Arg Thr Ala Tyr Ala Val 1540 1545 1550Asn His Gln Thr Trp Glu Leu Ser Ser Ser Ala Gly Thr Ser Glu Glu 1555 1560 1565Val Lys Pro Ile Arg Cys Leu Val Ser His Arg Trp Asn Gln Lys Gln 1570 1575 1580 7 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 7acacagcagg ttcgtcag 18 8 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 8ggcagaagca ctcaactc 18 9 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 9attgatagca cgcggcgacc 20 10 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 10ttgtaacgtg gagccgaact gg 22 11 37948 DNA Photorhabdus luminescens CDS (15171)..(18035) orf5 11tgttgctgga ccgtggagat tatgcctatc gtcagttaga acgagacacg ctcaatgaag 60ccaagatgtg gtatatgcaa gcactgcatc tgttaggcga taaacctcat ctatcgttca 120gttcagagtg gagcaaaccg agtttaggcg acgctgccgg aacggaaaga caaaagcaac 180acgcccaagc aatggccgct ctgcgacaag gtgatgttag tcggcacaac aacccgacag 240atcttttctt gccacaggtc aatgaagtga tgcaaaacta ttggcaaaaa ttggaacaac 300ggctgtataa cctgcgtcat aacctcacta ttgacggcca accgctacat ctgcctattt 360acgctacacc ggcagatcca aaagcattac ttagcgccgc tgtcgctagc tccgaaggtg 420gggtagctct ctcacagcca tttatgtcac tgtggcgttt cccacacatg ctggaaaacg 480cgcgtggtat ggtcagtcag ctcactcaat tcggctctac gctacaaaat attatcgaac 540gtcaggatgc ggaagcttta aacacgctct tgcagaatca agcagcagaa ctgatattga 600ctcatctcag catacaggac aaaaccatcg cagagctgga tgcggaaaaa atcgtactgg 660aaaaatccaa agccggggcg caatcacgct ttgacagcta caaaaagtta tacgacgaaa 720atatcaatgc gggtgaaaac cgggctatag cattgcatgc ctccgttgct ggcctcagca 780ctgccctgca agcatcacgt ctggcgggcg ctgcgcttga tctggcgccc aacattttcg 840gtctcgctga tggcggtagc cgttggggag cgattgccga agcgacaggt aatgttatgg 900aattctccgc cagtgtgatg aacaccgaag cggataaaat cagccagtca gaagcctatc 960gccgtcgccg tcaggaatgg gaaatccagc gtaatcatgc cgaagcagag ataaaacaga 1020tcgatgctca acttcaatca ctggcagtac gccgtgaagc cgcggtattg cagaaagcca 1080gcctaaaaac ccaacaggaa cagactcatg ctcaattgac tttcctgcaa cgtaaattca 1140gtaatcaagc gttgtactac tggctacgcg gtcggctagc tgctatttac ttccaatttt 1200acgatttggc cgtagcgcgt tgtctgatgg ctgaaatggc ttatcgttgg gagactaatg 1260agaccgcggc aagctttatc aaacccggcg cctggcaggg aacccatgcg ggtttactgg 1320ctggtgaaac cttgatgctg aatctggcgc aaatggaaga tgcccatttg aggtgggatc 1380aacgcgctct ggaagtggaa cggaccattt cattgacgca acactatgga gcactgccag 1440aaaaatcgtt taatttagcc acacggattt ctaccctgct agcaggtggt acaactgact 1500ccattgatga tcatcccgtt acattagaaa acgaccaact tagtgccaaa atctctctgt 1560caggtctgtc attagataat gactacccag atggcaacgg cgtaggcaac attcgacgca 1620ttaaacaaat cagtgtcacc ttgccagccc tgttaggacc atatcaggat gtacaagcta 1680ttctgtccta cggaggaagt gaaatcggat tagctgaaag ctgtaaatca ctggcgatct 1740ctcatgggat caatgacagt ggtcaattcc agttggattt taacaatggt aagttcctgc 1800cgtttgaagg gattgcgatt aacgatactg gcacattgac actcagtttc ccccaatgcg 1860actgtcaaac aagaaaacat gttgcagact ttgagtgata ttattctgca tattcgctat 1920accatccgcc aataaccacc tcaattaaat accaaaaaca ggctcctaaa cggggcctga 1980acttttcacg aatatatacc actcacagtc tgctctcttt acctgtctga cgctcgttat 2040aacagagata tttccttttc tcgtgagtcc catcacctac tataaaatat caaccctctt 2100ctttttcata atatgcaata tgtaacaaat gcaattattt catttagtta ttgttaacta 2160gttatattac ttatgatgta attataaatt ttgttattgc atcacaatag ccatttaaat 2220aaataataac gttgtgaaat agttgatagt taaatggtgt ttttatttag ccgttatttt 2280caacccaatt tcagaccgct atcagacgtt acctgtgttg cctttgtttt gatagatata 2340aataacctta tttatatcca cggtactcag accagcataa atgttttatt tacctaacat 2400ttaaaaggaa taaacatgaa cacactcaaa tccgaatatg aaaacgcgtt agtagcaggt 2460tttaataatc taaccgatat ttgtcatctc tcttttgacg aacttcgcaa aaaagtgaag 2520gacaaactct catggtcaca gacccaaagc ttatatcttg aagcacagca agtgcaaaag 2580gacaatctcc tgcatgaagc ccatattctg aaacgcgcca atcctcattt acaaagtgcg 2640gtccatcttg ccctgacaac acctcatgct gaccagcaag gttataatag cagatttggc 2700aatcgcgcca gcaaatatac agccccaggc gcaatttctt ccatgttttc tcctgcggct 2760tatttagctg aactttatcg tcaggcacgg aatttacatg atgaaaattc tatttatcat 2820ttggatacac ggcgtccgga tctaaaatca ttggtgctca gccagaaaaa tatggatacg 2880gagatttcca cactttctct gtctaatgac atgttgctag agggtattaa aactctgttc 2940aaggacaagc tgctggaggc tctgaagaat attaaatctc tgtccaagga cgagctgctg 3000gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat 3060attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc 3120aaggacgatc tgctggaggc tttgaagaat attaaacctc tgtccaagga cgatctgctg 3180gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat 3240attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc 3300aaggacgatc tacaggaatg tattgaaatt ctattcaatc tggacagcca cactaaagta 3360atgaaagcgt tatccaattt ccgcgtttct ggcatgatgc catatcacga tgcttatgaa 3420agcgtgcgta aggttgttca attacaggct ccggtgtttg aacacgttgt tagtacatca 3480ctagaaacga ctatcgatga actaaaatat caagcttctt tgttggaaat taattcttct 3540gtctcgccta aattatttac tatcttgact gaagaaatta ctacaatcaa tgcaagaagt 3600ctctatgagg aaaattttgg taatattaaa ccttctctaa taggaaaacc ggaatatctg 3660aaaagttatt acaatctgag tgatgaagag tttagcgatt tcattaaaat aagaactata 3720cttcttccag aagaagaaat agcaattact gatcttgcat cgcgtactac tagtacacaa 3780cagactatcg aaaatcctga ttatcgtgct ctattgaaaa ttaataagtt tattcgtcta 3840ttcaaagcta taaacttatc accgacggta ttaagtggaa tcctccgcag catcagcaca 3900gaattcaata tcaataaaga aatattacaa aaaatctttc gtgttaaata ctatatgcaa 3960cgttatggta ttgacactga gactgcatta atactatgca aggtaccaat ttcacaatat 4020atcaatgacg gacatctaag tcagtttgat cgtttattta attcccccaa actgaatggt 4080caagattttt ccgtcaatgg tactcagaat attgatttaa ccctaagcag taccaacaac 4140tggaataaaa cagtacttaa acgtgctttt aacctcgatg atatctcatt aaatcgacta 4200ctaaaaatta ccaatccggt caatactacc gaaatgataa ctaatgatat agagaatctt 4260tctcatctct ataggacaaa attactggca gatatccatc agttaactat tgatgaactg 4320gggttactgt tggaagccat aggtaaagga acaaccaatt tatctgagat tactcctgac 4380aatctggtta ctctaattaa caaactctat gctgtcacta gctggctacg tacacaaaag 4440tggagtgtct atcagttgtt tatgatgact actgataaat ataacaaaac cctaaccccg 4500gaaatacgga atttactgga taccgtctac aatggcttgc aagattttaa taagaagatg 4560ttgaaagctg aagaagatct agagaaaacc aaaaagaaat tgcagagcgc caaggaaaat 4620ctggaaaaat tcccggaaaa ccagccacaa ctccaagaag acaggaaaaa agcccagaga 4680agactgaata aagctgaaga gacccacgaa aaagccgaga aaaacctaga tgaggtcagg 4740aaaaatctgc caaaagccat atctccttat atcgccgccg ctctgcaatt accatctgaa 4800catgcggcat attccatact catctgggca gataatctgg aacccggcat aggaaaaatg 4860acagcggaaa aattatggaa ctggttgcgg aaaaatcccg ttacggctca acctgaattc 4920caaaaacaag ctgaacctgt ggtccagtat tgccagcgcc tggcacaact agcgttgatt 4980taccgttcta ccggccttaa cgaaaacacc ttaagtctgt ttgtgacaaa gccgcaacac 5040tttgttatta aaaccaaagc acccgaaaca actgaaacaa caccagcaca tgacgtatca 5100acactaatgt cactaacgcg ttttactgac tgggttaact cactaggtga aaacgcctct 5160tctgtactaa ccgaatttaa aaaaggaaca ttaacagcag aactattggc taaggctatg 5220aatcttgata aaaatctact ggagcaagcc aagattcagg caaaaactga tttgtccaac 5280tggccatcta tcgacaacct attgcagtgg attaacatct cacgtcaatt gaacatctct 5340ccacaaggca tttccacact gactcaagta ttgaccgcag aacctcccgc taactatacc 5400caatgggaaa acgccgctgc gatattaacc gccgggctgg acacccaaaa gactaacgcc 5460ctacatgcgt ttctggatga gtctcgcagt gctgcgttaa gcacatacta tatttattct 5520cataaccaaa aagatcgaga agcaagaaaa catacagtaa ttaaaaaccg tgatgatcta 5580tatcaatacc tattgatcga taaccaagtt tccgccgaca ttaaaactac agagatcgct 5640gaagctatcg ctagtatcca actgtatatt aaccgcgcgt tgaaaaatat ggagggagat 5700actgtcacaa gtgtcaccag ccgctcattc ttcaccaact gggataaata caataaacgc 5760tacagcactt gggccggtat ggctaaactc ctttactatc cagagaatta catcgatccg 5820acgctacgta ttgggcagac aaaaatgatg gatacgttgc tgcaatccat cagccaaagc 5880caattaaata tcgataccgt agaagatgcc tttaaatctt acctaacatc attcgaacag 5940gtggctaatc tggaaatcct cagcgcctac catgacaaca ttaataatga tcaaggatta 6000acttacttta tcggacgtag taaaacagaa gtgaatcaat attattggcg cagtgtggat 6060cacaataaat ccagcgaagg taaattcccc gctaatgcct ggagtgagtg gtacaaaatt 6120gattgtccaa ttaaccccta caaagatact attcacccgg taattttcca atctcgcctg 6180tatcttatct ggctggaaca aaaaaaggcg actaaacagg aaggtgataa aaccgcctcg 6240ggttattatt atgaactgaa attagcgcat atccgttatg acggcacctg gaatacacca 6300gtcacctttg atgtaaacca aaaaatatcc gatttaaatc tgggaaataa aacacctgga 6360ctttactgct caagctttca aggcagagat gaattgctgg tgatgtttta taaaaaacaa 6420gatcaattaa atcaatacac aaacacagta ccaataaaag gactatatat cacttccaat 6480atgtcttcta aggaaatgac acctgaaaat cacaaaccta acgcttataa acagtttgat 6540actaatagta ttattggtgt caataatcgc tatgcagaaa gctacgaaat cccttcatca 6600gtaaatagta ataacggtta tgattgggga gatggctatc tgagtatggt gtatggcgga 6660aatatttcag ccatcaaact ggagtcctca tcagataagt taaaactctc accaaggtta 6720agaattattc ataatggact tgtaggccga caacgcaacc aatgcaacct gatgaagaaa 6780tacggtcagc ttggtgataa atttattatt tatactactc taggtattaa ccccaataat 6840ttgtcgaata aaaaattcat ttaccctgtt tatcagtata gtgggaacac taccaataat 6900gagaaaggac gtctgctgtt ttatcgagaa agtactacta actttgtaag agcctggttc 6960cctaaccttc cctctggctc tcaagaaatg tccacaacca ctggcggtga cattagtggt 7020aactatggtt atattgataa caaacatagt gacgatgttc catttaaaca atatttctat 7080atggatgacc acggtggtat tgacactgat gtttcaggga tattatctat taatacgaac 7140attaatcatt caaaagttaa agtaatagtg aaagccgaag gtatcacaga gcaaactttt 7200gtagcgagcg aaaacagtaa tgtccccacc aatccgtccc gcttcgaaga aatgaattat 7260cagtttaaag agcttgaaat agatatctcc acactgacat ttcataataa tgaagcaagt 7320attgatatca cctttatcgc atttgctgag aaatttgacg ataatagtaa tgatcgtaac 7380ttaggcgaag aacatttcag tattcgtatt atcaaaaaag cggaaactga taatgccctg 7440accctgcacc ataatgcaaa cggggcgcaa tatatgcagt ggggaaactc ttgtattcgc 7500cttaatacgc tatttgcccg tcaattaatt agccgagcca acgcggggat agatactatt 7560ttgagcatgg acactcagaa tattcaggaa cctaaattag gagaagattc tcctgatgct 7620atggaaccaa tggacttcaa cggcgccaac agcctctatt tctgggaact gttctactac 7680accccgatgc tgattgctca acgtttgctg cacgaacaaa acttcgatga ggctaaccgt 7740tggctgaaat atgtctggaa cccatccggt tatattgtca atggtcaaat gcaacattac 7800cgctggaatg ttcgcccatt acaagaagac actagttgga acgatgatcc gttggattca 7860tttgatcctg ataccatagc tcaacatgat ccaatgcact acaaagtcgc cacctttatg 7920cgcaccctag atctgttgat cgaacgggga gattacgcct atcgccaatt ggagcgggac 7980acactcgctg aagccaaaat gtggtatatg caggcactgc atctattggg tgataaacct 8040catctaccac tcagttcagc atggaatgat ccagagctag aagaggccgc agctcttgaa 8100aaacaacagg cacatgccaa agaaatagca gatttacgac aaggacttcc tacatccaca 8160gggtctaaag atgaaatcaa aacagatctt ttcctgccgc aagtcaacga agtgatgctg 8220agctactggc agaaactaga acaacggttg tataacctgc gccataacct ctctattgat 8280ggtcaacctt tacatttgcc tattttcgca acaccagcag atccaaaagc gctgctcagc 8340gccgctgtcg ccagttcaca aggtggaagt aatcttccat cagaatttat atcagtgtgg 8400cgtttccctc atatgctgga aaacgcccgt agtatggtca gtcagctaac ccaattcggc 8460tccacattgc aaaatattat cgaacgtcaa gatgcggagg cattaaacac gctgttgcaa 8520aatcaggcgg cagaactgat attgaccaat ctcagcatac aggacaaaac catccaagag 8580ctggatgctg aaaaaactgt gctagaaaaa aaccgcgccg gaacccagtc gcgttttgat 8640agctacagca aattctacga tgaagacatc aacgcgggtg aaaaacaggc aatggcgttg 8700cgtgcttccg tcgctggcat ctctacagcc cttcaagcat cacatctggc gggcgcagca 8760cttgatctgg cgcccaacat cttcggcttc gctgatggtg gcagccgttg gggggcgatc 8820gcccaagcca caggtaatgt catggagttc tccgccagtg ttatgaacac cgaagcggat 8880aaaatcagcc aatctgaagc ctaccgtcgg cgtcgtcagg aatgggaaat tcagcgtaat 8940aacgccgagg cagagctgaa acaaatcgat gctcaacttg gttcgctggc agtgcgccgt 9000gaagccgcag tattgcagaa aaccagccta aaaacccaac aagagcagac tcatgcacaa 9060ctgaccttcc tgcaacataa gttcagtaat caggcgctgt acaactggct gcgtggtcga 9120ttgtccgcca tttacttcca gttctatgat ttaacggtag ctcgctgttt gatggcggaa 9180atggcctatc gctgggagac taacgatacc gcatcacgct ttatcaaacc cggcgcctgg 9240cagggaaccc atgccggttt gctcgcgggt gaaaccttaa tgctgaatct ggcacagatg 9300gaagatgccc acctgaaaca ggataaacgc gtactggagg tagaacgtac cgtttcgctg 9360gccgaagtct atgccaaatt accgcaagat aaatttatcc tgactcagga aatagagaag 9420ttggtgagta aaggttcagg cagggccggc aaggacaata ataagctggc gtttagtacc 9480aataccaata cctctctaga agcgtccatt tcgttatcta ccttgaacat tagcagcgat 9540tatcctgatt ctattggtaa aacccgtcgt attaaacaga tcagcgttac cctgccagca 9600ctgctaggac cctatcagga tgtgcaagca attctgtctt acagcggaaa agcctctgaa 9660ttggctgaaa gttgcaaatc attagcggtt tctcatggga tgaatgacag cggtcagttc 9720caactggatt tcaacgatgg caaattcctg ccgttcgaag gaatcaaaat cgatgaaggt 9780acgctgacat tgagcttccc aaatgcaatt agtaaagaag acaaaaaaga cgaaaaaggc 9840aaacaacaag ccatgctgga gagtctgaac gacatcattc tgcatattcg ctacaccatt 9900cgccaataac gattttaatt aagtgctaaa acaggcccct aagcggggcc tgcaaggagt 9960ctttcatgca aaattcacaa gatttcagta ttacagaact atcattgccc aaaggaggag 10020gcgctatcac gggaatgggg gaagctttaa cccccaccgg gccggatggg atggccgcgc 10080tgtctctgcc gttgcctatc tctgccgggc gcggttatgc tccgtcactc gccttaaact 10140acaacagcgg cgccggtaac agcccatttg gtctgggctg ggattgcaac gttatgacca 10200tccgccgccg cacccatttt ggcgttccac attatgatga aaccgatacc tttctggggc 10260cagatggcga ggtactggtg gtagcggatc aatcccgcga cgaatcgaca ttacagggta 10320tcaacttagg caccgccttt accgttaccg gataccgttc ccgtctggag agtcatttca 10380gccgattgga atattggcaa cccaaggcaa cacccaagac aactggcaaa acagattttt 10440ggctgatata tagcccagat ggacaagtac atttactggg taaatcacca caagcccgga 10500tcagcaaccc gtcagacatc actcaaacag cacaatggtt gctagaagcc tctgtgtcac 10560cacatggtga acaaatttat tatcaatatc gggccgagga taacaccggt tgcgaagctg 10620atgaaattac tctccatcca caggccgccg cgcaacgtta tctacacaca gtgtattacg 10680gcaaccggac agccagcaaa acgttacccg gtctggatgg cagcgcccca ccacaagcag 10740actggttatt ctatctggta tttgattacg gcgaacgcag taacaacctg agaacgccgc 10800cagcattttc gactacaggt agctggcttt gtcgccagga ccgtttttcc cgttatgaat 10860atggttttga gattcgtacc cgccgcttat gccgtcaggt attgatgtat caccacctgc 10920aagctctgga tagcgagata aaagaacaca acggaccaac gctggtttca cgcctgatac 10980tcaattatga cgaaagcgca atcgccagca cgctggtatt cgttcgtcga gtaggccacg 11040agcaagacgg tactgccgtc accctgccgc cattagaatt ggcgtatcag gatttttcac 11100cgcaacataa cgctcgctgg caatcgatga atgtgctggc aaacttcaat gccattcagc 11160gctggcaact agttgatcta aaaggcgaag gattccccgg tctgctatat caagataaag 11220gcgcctggtg gtaccgctcc gcacaacgtt ttggcaaaat tggctcagat gccgtcactt 11280gggaaaaaat gcaacctttg tcggttatcc cttccttgca aagtaatgcc tcgctggtgg 11340atatcaatgg agacggccaa cttgactggg ttatcaccgg accgggatta cggggatatc 11400atagtcagca tccagatggc agttggacac gttttacccc gctcaacgct ctgccagtgg 11460aatatactca tccacgcgcg caactcgccg atttaatggg agctggactt tctgatttag 11520tactgatcgg ccctaagagt gtacgtttat atgccaatac ccgcgacggc tttgccaaag 11580gaaaagatgt agtgcaatcc ggtgatatca cactgccagt accgggcgcc gatccgtgta 11640agttggtggc atttagtgat gtattgggtt ccggtcaggc acatctggtt gaagtgagcg 11700cgactaaagt cacctgctgg cctaatctgg ggcacggacg ttttggtcaa ccaattactc 11760ttccgggatt tagccaacca gaagcgacgt ttaatcctgc tcaagtttat ctggccgatc 11820tagatggcag cggcccgact gatctgattt atgttcacac agatcgtctg gatatcttcc 11880tgaataaaag cggcaacggc ttcgccgcac cagtaactct ccccttccca gccggagtgc 11940gttttgatca tacctgtcag ttacaagtgg ccgatgtaca agggttaggc gtcgccagcc 12000tgatattaag tgtgccgcat atgactcccc atcactggcg ttgcgatctg accaacacaa 12060aaccgtggtt actcagtgaa atgaacaaca atatgggggc tcatcacacc ctgcgttacc 12120gtagttccgc ccagttctgg ctggatgaaa aagccacggc actggatgcc ggacaaatac 12180cagtttgtta tctacccttc ccggtacaca ccctatggca aacggaaata gaggatgaaa 12240tcagcggcaa caaattagtc acaatactac gttatgcaca tggcgcctgg gatggacgtg 12300agcgagaatt tcgcggattt ggttatgttg aacagaaaga cagccatcaa ctggcccaag 12360gcagtgcgcc agaatgcaca ccacctgcac tgacccaagg caacgcgcct gaactcacat 12420cacccgcgct gacccaaggc aacgctccag aactcacacc acctgcgatg acccaaagca 12480acgcgcctga actcacatca cccgcgctga cccaaggcaa cgcgccagaa ttcacatcac 12540ccgcgctggc ccaaggcaat gcgccagaac tcacaccacc tgcgatgacc aaaaactggt 12600atgccaccgg aatacccatg atagataaca cattatcgac agagtattgg catggtgatc 12660accaagcttt tgccggtttt tcaccacgct ttacgacctg gcaagatggt caagatattc 12720tgctcacacc ggaaaatgat aacagtcagt actggctaaa ccgggcactg aaaggtcaac 12780tgctacgcag tgaactgtac ggcgaggatg gcagtacaca ggaaaaaatt ccctacacag 12840tcactgaatt tcgcccacag gtacgtcggt tacagcatac cgatagccga taccttgtgc 12900tttggtcatc tgtagttgaa agccgcaact atcattacga acgtatcgcc agcgatcctc 12960aatgcagcca aaagattacg ctatccagcg atctatttgg tcaaccgcta aaacaggttt 13020cggtacagta tccacgccgc cagcaaccgg caagcagtcc gtatcctgat acgttgcctg 13080ataagttatt tgctaacagc tatgatgacc agcaacacaa attacggctc acctatcaac 13140agttcagttg gcatcatctg accgacaata ccattctgat gttaggatta ccggatagta 13200cccgcagcga tatctttgct tatagcgctg aacatgtccc tactggtggt ctaaatctgg 13260aaatcctaaa tgataaaaat agtctgattg cggagaataa acctcgtgaa tacctcggcc 13320agcaaaaaac cgtttatacc gacgggcaaa atgcaacgcc atcgcaaacg ccaacacgac 13380aagcgctgat tgccttcacc gagacaacag tatttaatca atccacacta tcagcgtttg 13440atgggagtat ctcatctgct caattgtcaa cgacgctgga acaagccgga taccagcaaa 13500cagattatct attcccgcgc actggagaag ataaagtctg ggcagctcgt cgtggctata 13560ctgattacgg cacagccgaa cagttctggc ggccgcaaaa acagagcaac actcaactca 13620cgggcaaaat cacgctcact tgggatgcaa actattgcgt cgtcacacaa acccgggatg 13680cggctggact gacaacctca gccagatatg attggcgttt tctgaccccc gttcaactca 13740cggatatcaa cgacaatcag caccttacca cgctggatgc actgggccga ccaatcacac 13800tgcgcttttg gggaaccgaa aacggtaaga tgactggtta ttcttcaccg gaaaaaatat 13860cgttttctcc accatctgat gttgacgccg cgattaagtt aacaacgcca atccctgtag 13920cacagtgtca ggtctacgca cccgaaagct ggatgcccat attaaagaaa accctcaata 13980acctggcaga gcaagagcgg aaagagttat ataacacccg aatcatcacc gaagacggac 14040gcatctgtac cctagctcac cgccgctggg taaaaagcca aagtgcagtc acccagccaa 14100tcaatctgtc aaacggcagt ccccgtttac cccctcatag cctcacattg actacggatc 14160gttatgaccg cgatcttaag caacagattc gtcaacaagt agtattcagt gatggctttg 14220gccgtttact gcaagcatct gtacgacatg aagcaggcga agcctggcaa cgtaaccaag 14280acggcgctct ggtgacaaaa atggaagata ccaaaacgcg ctgggcggtt acgggacgca 14340ctgaatatga caataaggga caaccgatac gcacctatca accctatttc ctcaacgact 14400ggcaatacgt cagtaatgac agtgcccggc ggacagaaga agcctatgca gatacccatg 14460tctatgatcc cattggtcga gaaatcaagg tcactaccgc aaaaggctgg ttccgtcgaa 14520ccttgttcac tccttggttt actgtcaatg aagatgaaaa tgacacagct actgaggtga 14580aggtaaagaa gaaagaatgt aaagaaggta aagaaggtaa agatgtaatt tgatcaatcc 14640cgcccggttg aagggcggga aacataacat aatatagagg tgaaacgtgt cattcataat 14700gccgtcagat actcaactta tgagttggtt gatcattggt tttattgcgg cctggggcgg 14760attagtaagg tacctcattg atatacaaaa caaacaatgt aaatggaatt ggatcaacgt 14820actctgtcaa ctcattatct cctgttttac cggtatattg ggaggactgc tgagttttga 14880aagcggcggc agcccctata tgacttttgc gattgccggg ctatttggca ccacgggaag 14940ttctggattg aactggatct ggcgtcgcct ttttatgcat tatcgcgatg atggaggaaa 15000gcaataaggc attcccactg ccgcaaaaac catctgtctc cggcagttaa accgggaaat 15060tacctactac aactattgta agaaaacgaa tatatagaaa aactaacatg cagataaaaa 15120ctgcgattgc agaacagatg acacacaacg ccccaacaac gaggtaaatc atg aaa 15176 Met Lys 1aac atc gat cct aaa ctt tat caa aag acc cct gtc gtc aac atc tac 15224Asn Ile Asp Pro Lys Leu Tyr Gln Lys Thr Pro Val Val Asn Ile Tyr 5 10 15gat aac cga ggt cta acg atc cgt aac atc gac ttt cac cgt acc acc 15272Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His Arg Thr Thr 20 25 30gca aac ggc gat acc gat atc cgt att act cgc cat caa tat gac tcc 15320Ala Asn Gly Asp Thr Asp Ile Arg Ile Thr Arg His Gln Tyr Asp Ser 35 40 45 50ctt ggg cac cta agc caa agc acc gat ccg cgt cta tat gaa gcc aaa 15368Leu Gly His Leu Ser Gln Ser Thr Asp Pro Arg Leu Tyr Glu Ala Lys 55 60 65caa aaa tct aac ttt ctc tgg cag tat gat ttg acc ggt aat att ttg 15416Gln Lys Ser Asn Phe Leu Trp Gln Tyr Asp Leu Thr Gly Asn Ile Leu 70 75 80tgt aca gaa agc gtc gat gct ggt cgc act gtc acc ttg aat gat att 15464Cys Thr Glu Ser Val Asp Ala Gly Arg Thr Val Thr Leu Asn Asp Ile 85 90 95gaa ggc cgt ccg cta ctg aca gta act gca aca ggt gtc ata caa acc 15512Glu Gly Arg Pro Leu Leu Thr Val Thr Ala Thr Gly Val Ile Gln Thr 100 105 110cga caa tat gaa acg tct tcc cta ccc ggt cgt ctg ttg tct gtt acc 15560Arg Gln Tyr Glu Thr Ser Ser Leu Pro Gly Arg Leu Leu Ser Val Thr115 120 125 130gaa caa ata cca gaa aaa aca tcc cgt atc acc gaa cgc ctg att tgg 15608Glu Gln Ile Pro Glu Lys Thr Ser Arg Ile Thr Glu Arg Leu Ile Trp 135 140 145gct ggc aat agc gaa gca gag aaa aac cat aat ctt gcc agc cag tgc 15656Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser Gln Cys 150 155 160gtg cgc cac tat gac acg gcg gga gtc acc cga tta gag agt ttg tca 15704Val Arg His Tyr Asp Thr Ala Gly Val Thr Arg Leu Glu Ser Leu Ser 165 170 175ctg acc ggt act gtt tta tct caa tcc agc caa cta ttg agc gac act 15752Leu Thr Gly Thr Val Leu Ser Gln Ser Ser Gln Leu Leu Ser Asp Thr 180 185 190caa gaa gct agc tgg aca ggt gat aat gaa acc gtc tgg caa aac atg 15800Gln Glu Ala Ser Trp Thr Gly Asp Asn Glu Thr Val Trp Gln Asn Met195 200 205 210ctg gct gat gac atc tac aca acc ctg agc gcc ttt gat gcc acc ggc 15848Leu Ala Asp Asp Ile Tyr Thr Thr Leu Ser Ala Phe Asp Ala Thr Gly 215 220 225gct tta ctc act cag acc gat gcg aaa ggg aac att cag agg cta acc 15896Ala Leu Leu Thr Gln Thr Asp Ala Lys Gly Asn Ile Gln Arg Leu Thr 230 235 240tat gat gtg gcc ggg cag cta aac ggg agc tgg tta acc tta aaa gac 15944Tyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp Leu Thr Leu Lys Asp 245 250 255caa ccg gaa caa gtg att atc aga tcc ctg acc tat tcc gcc gcc gga 15992Gln Pro Glu Gln Val Ile Ile Arg Ser Leu Thr Tyr Ser Ala Ala Gly 260 265 270caa aaa tta cgc gag gaa cac ggc aat ggt gtt atc acc gaa tac agt 16040Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val Ile Thr Glu Tyr Ser275 280 285 290tat gaa ccg gaa acc caa cag ctt atc ggt acc aaa acc cac cgt ccg 16088Tyr Glu Pro Glu Thr Gln Gln Leu Ile Gly Thr Lys Thr His Arg Pro 295 300 305tca gat gcc aaa gtg ttg caa gat cta cgt tat gag tat gac ccg gta 16136Ser Asp Ala Lys Val Leu Gln Asp Leu Arg Tyr Glu Tyr Asp Pro Val 310 315 320ggc aat gtc atc agt atc cgt aat gac gca gaa gcc acc cgc ttc tgg 16184Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu Ala Thr Arg Phe Trp 325 330 335cac aat cag aaa gtg gcg ccg gaa aac act tat acc tac gac tcc ttg 16232His Asn Gln Lys Val Ala Pro Glu Asn Thr Tyr Thr Tyr Asp Ser Leu 340 345 350tat cag ctt atc agc gca acc ggg cgc gag atg gcg aat ata ggt cag 16280Tyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln355 360 365 370caa agt aac caa ctt ccc tcc ctc acc cta cct tct gat aac aac acc 16328Gln Ser Asn Gln Leu Pro Ser Leu Thr Leu Pro Ser Asp Asn Asn Thr 375 380 385tac acc aac tat acc cgt act tat act tat gac cgt ggc ggc aat ttg 16376Tyr Thr Asn Tyr Thr Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu 390 395 400act aaa atc cag cac agt tca ccg gcg acg caa aac aac tac acc aca 16424Thr Lys Ile Gln His Ser Ser Pro Ala Thr Gln Asn Asn Tyr Thr Thr 405 410 415aac atc acg gtt tct aac cgg agc aat cgc gca gta ctc agc act ctg 16472Asn Ile Thr Val Ser Asn Arg Ser Asn Arg Ala Val Leu Ser Thr Leu 420 425 430acc gaa gat ccg gcg caa gta gat gct tta ttt gat gca ggc gga cat 16520Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe Asp Ala Gly Gly His435 440 445 450cag aac acg ttg ata tca gga caa aac ctg aac tgg aat aca cgc ggt 16568Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn Trp Asn Thr Arg Gly 455 460 465gaa cta caa cat gtg aca ttg gtg aaa cgg gac aag ggc gcc aat gat 16616Glu Leu Gln His Val Thr Leu Val Lys Arg Asp Lys Gly Ala Asn Asp 470 475 480gat cgg gaa tgg tat cgc tat agt agt gac ggg aga agg ata tta aaa 16664Asp Arg Glu Trp Tyr Arg Tyr Ser Ser Asp Gly Arg Arg Ile Leu Lys 485 490 495atc aat gaa cag cag acc agc agc aac tct caa aca cag aga ata act 16712Ile Asn Glu Gln Gln Thr Ser Ser Asn Ser Gln Thr Gln Arg Ile Thr 500 505 510tat ttg ccg agc tta gaa ctt cgt cta aca caa aac agc acg atc aca 16760Tyr Leu Pro Ser Leu Glu Leu Arg Leu Thr Gln Asn Ser Thr Ile Thr515 520 525 530acc gaa gat ttg caa gtt atc aca gta gga gaa gcg ggt cgg gca cag 16808Thr Glu Asp Leu Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln 535 540 545gta cga gta tta cat tgg gat agc ggt caa ccg gaa gat atc gac aat 16856Val Arg Val Leu His Trp Asp Ser Gly Gln Pro Glu Asp Ile Asp Asn 550 555 560aat cag cta cgt tat agc tac gat aat ctt atc ggt tcc agt caa ctt 16904Asn Gln Leu Arg Tyr Ser Tyr Asp Asn Leu Ile Gly Ser Ser Gln Leu 565 570 575gaa tta gac agc aaa gga gaa att att agt gag gaa gag tac tat ccc 16952Glu Leu Asp Ser Lys Gly Glu Ile Ile Ser Glu Glu Glu Tyr Tyr Pro 580 585 590tat ggc ggc acg gca tta tgg gca aca agg aag cgg aca gaa gcc agt 17000Tyr Gly Gly Thr Ala Leu Trp Ala Thr Arg Lys Arg Thr Glu Ala Ser595 600 605 610tat aaa acc atc cgt tat tca ggt aaa gag cgg gat gcc acc gga cta 17048Tyr Lys Thr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr Gly Leu 615 620 625tat tat tac ggt tac cga tat tat cag cct tgg gta gga cga tgg tta 17096Tyr Tyr Tyr Gly Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu 630 635 640agt gcc gat ccg gca gga aca gta gat ggg ttg aat tta tat cgg atg 17144Ser Ala Asp Pro Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met 645 650 655gta agg aat aat ccg gtt act ctg ctt gat cct gat gga tta atg cca 17192Val Arg Asn Asn Pro Val Thr Leu Leu Asp Pro Asp Gly Leu Met Pro 660 665 670aca att gca gaa cgc ata gca gca ctg caa aaa aat aaa gta gca gat 17240Thr Ile Ala Glu Arg Ile Ala Ala Leu Gln Lys Asn Lys Val Ala Asp675 680 685 690tca gcg cct tcg cca aca aat gcc aca aac gta gcg ata aac atc cgc 17288Ser Ala Pro Ser Pro Thr Asn Ala Thr Asn Val Ala Ile Asn Ile Arg 695 700 705ccg ccc gta gca cca aaa cct acc tta ccc aaa gca tca acg agt agc 17336Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys Ala Ser Thr Ser Ser 710 715 720caa tca act aca tac ccc atc aaa tct gca agc ata aaa cca acg acg 17384Gln Ser Thr Thr Tyr Pro Ile Lys Ser Ala Ser Ile Lys Pro Thr Thr 725 730 735tcg gga tca tcc att act gct cca ctg agt cca gta gga aat aaa tct 17432Ser Gly Ser Ser Ile Thr Ala Pro Leu Ser Pro Val Gly Asn Lys Ser 740 745 750act cct gaa ata tct ctt cca gaa agc act caa agc aat tct tca agc 17480Thr Pro Glu Ile Ser Leu Pro Glu Ser Thr Gln Ser Asn Ser Ser Ser755 760 765 770gct att tca aca aat cta cag aaa aag tca ttt act tta tat aga gcg 17528Ala Ile Ser Thr Asn Leu Gln Lys Lys Ser Phe Thr Leu Tyr Arg Ala 775 780 785gat aat aga tcc ttt gaa gac atg cag agt aaa ttc cct gaa gga ttt 17576Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys Phe Pro Glu Gly Phe 790 795 800aaa gcc tgg act cct cta gat act aag atg gca agg cag ttt gct agt 17624Lys Ala Trp Thr Pro Leu Asp Thr Lys Met Ala Arg Gln Phe Ala Ser 805 810 815gtc ttt att ggt cag aaa gat act tct aat tta cct aaa gaa aca gtc 17672Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu Pro Lys Glu Thr Val 820 825 830aag aat ata aac aca tgg gga aca aaa cca aaa tta aat gat ctc tca 17720Lys Asn Ile Asn Thr Trp Gly Thr Lys Pro Lys Leu Asn Asp Leu Ser835 840 845 850act tac ata aaa tat acc aag gac aaa tct aca gta tgg gtc tct act 17768Thr Tyr Ile Lys Tyr Thr Lys Asp Lys Ser Thr Val Trp Val Ser Thr 855 860 865gca att aat act gaa gca ggt gga caa agt tca ggg gct cca ctc cat 17816Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser Gly Ala Pro Leu His 870 875 880gaa att aat atg gat ctt tat gag ttt acc att gac gga caa aag cta 17864Glu Ile Asn Met Asp Leu Tyr Glu Phe Thr Ile Asp Gly Gln Lys Leu 885 890 895aat cca cta cca agg gga aga tct aaa gac agg gtg cct tca cta tta 17912Asn Pro Leu Pro Arg Gly Arg Ser Lys Asp Arg Val Pro Ser Leu Leu 900 905 910ctt gac aca cca gaa ata gaa aca gca tcc ata att gca ctt aat cat 17960Leu Asp Thr Pro Glu Ile Glu Thr Ala Ser Ile Ile Ala Leu Asn His915 920 925 930gga ccg gta aat gat gca gaa gtt tca ttc cta aca aca att ccg ctt 18008Gly Pro Val Asn Asp Ala Glu Val Ser Phe Leu Thr Thr Ile Pro Leu 935 940 945aaa aat gta aaa cct tat aag aga taa cgaaaaatta atattcttta 18055Lys Asn Val Lys Pro Tyr Lys Arg 950 955tctactttta atagccctct tgaacttaca ctcaaggggg ggaaaccaaa taagaaacca 18115tctttaataa caagccatga aagaatattt atttcatggc ttgattactt ttaacattca 18175atattaaata attaaaacaa tatctaacca attaaaataa caatacctta tttatcatat 18235taaaatatca aatcagaaat taatgaattt aagggttctt tatatttatt tctgagagca 18295taggcacaat accttaccga tggcgctgga cgtgattcaa aatccagaaa tgctatattt 18355tcatcaatat gggcagaata gcgcatttca ttgggagtca ttaaacttat cgcgacaccc 18415gcttttacca gatccaatct attagtaaaa tcagggaccg tcaataacgc taaattttgg 18475tattcaggga gataattcaa tggcataaaa ttattgcatt gttttaaaaa agcactatta 18535tgctgaacaa aaggaaaact agatattatt tcatcagcgt gactttctgg ttctaaaata 18595tcatgggata cagcaagaga cagcatttga taagcaccat ctatcctgat gatatcatca 18655ttatctggat aacattcagt cgtcacataa actgttatat cccctttcat taaggaagaa 18715aataccgcat cttgccttat taaatcatca attagaaaat tgttgattat acaaatatcg 18775cgataatgat aacgttgcac cgctcttttt acgaccgtag atattttatt aacatattct 18835ccacttgtgc caataaccag tttgtctctg tttgataatt tataatttct acgacaattc 18895caattattct caactttcag gatcctttca taacacggca gcaactcttg atatagtgcc 18955tttccctctt ctgtgagctt ggtttttccc ggtagtcgct caaacaattg acaccccaca 19015cgctgttcca gttgatatac gagcctgcta agtggagaag gggtaataca aagcgtatcc 19075gccgctaacg tgaatgactc tttcttagct gattccataa aatactttag ttgctttgaa 19135caaaatatca tcacataccc tcttgttttc attccagaaa tagaatatta accatagaac 19195atgacaacga tgtttctact ttgcattctt ttacattagg acatgcgtta atggacattg 19255aatttcacta catcaattgt taatatttat ttaatacttg cacaataatt ataaaataaa 19315tataacttag ttaattattt cttgatattg atcatggtaa gttttcctca atacctacag 19375aagtagatat tattttatct tccagtaatc tatcgtttgg cgacggaggt cgattcttcc 19435attgggatat tcaacccatt cgccgccttt cttattaatt acagtgattt ttggcatttt 19495ggtttcatcc aacttaggtt tataggtgat tttccattta gcacccggtg ttaacttcaa 19555cctaaaggga tacataccaa cttcaccttg taagaatatt ctgtttggtc taccttcaac 19615gactttcaaa atggggtaaa taaccgggct aaaatcaatc gtatccaatg catcaatttc 19675gctgatattt gtccgggctg catcattgat aaatgcgatt aaatcggttg ctgaatacgg 19735aatagcatct ttcactagat gacggacatc ggtataactc actgacacaa aggctcggtc 19795aatcttccac ttacatcgac cgccaccatt aaaaggtagt tttgcctgaa agtaaccggt 19855tttcggatca gcttttacat ccagacgtaa tccgttataa gttggtacct taaaaggcga 19915catattggaa tctaaacgat atttaaggca atcttttgag atatacacag cggatacatg 19975cggctgtgtg tatttaggtg cgactccttc tacagtaatc cactgattct ctttgggagg 20035agagagcggc tcatttgggt cagcacagcc tgatattaaa atcacggata agacagataa 20095gtatttcttg atatttatca tggtaagttt tcctcaactc ctacagcgtt atctgcatgt 20155gtgtccaatt ccagatcttc ctgtttatct atttagaaat aaataagcta cgctgatagc 20215attacttcat atttccatac atgaatcgaa aatcgacttc ttgagtgccg ttatcaattt 20275tgccgcccgg atattcaacc cactcgccgc ctttcttatt agtcaccgtg accttcgcca 20335ttttggtttc atccagctta ggcttaaaaa taattttcca tttagctcct ggagttaacg 20395tgagttgaaa aggacgcatt tttaatactt caccttgtaa gaatattctg ttcgggcgac 20455cttcaacgac tttcaaaaca gggtaaataa ccgggctaaa atcaatcgta ttcaatgtcg 20515agattttgct aatattcatc tggactatgc cattgataga tgcgattaaa ccggttgctg 20575aatacggaat agcatctttc accagatggc tgacatcagt ataactcacc gatacaaagg 20635cccggttaat tttccattta catcgtcccc ctccattaaa aggtagtttt gcttgaaaat 20695aaccggtttg tggatcggcc ttcactttca gacgaagccc attataggtc ggcactttaa 20755aaggcgacat attggaatcc agacgatact caaggcaatc ctttgatatg tattctgcgg 20815atacatgtgg ttcggtatat ttcggcgcta ccccttctac cgtgatccat tgattttctt 20875taggagggga aagcggctca tttgggtcag cacagcctga tattaaaatc actgacaaga 20935caaataagta ttttttaaca tttatcatgg taagttttcc tcaattccta cagcattatc 20995cgcataaata tcctgtcaag aatagcgttc attgatttcg tcaccaaaga aacaagatag 21055taaaaatcct attaccacag ataaaaaaca ccgcttatgc cgtgagtaat agtgagttga 21115gcgacaggga tacagcagtg catccccatc aattagtccc tttgaataaa gggaacagaa 21175tttgaaattt ccgtcatacc gtccatatta cggaacttag attatgatta ttaaatcacc 21235accaaatggc aagaaaaatt ttcatttttt aatttacgaa gaatgaattt gtaagaaagt 21295gttacaaact taatagaaat taatttactg ttaatctaat gaaggatgaa attataaaaa 21355taacccattt ctcagggaca acaatccaca atatatagaa ccactggtcc tcacttaatt 21415tcctgtcagg agtagaaata tcctgatgac tcagtcgatg acatacagca atgtcattgg 21475tattgagact accgactgtt taataaattt cttttgtctt taatggcgag atacaagtga 21535ttcactattt aagcactatc gataaataag attccaaaat agcgccatat cttacaccac 21595tcataattct atgtataaca attggttaaa taggatcatg tgtaacagga ttatgaaacg 21655ttatttatat caaatctatc aattatttta tatatagttt cacagtcaca ctcgctatct 21715ggtaccttca taaccaactg ccctccctgc gctaccttct gataacaaca gctacactaa 21775ctatacccgc gcctataatt atgaccgtgt gaaaattcag cgtagttcac cggccacgca 21835aaataactac acgaaatatt gctccccaga gaaacaccgt tcgaggttgt ttcaatgaaa 21895catcaaggta gagacaccta tgtattatta caagatatta aaccctctgc gattactcat 21955aggaatgtac gtaatactta tacaggcaac ttcacgtcat ccagagaaaa ttaagttgta 22015caaaatagac atcaactaat atagtaatag aaaatcccct gaaaatagat tcaggggatt 22075taataaatta accaaaaatc ataataaaaa tttatttcat tattttagga taaatattta 22135attagcctaa taatgaatta ttacttaaag taattcctaa acaatcaaat cggaaattaa 22195taaattcaat ggttcttgat atttatgcct gagagtataa gcacaatatt tcactgaggg 22255tgtcggatgc gatttaaaat tcaaaaaggt aatgccttta tgaaggtcag cagaacagag 22315cacttcattc ggtgtcatta aacttatcgc gatacctgat ttcactaagt ccaaccgatt 22375agtaaaatca ggcacagtta gcaaagttaa attctggtat tcaggtagac aattcagtga 22435aataaaattg ccgcactgtt taaagaaagc actattatgc tgagccaaag gcaatgtata 22495tataatatta tcagcgttac tttccgatcc taaaatatca ttagctacag caaggcgcaa 22555agtctgataa gttccatcca ctctgataat atcatcgttg tcaggataat gttcagtcgt 22615cacatagacg gttatatctt ccttcattaa agaagaaaat attgtctctt ttcttgccga 22675atcatcaacc agaaaattgt tttttatgta aatatcacga taatgataac gttgtaccgc 22735tctttttatc actgccgaaa ttttattaat atattctcca cttgtcccga tgaccagttt 22795gccggtgttt gctaattttc cgcttctacg ataatgccaa ttatcctcaa ctcgctgaat 22855tctctcataa cacggcaata gctcctgata taatgccttc ccctcttcag tgagtttggt 22915ccttcccggt agtcgttcaa atagttgaca gcccacacgt tgttccagtt gatatatgat 22975cctacttaga ggagagggag taatacaaag cgtatccgcg gctaaagtaa atgactcttt 23035tttcgctgac tccataaaat atttcaagct ctttgaacaa aatagcatca tatatccttc 23095ttattttaat tcattgttcc atccgaaata gaatggaatg ttaacaagaa aacattacaa 23155ctacttttct tctttgcatt atttaacatc aaagtatgca ttaactgaga ttgagtttta 23215tcatctttat tcttaacagt tatcaaacaa ttttcattat tattgcaaaa taaatacaac 23275cccttcttat gttacaataa tgattataaa gaaatttcac atattatcat taagtaataa 23335tgggcacaat taaccattta attaaacatt tcaattggtt gacaaagact cattatgttc 23395aacatgtaat gagcgcaatt ttaacattaa ataaattaca tagttcatat tcattatcac 23455tgagatcagc ttttttcgta tagtacatca tgtgaacaat accgtgccat ttcctgccaa 23515atcttattaa aaagtcagtt gcaaattttg catctgcttt ttttgcaaca gctatttaaa 23575gaaaacagtg agatagtgat tatccgagag atcaagatat gtctgctctt tacgcacaaa 23635ctgcaaacca tttctatgca tatctcagct atttctcaaa acctgtattt aatcatctct 23695tattccgatg gaacggaatc attctctgat tgattcatga tgtaaagaca atatggatgt 23755ttcatttact 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 965gag ata cct gat gta tta aaa atc cag tgt ggt ttt cag tgt ctg aca 23854Glu Ile Pro Asp Val Leu Lys Ile Gln Cys Gly Phe Gln Cys Leu Thr 970 975 980gat att agc cac agc tct ttt aac gaa ttt cac cag caa gta tcc gaa 23902Asp Ile Ser His Ser Ser Phe Asn Glu Phe His Gln Gln Val Ser Glu985 990 995 1000cac ctc tcc tgg tcc gaa gca cac gac tta tat cat gat gca caa cag 23950His Leu Ser Trp Ser Glu Ala His Asp Leu Tyr His Asp Ala Gln Gln 1005 1010 1015gcc caa aag gat aat cgg ctg tat gaa gcg cgt att ctt aaa cgc acg 23998Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Thr 1020 1025 1030aat cct caa tta caa aat gct gta cat ctt gcc atc gta gcg cct aat 24046Asn Pro Gln Leu Gln Asn Ala Val His Leu Ala Ile Val Ala Pro Asn 1035 1040 1045gct gaa ctg ata ggc tat aac aac caa ttt agc ggc agg gcc agt caa 24094Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln 1050 1055 1060tat gtc gcg ccg ggt acc gtt tcc tcc atg ttc tcc ccc gcc gct tat 24142Tyr Val Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr1065 1070 1075 1080ttg act gag ctt tat cgt gaa gca cgc aat tta cac gcc agc gat tcc 24190Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser 1085 1090 1095gtt tat cgc ctg gat act cgc cgc cca gat ctc aaa tca atg gcg ctc 24238Val Tyr Arg Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu 1100 1105 1110agt caa caa aat atg gat acg gaa ctt tcc act ctc tct tta tcc aat 24286Ser Gln Gln Asn Met Asp Thr Glu Leu Ser Thr Leu Ser Leu Ser Asn 1115 1120 1125gag cta tta ttg gaa agc att aaa act gag tct aag ctg gat aat tat 24334Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Asp Asn Tyr 1130 1135 1140act caa gtg atg gaa atg ctc tcc gct ttc cgt cct tcc ggc gcg acg 24382Thr Gln Val Met Glu Met Leu Ser Ala Phe Arg Pro Ser Gly Ala Thr1145 1150 1155 1160cct tat cac gat gct tac gaa aat gtg cgt aaa gtt atc cag cta caa 24430Pro Tyr His Asp Ala Tyr Glu Asn Val Arg Lys Val Ile Gln Leu Gln 1165 1170 1175gat cct ggg ctt gag caa tta aat gct tca cca gcc att gcc ggg ctg 24478Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu 1180 1185 1190atg cat caa gct tcc cta tta ggt att aac gct tca atc tca cct gag 24526Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu 1195 1200 1205ttg ttt aat att ctg acg gag gag att act gaa ggt aat gct gag gaa 24574Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu 1210 1215 1220ctt tat aag aaa aat ttt ggt aat atc gaa ccg gct tca ctg gct atg 24622Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met1225 1230 1235 1240ccg gaa tac ctt aga cgt tat tac aat tta agt gat gaa gaa ctc agc 24670Pro Glu Tyr Leu Arg Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser 1245 1250 1255cag ttt att ggt aaa gcc agc aat ttc ggc caa caa gaa tat agt aat 24718Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn 1260 1265 1270aac caa ctc att act ccg ata gtc aac agc aat gat ggc aca gtc aag 24766Asn Gln Leu Ile Thr Pro Ile Val Asn Ser Asn Asp Gly Thr Val Lys 1275 1280 1285gta tat cga att acc cgc gaa tat aca aca aat gcc aat caa gta gac 24814Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Asn Gln Val Asp 1290 1295 1300gtg gag ctg ttt ccc tac ggt gga gaa aat tat cag tta aat tac aaa 24862Val Glu Leu Phe Pro Tyr Gly Gly Glu Asn Tyr Gln Leu Asn Tyr Lys1305 1310 1315 1320ttc aaa gat tct cgt cag gat gtc tcc tat tta tcc atc aaa tta aat 24910Phe Lys Asp Ser Arg Gln Asp Val Ser Tyr Leu Ser Ile Lys Leu Asn 1325 1330 1335gac aaa aga gaa ctt atc cga att gaa gga gcg cct cag gtc aac atc 24958Asp Lys Arg Glu Leu Ile Arg Ile Glu Gly Ala Pro Gln Val Asn Ile 1340 1345 1350gaa tat tca gaa cat atc aca tta agt aca act gat atc agt caa cct 25006Glu Tyr Ser Glu His Ile Thr Leu Ser Thr Thr Asp Ile Ser Gln Pro 1355 1360 1365ttt gaa atc ggc cta aca cga gta tat cct tct agt tct tgg gca tat 25054Phe Glu Ile Gly Leu Thr Arg Val Tyr Pro Ser Ser Ser Trp Ala Tyr 1370 1375 1380gca gcc gca aaa ttt acc att gag gaa tat aac caa tac tct ttc ctg 25102Ala Ala Ala Lys Phe Thr Ile Glu Glu Tyr Asn Gln Tyr Ser Phe Leu1385 1390 1395 1400tta aaa ctc aat aaa gct att cgt cta tct cgt gcg aca gaa tta tca 25150Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser 1405 1410 1415ccc acc att ctg gaa agt att gtg cgt agt gtt aat cag caa ctg gat 25198Pro Thr Ile Leu Glu Ser Ile Val Arg Ser Val Asn Gln Gln Leu Asp 1420 1425 1430atc aac gca gaa gta tta ggt aaa gtt ttt ctg act aaa tat tat atg 25246Ile Asn Ala Glu Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met 1435 1440 1445caa cgt tat gct att aat gct gaa act gcc cta ata cta tgc aat gca 25294Gln Arg Tyr Ala Ile Asn Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala 1450 1455 1460ctt att tca caa cgt tca tat gat aat caa cct agc caa ttt gat cgc 25342Leu Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg1465 1470 1475 1480ctg ttt aat acg cca tta ctg aac ggc caa tat ttt tct acc gga gat 25390Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp 1485 1490 1495gaa gag att gat tta aat cca ggt agt act ggc gat tgg cgt aaa tcc 25438Glu Glu Ile Asp Leu Asn Pro Gly Ser Thr Gly Asp Trp Arg Lys Ser 1500 1505 1510gtg ctt aaa cgt gca ttt aat atc gat gat att tcc ctc tac cgc ctg 25486Val Leu Lys Arg Ala Phe Asn Ile Asp Asp Ile Ser Leu Tyr Arg Leu 1515 1520 1525ctt aaa att acc aac cat aat aat caa gat gga aag att aaa aat aac 25534Leu Lys Ile Thr Asn His Asn Asn Gln Asp Gly Lys Ile Lys Asn Asn 1530 1535 1540tta aat aat ctt tct gat tta tat att ggg aaa tta ctg gca gaa att 25582Leu Asn Asn Leu Ser Asp Leu Tyr Ile Gly Lys Leu Leu Ala Glu Ile1545 1550 1555 1560cat caa tta acc att gat gaa ttg gat tta ttg ctg gtt gcc gtg ggt 25630His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu Leu Val Ala Val Gly 1565 1570 1575gaa gga gaa act aat tta tcc gct atc agt gat aaa caa ctg gcg gca 25678Glu Gly Glu Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Ala 1580 1585 1590ctg atc aga aaa ctc aat acc att acc gtc tgg cta cag aca cag aag 25726Leu Ile Arg Lys Leu Asn Thr Ile Thr Val Trp Leu Gln Thr Gln Lys 1595 1600 1605tgg agt gcg ttc caa tta ttt gtt atg act tcc acc agc tat aac aaa 25774Trp Ser Ala Phe Gln Leu Phe Val Met Thr Ser Thr Ser Tyr Asn Lys 1610 1615 1620acg ctg acg cct gaa att aag aat ctg ctg gat acc gtc tac cac ggt 25822Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly1625 1630 1635 1640tta caa ggc ttt gat aaa gac aag gca aat tta ctg cat gtt atg gcg 25870Leu Gln Gly Phe Asp Lys Asp Lys Ala Asn Leu Leu His Val Met Ala 1645 1650 1655ccc tat att gcg gcc acc tta caa tta tca tcg gaa aat gtc gcc cat 25918Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His 1660 1665 1670tct gtg ctg ctt tgg gca gac aag tta aag ccc ggc gac ggc gca atg 25966Ser Val Leu Leu Trp Ala Asp Lys Leu Lys Pro Gly Asp Gly Ala Met 1675 1680 1685aca gcc gaa aaa ttc tgg gac tgg ttg aat act caa tat acg cca gat 26014Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr Gln Tyr Thr Pro Asp 1690 1695 1700tca tcg gaa gta tta gca aca cag gaa cat att gtt cag tat tgt cag 26062Ser Ser Glu Val Leu Ala Thr Gln Glu His Ile Val Gln Tyr Cys Gln1705 1710 1715 1720gcg ttg gcg caa tta gaa atg gtt tac cat tcc acc ggt atc aat gaa 26110Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu 1725 1730 1735aac gcc ttc cgc ctg ttt gtg aca aaa cca gag atg ttt ggc tcg tca 26158Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ser Ser 1740 1745 1750act gag gca gta cct gcg cat gat gca ctt tca ctg atc atg ctg acg 26206Thr Glu Ala Val Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr 1755 1760 1765cgt ttt gca gat tgg gtt aat gcg tta ggc gaa aaa gcc tct tcc gta 26254Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val 1770 1775 1780cta gcg gca ttt gaa gct aac agt tta acg gca gaa caa ttg gct gat 26302Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp1785 1790 1795 1800gcc atg aat ctt gat gct aat ttg cta ttg caa gcc agt act caa gca 26350Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Thr Gln Ala 1805 1810 1815caa aac cat caa cat ctt ccc cca gtg acg caa aaa aat gct ttc tcc 26398Gln Asn His Gln His Leu Pro Pro Val Thr Gln Lys Asn Ala Phe Ser 1820 1825 1830tgt tgg aca tct atc gac act atc ctg caa tgg gtt aat gtt gca caa 26446Cys Trp Thr Ser Ile Asp Thr Ile Leu Gln Trp Val Asn Val Ala Gln 1835 1840 1845caa ttg aat gtc gcc cca cag gga gtt tcc gct ttg gtc ggg ctg gat 26494Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp 1850 1855 1860tat att caa tta aat caa aaa atc ccc acc tat gcc cag tgg gaa agt 26542Tyr Ile Gln Leu Asn Gln Lys Ile Pro Thr Tyr Ala Gln Trp Glu Ser1865 1870 1875 1880gct ggg gaa ata ttg act gcc gga ttg aat tca caa cag gct gat ata 26590Ala Gly Glu Ile Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asp Ile 1885 1890 1895tta cac gct ttt ttg gac gaa tct cgc agt gcc gca tta agc acc tac 26638Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr 1900 1905 1910tat atc cgt caa gtc gcc aag cca gcg gca gcc ata aaa agc cgt gat 26686Tyr Ile Arg Gln Val Ala Lys Pro Ala Ala Ala Ile Lys Ser Arg Asp 1915 1920 1925gac ttg tac caa tac tta cta att gat aat cag gtt tcc gct gca atc 26734Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile 1930 1935 1940aaa act acc cgg att gcc gaa gcc att gcc agc att caa ctg tac gtc 26782Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val1945 1950 1955 1960aac cgc acg ctg gaa aat gta gaa gaa aat gcc cat tca ggg gtt atc 26830Asn Arg Thr Leu Glu Asn Val Glu Glu Asn Ala His Ser Gly Val Ile 1965 1970 1975agc cgt cag ttc ttt atc gac tgg gac aaa tat aac aaa cgc tac agc 26878Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser 1980 1985 1990acc tgg gcg ggt gtt tct caa tta gtt tac tac ccg gaa aac tat att 26926Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile 1995 2000 2005gat ccc acc atg cgt atc gga caa acc aaa atg atg gac gca tta ttg 26974Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu 2010 2015 2020caa tcc gtc agc caa agc caa tta aat gcc gat act gtc gaa gac gcc 27022Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala2025 2030 2035 2040ttt atg tct tat ctg aca tcg ttt gag caa gtg gct aat ctt aaa gtt 27070Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val 2045 2050 2055att agc gcg tat cac gat aat att aac aac gat caa ggg ctg acc tat 27118Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr 2060 2065 2070ttt atc ggc ctc agt gaa act gat acc ggt gaa tac tat tgg cgc agt 27166Phe Ile Gly Leu Ser Glu Thr Asp Thr Gly Glu Tyr Tyr Trp Arg Ser 2075 2080 2085gtc gat cac agt aaa ttc agc gac ggt aaa ttc gcc gct aat gcc tgg 27214Val Asp His Ser Lys Phe Ser Asp Gly Lys Phe Ala Ala Asn Ala Trp 2090 2095 2100agt gaa tgg cac aaa att gat tgt cca att aat cct tac cga agc act 27262Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Arg Ser Thr2105 2110 2115 2120atc cgt cct gtg atg tac aaa tcc cgc ttg tat ctg ctc tgg ttg gaa 27310Ile Arg Pro Val Met Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu 2125 2130 2135caa aag gag atc act aaa caa aca gga aat agc aaa gat ggc tat caa 27358Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln 2140 2145 2150acc gag aca gat tat cgt tat gag cta aaa ttg gcg cat atc cgt tat 27406Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr 2155 2160 2165gac ggt acc tgg aat acg cca atc act ttt gat gtc aat gaa aaa ata 27454Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Glu Lys Ile 2170 2175 2180tcc aag cta gaa ctg gca aaa aat aaa gcg cct ggg ctc tat tgt gct 27502Ser Lys Leu Glu Leu Ala Lys Asn Lys Ala Pro Gly Leu Tyr Cys Ala2185 2190 2195 2200ggt tat caa ggt gaa gat acg ttg ctg gtt atg ttt tat aac caa caa 27550Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln 2205 2210 2215gat aca ctc gat agt tat aaa acc gct tca atg caa ggg cta tat atc 27598Asp Thr Leu Asp Ser Tyr Lys Thr Ala Ser Met Gln Gly Leu Tyr Ile 2220 2225 2230ttt gcc gat atg gaa tat aaa gat atg acc gat gga caa tac aaa tct 27646Phe Ala Asp Met Glu Tyr Lys Asp Met Thr Asp Gly Gln Tyr Lys Ser 2235 2240 2245tat cgg gac aac agc tat aaa caa ttc gat act aat agt gtc aga aga 27694Tyr Arg Asp Asn Ser Tyr Lys Gln Phe Asp Thr Asn Ser Val Arg Arg 2250 2255 2260gtg aat aac cgc tat gca gag gat tat gaa att ccc tca tcg gta aat 27742Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Asn2265 2270 2275 2280agc cgt aaa ggc tat gat tgg gga gat tat tat ctc agt atg gta tat 27790Ser Arg Lys Gly Tyr Asp Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr 2285 2290 2295aac gga gat att cca act att agt tac aaa gcc aca tca agt gat tta 27838Asn Gly Asp Ile Pro Thr Ile Ser Tyr Lys Ala Thr Ser Ser Asp Leu 2300 2305 2310aaa atc tat atc tcg cca aaa tta aga att att cat aat gga tat gaa 27886Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu 2315 2320 2325ggg cag caa cgc aat caa tgc aat cta atg aat aaa tat ggc aaa cta 27934Gly Gln Gln Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu 2330 2335 2340ggt gat aaa ttt att gtt tat act agc ttg gga gtt aat cca aat aat 27982Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn2345 2350 2355 2360tcg tca aat aag ctg atg ttt tac ccc gtt tat caa tat aac gga aat 28030Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Asn Gly Asn 2365 2370 2375gtc agt ggg ctt agt caa ggg aga tta cta ttc cac cgt gac acc aat 28078Val Ser Gly Leu Ser Gln Gly Arg Leu Leu Phe His Arg Asp Thr Asn 2380 2385 2390tat tca tct aaa gta gaa gct tgg att cct gga gca gga cgt tct cta 28126Tyr Ser Ser Lys Val Glu Ala Trp Ile Pro Gly Ala Gly Arg Ser Leu 2395 2400 2405acc aat ccg aat gct gcc att ggt gat gat tat gct aca gac tcg tta 28174Thr Asn Pro Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu 2410 2415 2420aac aaa ccg aat gat ctt aag caa tac gtc tat atg act gac agt aaa 28222Asn Lys Pro Asn Asp Leu Lys Gln Tyr Val Tyr Met Thr Asp Ser Lys2425 2430 2435 2440ggt act gct acc gat gtc tca gga cca gta gat atc aat act gca att 28270Gly Thr Ala Thr Asp Val Ser Gly Pro Val Asp Ile Asn Thr Ala Ile 2445 2450 2455tcc ccg gca aaa gtt cag gta aca gta aaa gcc ggt agc aaa gaa caa 28318Ser Pro Ala Lys Val Gln Val Thr Val Lys Ala Gly Ser Lys Glu Gln 2460 2465 2470acg ttt acc gcg gat aaa aat gtc tcc att cag cca tcc cct agc ttt 28366Thr Phe Thr Ala Asp Lys Asn Val Ser Ile Gln Pro Ser Pro Ser Phe 2475 2480 2485gat gaa atg aat tat caa ttt aat gct ctc gaa ata gat ggc tca agt 28414Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Ser 2490 2495 2500ctg aat ttt act aac aat tca gcc agt att gat att acc ttt acc gca 28462Leu Asn Phe Thr Asn Asn Ser Ala Ser Ile Asp Ile Thr Phe Thr Ala2505 2510 2515 2520ttt gca gag gat gga cgt aaa ctg ggt tat gaa agt ttc agt att cct 28510Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro 2525 2530 2535att acc cgc aag gtg agt act gat aat tcc ctg acc ctg cgc cat aat 28558Ile Thr Arg Lys Val Ser Thr Asp Asn Ser Leu Thr Leu Arg His Asn 2540 2545 2550gaa aat ggt gcg caa tat atg caa tgg gga gtc tat cgc att cgt ctt 28606Glu Asn Gly Ala Gln Tyr Met Gln Trp Gly Val Tyr Arg Ile Arg Leu 2555 2560 2565aat act tta ttt gct cgc caa tta gtt gcg cga gcc act acc ggt att 28654Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile 2570 2575 2580gat acg att ctg agt atg gaa act cag aat att cag gaa cca cag tta 28702Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu2585 2590 2595 2600ggc aaa ggt ttc tac gct acg ttc gtg ata cct ccg tat aac cca tca 28750Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Pro Ser 2605 2610 2615act cat ggt gat gaa cgt tgg ttt aag ctt tat atc aaa cat gtt gtt 28798Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val 2620 2625 2630gat aat aat tca cat att atc tat tca ggt cag cta aaa gat aca aat 28846Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln Leu Lys Asp Thr Asn 2635 2640 2645ata agc acc acg tta ttt atc cct ctt gat gat gtt cca ttg aac caa 28894Ile Ser Thr Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln 2650 2655 2660gat tac agc gcc aag gtt tac atg acc ttc aag aaa tca cca tca gat 28942Asp Tyr Ser Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp2665 2670 2675 2680ggt acc tgg tgg ggc cct cac ttt gtt aga gat gat aaa gga ata gta 28990Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val 2685 2690 2695aca ata aac cct aaa tcc att ttg acc cac ttt gag agc gtc aat gtc 29038Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val 2700 2705 2710ctg aat aat att agt agc gaa cca atg gat ttc agc ggc gct aac agc 29086Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser 2715 2720 2725ctc tat ttt tgg gaa ctg ttc tac tat acc ccg atg ctg gtt gcc caa 29134Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln 2730 2735 2740cgt ttg ttg cat gag caa aac ttt gat gaa gcg aac cgc tgg ctg aaa 29182Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys2745 2750 2755 2760tat gtc tgg agc cca tcc ggg tat att gtt cac ggc cag att cag aat 29230Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn 2765 2770 2775tat caa tgg aac gtc cgc ccg tta ttg gaa gat acc agt tgg aac agt 29278Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser 2780 2785 2790gat cct ttg gat tcc gtc gat cct gac gcg gta gcg cag cac gat ccg 29326Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro 2795 2800 2805atg cac tat aaa gtt tca acc ttt atg cgc acc ctt gat ctg ttg atc 29374Met His Tyr Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile 2810 2815 2820gcg cgc ggc gac cat gct tac cgc caa ttg gag cgc gat acg ctt aac 29422Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn2825 2830 2835 2840gaa gcg aag atg tgg tat atg caa gcg ctg cat ctg tta ggc gat aaa 29470Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys 2845 2850 2855cct tat ctg ccg ctg agt acc aca tgg aat gat cca cga ctg gac aaa 29518Pro Tyr Leu Pro Leu Ser Thr Thr Trp Asn Asp Pro Arg Leu Asp Lys 2860 2865 2870gcc gcg gat att act acc caa agt gct cat tcc agc tca ata gtc gct 29566Ala Ala Asp Ile Thr Thr Gln Ser Ala His Ser Ser Ser Ile Val Ala 2875 2880 2885ttg cgg cag agt aca ccg gcg ctt tta tca ttg cgc agc gcc aat acc 29614Leu Arg Gln Ser Thr Pro Ala Leu Leu Ser Leu Arg Ser Ala Asn Thr 2890 2895 2900ctg acc gat ctc ttc ctg ccg caa atc aat gaa gtg atg atg aat tac 29662Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr2905 2910 2915 2920tgg caa aca tta gct cag aga gta tac aac ctg cgc cac aac ctc tct 29710Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser 2925 2930 2935atc gac ggt cag ccg tta tat ctg cca atc tat gcc aca ccg gcg gac 29758Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp 2940 2945 2950ccg aaa gcg tta ctc agc gcc gct gtt gcc act tct caa ggt gga ggc 29806Pro Lys Ala Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly 2955 2960 2965aag ctg ccg gag tca ttt atg tcc ctg tgg cgt ttc ccg cac atg ctg 29854Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu 2970 2975 2980gaa aat gct cgc agc atg gtt agc cag ctc acc caa ttc ggc tcc acg 29902Glu Asn Ala Arg Ser Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr2985 2990 2995 3000tta caa aat att atc gaa cgt cag gac gca gaa gcg ctc aat gcg tta 29950Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu 3005 3010 3015tta caa aat cag gcc gca gag ctg ata ttg act aac ctg agt att caa 29998Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln 3020 3025 3030gac aaa acc att gaa gaa ctg gat gcc gag aaa acc gtg ctg gaa aaa 30046Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys 3035 3040 3045tcc aaa gcg gga gca caa tcg cgc ttt gat agc tat agc aaa ctg cat 30094Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser Tyr Ser Lys Leu His 3050 3055 3060gat gaa aac atc aac gcc ggt gaa aac caa gct atg acg cta cga gcg 30142Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala3065 3070 3075 3080tcc gca gcc ggg ctt acc acg gcg gtt cag gca tcc cgt ctg gcc ggc 30190Ser Ala Ala Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly 3085 3090 3095gca gcg gct gat ctg gtg cct aac atc ttc ggc ttc gcc ggt ggt ggt 30238Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly 3100 3105 3110agc cgt tgg ggg gct atc gct gag gcg acc ggc tat gta atg gaa ttt 30286Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe 3115 3120 3125tcc gct aat gtt atg aat acc gaa gcg gat aaa att agc caa tct gaa 30334Ser Ala Asn Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu 3130 3135 3140acc tac cgt cgt cgc cgt cag gag tgg gaa att cag cgt aat aat gcc 30382Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala3145 3150 3155 3160gaa gcg gag ctg aaa caa ctc gat gcc caa ctt aaa tcg ctg gca gta 30430Glu Ala Glu Leu Lys Gln Leu Asp Ala Gln Leu Lys Ser Leu Ala Val 3165 3170 3175cgc cgt gaa gcc gcc gta ttg caa aaa acc agc ctg aaa acc caa caa 30478Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln 3180 3185 3190gag cag acc caa gcc caa ttg gcc ttc ctg caa cgt aag ttc agc aat 30526Glu Gln Thr Gln Ala Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn 3195 3200 3205caa gcg ttg tac aac tgg cta cgt ggc cga ctg gca gca att tac ttc 30574Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe 3210 3215 3220caa ttc tac gac ttg gct atc gcg cgt tgt tta atg gca gag cag gct 30622Gln Phe Tyr Asp Leu Ala Ile Ala Arg Cys Leu Met Ala Glu Gln Ala3225 3230 3235 3240tac cgt tgg gaa att agc gat gac tct gct cgc ttt att aaa ccg ggc 30670Tyr Arg Trp Glu Ile Ser Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly 3245 3250 3255gcc tgg caa gga acc tat gca ggt ctg ctg gca ggt gaa acc ttg atg 30718Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met 3260 3265 3270cta agt ttg gca caa atg gaa gac gcc cat tta aga cgc gat aaa cgc 30766Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu Arg Arg Asp Lys Arg 3275 3280 3285gca tta gag gtc gaa cgt aca gta tcg ctg gcc gaa att tat gct ggt 30814Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Ile Tyr Ala Gly 3290 3295 3300tta ccg caa gat aaa ggc cca ttc tcc ctg acg caa gaa atc gag aag 30862Leu Pro Gln Asp Lys Gly Pro Phe Ser Leu Thr Gln Glu Ile Glu Lys3305 3310 3315 3320ctg gtg aat gca ggt tca ggc agc gcc ggc agt ggt aat aat aat ttg 30910Leu Val Asn Ala Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu 3325 3330 3335gca ttt ggc gcc ggc acg gac act aaa act tct ttg cag gca tcc att 30958Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Ile 3340 3345 3350tca tta gct gat tta aaa att cgt gag gat tac ccg gaa tct att ggc 31006Ser Leu Ala Asp Leu Lys Ile Arg Glu Asp Tyr Pro Glu Ser Ile Gly 3355 3360 3365aaa atc cga cgc atc aaa cag atc agc gtt acc ctg ccg gcg cta ttg 31054Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu 3370 3375 3380gga cct tat cag gat gtg cag gca ata tta tct tac ggc gat aaa gcc 31102Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala3385 3390 3395 3400gga tta gcg aac ggc tgt gca gcg ctg gcc gtt tcc cac ggt acg aat 31150Gly Leu Ala Asn Gly Cys Ala Ala Leu Ala Val Ser His Gly Thr Asn 3405 3410 3415gac agc ggt caa ttc cag ctc gat ttc aac gat ggc aaa ttc ctg ccg 31198Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro 3420 3425 3430ttt gaa ggt atc gcc att gat caa ggt acg cta aca ctg agt ttt cct 31246Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro 3435 3440 3445aat gca tca acg cca gcc aaa ggt aaa caa gcc act atg tta aaa acc 31294Asn Ala Ser Thr Pro Ala Lys Gly Lys Gln Ala Thr Met Leu Lys Thr 3450 3455 3460ctg aac gat atc att ttg cat att cgc tac acc att aag taa 31336Leu Asn Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Lys3465 3470 3475ccatcccaac acagaactaa gacaggcccc gaatcggggt ctggtaagga gtttct atg 31395 Metcag aat tca cag aca ttc agc atg acc gag ctg tca tta cct aag ggc 31443Gln Asn Ser Gln Thr Phe Ser Met Thr Glu Leu Ser Leu Pro Lys Gly3480 3485 3490 3495ggc ggc gcc att acc ggt atg ggt gaa gca tta acg ccg gcc ggg ccg 31491Gly Gly Ala Ile Thr Gly Met Gly Glu Ala Leu Thr Pro Ala Gly Pro 3500 3505 3510gat ggt atg gca gcc tta tcg ctg cca ttg ccc att tct gcc gga cgt 31539Asp Gly Met Ala Ala Leu Ser Leu Pro Leu Pro Ile Ser Ala Gly Arg 3515 3520 3525ggt tat gcc ccc tcg ctc acg ctg aac tac aac agc gga acc ggt aac 31587Gly Tyr Ala Pro Ser Leu Thr Leu Asn Tyr Asn Ser Gly Thr Gly Asn 3530 3535 3540agc ccg ttc ggt ctc ggt tgg gac tgt aac gtc atg aca att cgt cgt 31635Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val Met Thr Ile Arg Arg 3545 3550 3555cgc acc agt acc ggc gtg ccg aat tat gat gaa acc gat act ttt ctg 31683Arg Thr Ser Thr Gly Val Pro Asn Tyr Asp Glu Thr Asp Thr Phe Leu3560 3565 3570 3575ggg ccg gaa ggt gaa gtg ttg gtc gta gca tta aat gag gca ggt caa 31731Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu Asn Glu Ala Gly Gln 3580 3585 3590gct gat atc cgc agt gaa tcc tca tta cag ggc atc aat ttg ggg atg 31779Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly Ile Asn Leu Gly Met 3595 3600 3605acc ttc acc gtt acc ggt tat cgc tcc cgt ttg gaa agc cac ttt agc 31827Thr Phe Thr Val Thr Gly Tyr Arg Ser Arg Leu Glu Ser His Phe Ser 3610 3615 3620cgg ttg gaa tac tgg caa ccc caa aca aca ggc gca acc gat ttc tgg 31875Arg Leu Glu Tyr Trp Gln Pro Gln Thr Thr Gly Ala Thr Asp Phe Trp 3625 3630 3635ctg ata tac agc ccc gac gga caa gcc cat tta ctg ggc aaa aat cct 31923Leu Ile Tyr Ser Pro Asp Gly Gln Ala His Leu Leu Gly Lys Asn Pro3640 3645 3650 3655caa gca cgc atc agc aat cca cta aat gtt aac caa aca gcg caa tgg 31971Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn Gln Thr Ala Gln Trp 3660 3665 3670cta ttg gaa gcc tcg gta tca tcc cac ggc gag cag att tat tat cag 32019Leu Leu Glu Ala Ser Val Ser Ser His Gly Glu Gln Ile Tyr Tyr Gln 3675 3680 3685tat cga gcc gaa gat gaa act gat tgc gaa act gac gaa ctc aca gcc 32067Tyr Arg Ala Glu Asp Glu Thr Asp Cys Glu Thr Asp Glu Leu Thr Ala 3690 3695 3700cac ccg aac aca acc gtc cag cgc tac ctg caa gta gta cat tac ggt 32115His Pro Asn Thr Thr Val Gln Arg Tyr Leu Gln Val Val His Tyr Gly 3705 3710 3715aat cta acc gcc agc gaa gta ttt ccc acg cta aat gga gat gat cca 32163Asn Leu Thr Ala Ser Glu Val Phe Pro Thr Leu Asn Gly Asp Asp Pro3720 3725 3730 3735ctc aaa tct ggc tgg ttg ttc tgt tta gta ttt gat tac ggt gag cgc 32211Leu Lys Ser Gly Trp Leu Phe Cys Leu Val Phe Asp Tyr Gly Glu Arg 3740 3745 3750aaa aac agc tta tct gaa atg ccg cca ttt aaa gcc aca agt aac tgg 32259Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys Ala Thr Ser Asn Trp 3755 3760 3765ctt tgc cgc aaa gac cgt ttt tcc cgt tat gaa tac ggt ttt gca ttg 32307Leu Cys Arg Lys Asp Arg Phe Ser Arg Tyr Glu Tyr Gly Phe Ala Leu 3770 3775 3780cgc acc cgg cgc tta tgt cgc caa ata ctg atg ttt cac cgt ctg caa 32355Arg Thr Arg Arg Leu Cys Arg Gln Ile Leu Met Phe His Arg Leu Gln 3785 3790 3795acc ctg tct ggt cag gca aaa ggc gac gat gaa ccc gca tta gtt tca 32403Thr Leu Ser Gly Gln Ala Lys Gly Asp Asp Glu Pro Ala Leu Val Ser3800 3805 3810 3815cgt ctg ata ctg gat tat gac gaa aac gcg gtg gtc agt acg ctc gtt 32451Arg Leu Ile Leu Asp Tyr Asp Glu Asn Ala Val Val Ser Thr Leu Val 3820 3825 3830tct gtc cgc cga gtg gga cat gag caa gat ggc aca acg gcg gtc gcc 32499Ser Val Arg Arg Val Gly His Glu Gln Asp Gly Thr Thr Ala Val Ala 3835 3840 3845ctg ccg cca ttg gaa ctg gct tat cag cct ttt gaa cca gaa caa aaa 32547Leu Pro Pro Leu Glu Leu Ala Tyr Gln Pro Phe Glu Pro Glu Gln Lys 3850 3855 3860gca ctc tgg cga cca atg gat gta ctg gcg aat ttc aac acc atc caa 32595Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn Phe Asn Thr Ile Gln 3865 3870 3875cgc tgg caa ctg ctt gat ctg caa ggc gaa ggc gta ccc ggt att ctg 32643Arg Trp Gln Leu Leu Asp Leu Gln Gly Glu Gly Val Pro Gly Ile Leu3880 3885 3890 3895tat cag gat aaa aat ggc tgg tgg tat cga tct gct caa cgt cag aca 32691Tyr Gln Asp Lys Asn Gly Trp Trp Tyr Arg Ser Ala Gln Arg Gln Thr 3900 3905 3910ggg gaa gag atg aat gcg gtc acc tgg ggc aaa atg caa ctc ctt cct 32739Gly Glu Glu Met Asn Ala Val Thr Trp Gly Lys Met Gln Leu Leu Pro 3915 3920 3925atc acg ccc gct att cag gat aac gcc tca ctg atg gat att aat ggt 32787Ile Thr Pro Ala Ile Gln Asp Asn Ala Ser Leu Met Asp Ile Asn Gly 3930 3935 3940gat ggg caa ctg gat tgg gtt atc acc ggt ccg ggg cta agg ggt tat 32835Asp Gly Gln Leu Asp Trp Val Ile Thr Gly Pro Gly Leu Arg Gly Tyr 3945 3950 3955cac agc cag cat cca gat ggc agt tgg aca cgt ttt acg ccg ttg cac 32883His Ser Gln His Pro Asp Gly Ser Trp Thr Arg Phe Thr Pro Leu His3960 3965 3970 3975gcc tta ccg ata gaa tat acc cat ccc cgc gcc caa ctt gcg gat tta 32931Ala Leu Pro Ile Glu Tyr Thr His Pro Arg Ala Gln Leu Ala Asp Leu 3980 3985 3990atg ggg gcc ggg ctg tcc gat tta gtg ctg att ggt ccc aaa agc gtg 32979Met Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser Val 3995 4000 4005cgt ttg tat gcc aat aac cgt gat ggt ttt acc gaa gga cgg gat gtg 33027Arg Leu Tyr Ala Asn Asn Arg Asp Gly Phe Thr Glu Gly Arg Asp Val 4010 4015 4020gtg caa tcc ggt ggt atc acc ctg ccg tta ccg ggc gcc gat gcg cgt 33075Val Gln Ser Gly Gly Ile Thr Leu Pro Leu Pro Gly Ala Asp Ala Arg 4025 4030 4035aag tta gtg gcc ttt agc gac gta ctc ggt tca ggc caa gca cat ttg 33123Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser Gly Gln Ala His Leu4040 4045 4050 4055gtt gaa gtt agt gcg acg aaa gtc acc tgc tgg cca aat ctg gga cat 33171Val Glu Val Ser Ala Thr Lys Val Thr Cys Trp Pro Asn Leu Gly His 4060 4065 4070ggc cgt ttt ggt cag cca atc aca ttg ccg gga ttt agc caa tcc gcc 33219Gly Arg Phe Gly Gln Pro Ile Thr Leu Pro Gly Phe Ser Gln Ser Ala 4075 4080 4085gcc aat ttt aat cct gat cga gtt cat ctg gcc gat ctg gac ggt agt 33267Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala Asp Leu Asp Gly Ser 4090 4095 4100ggt cct gcc gat ctg att tat gtt cat gct gac cat ctg gat att ttc 33315Gly Pro Ala Asp Leu Ile Tyr Val His Ala Asp His Leu Asp Ile Phe 4105 4110 4115agc aat gaa agt ggt aac ggt ttt gca caa cca ttc aca ctc cgt ttt 33363Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro Phe Thr Leu Arg Phe4120 4125 4130 4135cct gac ggc ctg cgt ttt gat gat act tgc cag cta caa gtg gct gat 33411Pro Asp Gly Leu Arg Phe Asp Asp Thr Cys Gln Leu Gln Val Ala Asp 4140 4145 4150gta cag gga tta ggg gtt gtc agc ctg atc ctg agc gta ccg cat atg 33459Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu Ser Val Pro His Met 4155 4160 4165gcg cca cac cat tgg cgc tgc gat ctg acc aac gcg aaa ccg tgg tta 33507Ala Pro His His Trp Arg Cys Asp Leu Thr Asn Ala Lys Pro Trp Leu 4170 4175 4180ctc agt gaa atg aac aac aac atg gga gcc cat cac acc ctg cat tac 33555Leu Ser Glu Met Asn Asn Asn Met Gly Ala His His Thr Leu His Tyr 4185 4190 4195cgt agc tcc gtc cag ttt tgg ctg gat gaa aaa gcc gca gcc tta gct 33603Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys Ala Ala Ala Leu Ala4200 4205 4210 4215acc gga caa aca ccg gtc tgt tac ctg ccc ttc ccg gtc cat acc ctg 33651Thr Gly Gln Thr Pro Val Cys Tyr Leu Pro Phe Pro Val His Thr Leu 4220 4225 4230tgg caa aca gaa acc gag gat gaa atc agc ggc aat aaa tta gtg acc 33699Trp Gln Thr Glu Thr Glu Asp Glu Ile Ser Gly Asn Lys Leu Val Thr 4235 4240 4245act tta cgt tac gct cac ggc gcc tgg gat gga cgt gag cgg gaa ttt 33747Thr Leu Arg Tyr Ala His Gly Ala Trp Asp Gly Arg Glu Arg Glu Phe 4250 4255 4260cgc ggc ttt ggc tat gtt gag cag aca gac agc cat caa ctg gct caa 33795Arg Gly Phe Gly Tyr Val Glu Gln Thr Asp Ser His Gln Leu Ala Gln 4265 4270 4275ggc aat gcg ccg gaa cgt aca tca ccg gca ctt acc aaa aac tgg tat 33843Gly Asn Ala Pro Glu Arg Thr Ser Pro Ala Leu Thr Lys Asn Trp Tyr4280 4285 4290 4295gcc acc gga atc cct gag gta gac aat acg cta tct gcc ggg tat tgg 33891Ala Thr Gly Ile Pro Glu Val Asp Asn Thr Leu Ser Ala Gly Tyr Trp 4300 4305 4310cgc ggt gat acg cag gct ttc act ggt ttt acg cca cac ttt act ctc 33939Arg Gly Asp Thr Gln Ala Phe Thr Gly Phe Thr Pro His Phe Thr Leu 4315 4320 4325tgg aaa gag ggc aaa gat gtt cca ctg aca ccg gaa gat gac cac aat 33987Trp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro Glu Asp Asp His Asn 4330 4335 4340ctg tac tgg tta aac cgg gca cta aaa ggt caa cca ctg cgt agt gaa 34035Leu Tyr Trp Leu Asn Arg Ala Leu Lys Gly Gln Pro Leu Arg Ser Glu 4345 4350 4355ctc tac ggg cta gat ggc agc gca cag cag aag atc ccc tat aca gtg 34083Leu Tyr Gly Leu Asp Gly Ser Ala Gln Gln Lys Ile Pro Tyr Thr Val4360 4365 4370 4375act gaa tcc cgc cca caa gtg cgc caa tta caa gat aac act acc ctt 34131Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln Asp Asn Thr Thr Leu 4380 4385 4390tcc ccg gtg ctc tgg gcc tca gtg gtg gaa agt cgt agt tat cac tat 34179Ser Pro Val Leu Trp Ala Ser Val Val Glu Ser Arg Ser Tyr His Tyr 4395 4400 4405gaa cgt atc atc agc gat ccc caa tgc aat cag gat atc act ctg tcc 34227Glu Arg Ile Ile Ser Asp Pro Gln Cys Asn Gln Asp Ile Thr Leu Ser 4410 4415 4420agt gac cta ttc ggg caa ccg ctg aaa cag gtt tca gtg caa tat ccc 34275Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val Ser Val Gln Tyr Pro 4425 4430 4435cgc cgc aat aaa cca aca acc aat ccg tat ccc gat aca cta cca gat 34323Arg Arg Asn Lys Pro Thr Thr Asn Pro Tyr Pro Asp Thr Leu Pro Asp4440 4445 4450 4455act ctg ttt gcc agc agt tat gac gac caa caa caa cta ttg cgg tta 34371Thr Leu Phe Ala Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg Leu 4460 4465 4470acc tac cag caa tcc agt tgg cat cat cta att gct aat gaa ctc aga 34419Thr Tyr Gln Gln Ser Ser Trp His His Leu Ile Ala Asn Glu Leu Arg 4475 4480 4485gtg tta gga tta ccg gat ggt aca cgc agt gat gct ttc act tac gat 34467Val Leu Gly Leu Pro Asp Gly Thr Arg Ser Asp Ala Phe Thr Tyr Asp 4490 4495 4500gct aaa cac gtg cct gtt gat ggt tta aat ctg gaa gct cta tgt gct 34515Ala Lys His Val Pro Val Asp Gly Leu Asn Leu Glu Ala Leu Cys Ala 4505 4510 4515gaa aat agc ctg att gcc gat gat aaa cct cgc gaa tac ctc aac cag 34563Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg Glu Tyr Leu Asn Gln4520 4525 4530 4535caa cga acg ttc tat acc gat ggg aaa acc gat gga aaa aat cca acg 34611Gln Arg Thr Phe Tyr Thr Asp Gly Lys Thr Asp Gly Lys Asn Pro Thr 4540 4545 4550cca ctg aaa aca ccg aca cga cag gct tta atc gcc ttt acc gaa acg 34659Pro Leu Lys Thr Pro Thr Arg Gln Ala Leu Ile Ala Phe Thr Glu Thr 4555 4560 4565gcg gta tta acg gaa tct ctg tta tcc gca ttt gat ggc ggt atc acg 34707Ala Val Leu Thr Glu Ser Leu Leu Ser Ala Phe Asp Gly Gly Ile Thr 4570 4575 4580cca gat gaa tta ccc ggc ctt ctg aca caa gca gga tac caa caa gaa 34755Pro Asp Glu Leu Pro Gly Leu Leu Thr Gln Ala Gly Tyr Gln Gln Glu 4585 4590 4595cct tat ctg ttc cca ctc agt ggc gaa aac caa gtc tgg gta gca cgc 34803Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln Val Trp Val Ala Arg4600 4605 4610 4615aaa ggc tat acc gat tac gga act gag gta caa ttt tgg cgt cct gtc 34851Lys Gly Tyr Thr Asp Tyr Gly Thr Glu Val Gln Phe Trp Arg Pro Val 4620 4625 4630gca caa cgt aac acc cag tta acc ggg aaa acg act cta aaa tgg gat 34899Ala Gln Arg Asn Thr Gln Leu Thr Gly Lys Thr Thr Leu Lys Trp Asp 4635 4640 4645acc cac tac tgt gtc atc act caa acc caa gac gcg gct ggt ttg act 34947Thr His Tyr Cys Val Ile Thr Gln Thr Gln Asp Ala Ala Gly Leu Thr 4650 4655 4660gtc tca gcc aat tat gac tgg cgt ttt ctc aca cct atg caa ctg act 34995Val Ser Ala Asn Tyr Asp Trp Arg Phe Leu Thr Pro Met Gln Leu Thr 4665 4670 4675gat atc aac gat aat gtg cat atc ata acc ttg gat gcg cta gga cgc 35043Asp Ile Asn Asp Asn Val His Ile Ile Thr Leu Asp Ala Leu Gly Arg4680 4685 4690 4695cct gtc act caa cgt ttc tgg gga atc gaa aat ggt gtg gca aca ggt 35091Pro Val Thr Gln Arg Phe Trp Gly Ile Glu Asn Gly Val Ala Thr Gly 4700 4705 4710tac tct tca cca gaa gca aaa cca ttc act cca cca gtc gat gtc aat 35139Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro Pro Val Asp Val Asn 4715 4720 4725gct gcc att gct ctg acc gga cca ctc cct gtc gcg cag tgt ctg gtc 35187Ala Ala Ile Ala Leu Thr Gly Pro Leu Pro Val Ala Gln Cys Leu Val 4730 4735 4740tat gcg ccg gac agt tgg atg ccg cta ttc ggt cag gaa acc ttc aac 35235Tyr Ala Pro Asp Ser Trp Met Pro Leu Phe Gly Gln Glu Thr Phe Asn 4745 4750 4755aca tta acg cag gaa gag caa aag aca ctg cgt gat tta cgg att atc 35283Thr Leu Thr Gln Glu Glu Gln Lys Thr Leu Arg Asp Leu Arg Ile Ile4760 4765 4770 4775aca gaa gat tgg cgt att tgc gca ctg gct cgc cgc cgt tgg cta caa 35331Thr Glu Asp Trp Arg Ile Cys Ala Leu Ala Arg Arg Arg Trp Leu Gln 4780 4785 4790agt caa aaa gcc ggc aca cca ttg gtt aag ctg tta acc aac agc atc 35379Ser Gln Lys Ala Gly Thr Pro Leu Val Lys Leu Leu Thr Asn Ser Ile 4795 4800 4805ggt tta cct ccc cac aac ctc atg ctg gct acg gac cgt tat gac cgt 35427Gly Leu Pro Pro His Asn Leu Met Leu Ala Thr Asp Arg Tyr Asp Arg 4810 4815 4820gat tct gaa cag caa att cgt caa caa gtc gca ttc agt gat ggt ttt 35475Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala Phe Ser Asp Gly Phe 4825 4830 4835ggc cgt ttg ttg caa gcg gct gtg cgg cat gag gca ggc gaa gcc tgg 35523Gly Arg Leu Leu Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala Trp4840 4845 4850 4855caa cgt aac caa gac ggt tct ctg gtg aca aaa atg gaa gat acc aaa 35571Gln Arg Asn Gln Asp Gly Ser Leu Val Thr Lys Met Glu Asp Thr Lys 4860 4865 4870acg cgc tgg gcg att acg gga cgc act gaa tat gac aat aag ggg cag 35619Thr Arg Trp Ala Ile Thr Gly Arg Thr Glu Tyr Asp Asn Lys Gly Gln 4875 4880 4885gcg ata cga act tat cag ccc tat ttc ctc aat gac tgg cga tat gtg 35667Ala Ile Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr Val 4890 4895 4900agt gat gac agc gcc aga aaa gag gcc tat gcc gat act cat atc tat 35715Ser Asp Asp Ser Ala Arg Lys Glu Ala Tyr Ala Asp Thr His Ile Tyr 4905 4910 4915gat ccg att ggg cgg gaa atc caa gtt atc acg gca aaa ggc tgg ctg 35763Asp Pro Ile Gly Arg Glu Ile Gln Val Ile Thr Ala Lys Gly Trp Leu4920 4925 4930 4935cgg cag aac caa tat ttc ccg tgg ttt acc gtg agt gaa gat gaa aat 35811Arg Gln Asn Gln Tyr Phe Pro Trp Phe Thr Val Ser Glu Asp Glu Asn 4940 4945 4950gat ttg tcc gct gac gcg ctc gtg taa ttgaatcaag attcgctcgt 35858Asp Leu Ser Ala Asp Ala Leu Val 4955 4960ttaatgttaa cgagcgaata taatatacct aatagatttc gagttgcagc gcggcggcaa 35918gtgaacgaat ccccaggagc atagataact atgtgactgg ggtgagtgaa agcagccaac 35978aaagcagcag cttgaaagat gaagggtata aataagaaac tgcattgtga gttctaaata 36038gagtagcagc atattttatt gccttttatt tcataggtaa taaaattcaa ttgctgtaaa 36098aatctgtcat catgagaact aaaaataaca actttctctt ctgcaagaga aatcaataat 36158tcaattaaaa atgttataga atctgaatca agaccatttg ttggctcatc aaaaatataa 36218acatccgcat cggtaataaa agctgatgtc aatagaaatt tcttttttat cccaagtgac 36278atatgtccat actcaatacc agaataatta gatataccaa aaccatttaa atagtaatct 36338aattgatatt ttaaattact tttcctataa cgctgactta aattaatcac atccattccc 36398gtgatgaaat tataaaagtt aacattatcc gatagataaa aaccatgctg ttgcaaatta 36458aatcggctct tttctccctt ttttataaaa ttaaccattc cttttttaac cttatttaca 36518ccagcaatac ttgaaagaaa agtcgtttta cccgccccat taactcccgc aatacggttt 36578aatccaaccc gaaaatcaca attgactcct gaaaaaatag tcttaccatt aataacaacc 36638tctaacccaa taacttcaag cataaataac ccctaaaaat aacgtaaaaa agaaaataac 36698accaacaata ataattttcg tgtattgcgt tctcaacaga gaaatagaag aaacaataat 36758agaaagaaaa gcataagata aaaatataat cacaggaaaa gatttaacaa caagaaagca 36818aaaaataaaa aaacaaagca aataaaaaaa caaagaaata ccataattaa aaaagaatat 36878tttccgcaca gataaaaagt tggacaaata tgaaagataa tttatttcaa tatatgatag 36938attataaaat aacaacatgc atatatataa aacaacactg gcatatatta atgatatata 36998atcagccttg tttgggattt gagaaaaggc actttcacat aatagatata aaagcagaac 37058agataatgcc ataataagca cagacatttt atttttatta aaacaataac gaagattcat 37118tatataaggc aatgaaaaaa aacctgatga aaataatttt ttatttctat taattatata 37178acatggtgtg aaattcaaat ataatatcaa tgctactaat ggaataacta atgtaaaaat 37238caaatcatat aatattccac tcctgaatga tgccgccaga agaaagaaca cagcaacaat 37298aaaaaaatgc aaaaaactta attcaaataa gcaaaatcca attacagcaa aagaaactat 37358caaaaaaaac acagatgaaa ggtaatgcaa ataattaaca ttttcgtaaa aaaaacctat 37418aaagaagaaa ataactatcg gaaaagcact ataaataaaa aaaacgatac gactaaaaaa 37478caacgttttt ttacctacca aagaaacgat gattgaattc tcctttgcag aaggaaaaaa 37538ccttatgtta atcaaataaa ataccatata taccattaaa gatatggcag taaaataaaa 37598tgattttatg tagccatctg gaataataat attggaagat aaagttatta aaacctcaaa 37658gataccactg aactttgccg gaagtaataa aagaaaaagg aatataatga catttttatt 37718cccagacgca aatttcttta tcctaccttt atattccaag gcatcagcga ttattaaatt 37778catactgcct ctctaaaacc aaaatctaaa taatgtcctt ggtgaatctt tagggaattt 37838cgtcctggaa tgcaaatata aatagttact gaaaacaata cattgatttt taattaaata 37898ctggcgatat gaccttaatg atgctacttt attttccagt attcaattcg 37948 12 954 PRT Photorhabdus luminescens 12Met Lys Asn Ile Asp Pro Lys Leu Tyr Gln Lys Thr Pro Val Val Asn 1 5 10 15Ile Tyr Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His Arg 20 25 30Thr Thr Ala Asn Gly Asp Thr Asp Ile Arg Ile Thr Arg His Gln Tyr 35 40 45Asp Ser Leu Gly His Leu Ser Gln Ser Thr Asp Pro Arg Leu Tyr Glu 50 55 60Ala Lys Gln Lys Ser Asn Phe Leu Trp Gln Tyr Asp Leu Thr Gly Asn 65 70 75 80Ile Leu Cys Thr Glu Ser Val Asp Ala Gly Arg Thr Val Thr Leu Asn 85 90 95Asp Ile Glu Gly Arg Pro Leu Leu Thr Val Thr Ala Thr Gly Val Ile 100 105 110Gln Thr Arg Gln Tyr Glu Thr Ser Ser Leu Pro Gly Arg Leu Leu Ser 115 120 125Val Thr Glu Gln Ile Pro Glu Lys Thr Ser Arg Ile Thr Glu Arg Leu 130 135 140Ile Trp Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser145 150 155 160Gln Cys Val Arg His Tyr Asp Thr Ala Gly Val Thr Arg Leu Glu Ser 165 170 175Leu Ser Leu Thr Gly Thr Val Leu Ser Gln Ser Ser Gln Leu Leu Ser 180 185 190Asp Thr Gln Glu Ala Ser Trp Thr Gly Asp Asn Glu Thr Val Trp Gln 195 200 205Asn Met Leu Ala Asp Asp Ile Tyr Thr Thr Leu Ser Ala Phe Asp Ala 210 215 220Thr Gly Ala Leu Leu Thr Gln Thr Asp Ala Lys Gly Asn Ile Gln Arg225 230 235 240Leu Thr Tyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp Leu Thr Leu 245 250 255Lys Asp Gln Pro Glu Gln Val Ile Ile Arg Ser Leu Thr Tyr Ser Ala 260 265 270Ala Gly Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val Ile Thr Glu 275 280 285Tyr Ser Tyr Glu Pro Glu Thr Gln Gln Leu Ile Gly Thr Lys Thr His 290 295 300Arg Pro Ser Asp Ala Lys Val Leu Gln Asp Leu Arg Tyr Glu Tyr Asp305 310 315 320Pro Val Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu Ala Thr Arg 325 330 335Phe Trp His Asn Gln Lys Val Ala Pro Glu Asn Thr Tyr Thr Tyr Asp 340 345 350Ser Leu Tyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met Ala Asn Ile 355 360 365Gly Gln Gln Ser Asn Gln Leu Pro Ser Leu Thr Leu Pro Ser Asp Asn 370 375 380Asn Thr Tyr Thr Asn Tyr Thr Arg Thr Tyr Thr Tyr Asp Arg Gly Gly385 390 395 400Asn Leu Thr Lys Ile Gln His Ser Ser Pro Ala Thr Gln Asn Asn Tyr 405 410 415Thr Thr Asn Ile Thr Val Ser Asn Arg Ser Asn Arg Ala Val Leu Ser 420 425 430Thr Leu Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe Asp Ala Gly 435 440 445Gly His Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn Trp Asn Thr 450 455 460Arg Gly Glu Leu Gln His Val Thr Leu Val Lys Arg Asp Lys Gly Ala465 470 475 480Asn Asp Asp Arg Glu Trp Tyr Arg Tyr Ser Ser Asp Gly Arg Arg Ile 485 490 495Leu Lys Ile Asn Glu Gln Gln Thr Ser Ser Asn Ser Gln Thr Gln Arg 500 505 510Ile Thr Tyr Leu Pro Ser Leu Glu Leu Arg Leu Thr Gln Asn Ser Thr 515 520 525Ile Thr Thr Glu Asp Leu Gln Val Ile Thr Val Gly Glu Ala Gly Arg 530 535 540Ala Gln Val Arg Val Leu His Trp Asp Ser Gly Gln Pro Glu Asp Ile545 550 555 560Asp Asn Asn Gln Leu Arg Tyr Ser Tyr Asp Asn Leu Ile Gly Ser Ser 565 570 575Gln Leu Glu Leu Asp Ser Lys Gly Glu Ile Ile Ser Glu Glu Glu Tyr 580 585 590Tyr Pro Tyr Gly Gly Thr Ala Leu Trp Ala Thr Arg Lys Arg Thr Glu 595 600 605Ala Ser Tyr Lys Thr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr 610 615 620Gly Leu Tyr Tyr Tyr Gly Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg625 630 635 640Trp Leu Ser Ala Asp Pro Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr 645 650 655Arg Met Val Arg Asn Asn Pro Val Thr Leu Leu Asp Pro Asp Gly Leu 660 665 670Met Pro Thr Ile Ala Glu Arg Ile Ala Ala Leu Gln Lys Asn Lys Val 675 680 685Ala Asp Ser Ala Pro Ser Pro Thr Asn Ala Thr Asn Val Ala Ile Asn 690 695 700Ile Arg Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys Ala Ser Thr705 710 715 720Ser Ser Gln Ser Thr Thr Tyr Pro Ile Lys Ser Ala Ser Ile Lys Pro 725 730 735Thr Thr Ser Gly Ser Ser Ile Thr Ala Pro Leu Ser Pro Val Gly Asn 740 745 750Lys Ser Thr Pro Glu Ile Ser Leu Pro Glu Ser Thr Gln Ser Asn Ser 755 760 765Ser Ser Ala Ile Ser Thr Asn Leu Gln Lys Lys Ser Phe Thr Leu Tyr 770 775 780Arg Ala Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys Phe Pro Glu785 790 795 800Gly Phe Lys Ala Trp Thr Pro Leu Asp Thr Lys Met Ala Arg Gln Phe 805 810 815Ala Ser Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu Pro Lys Glu 820 825 830Thr Val Lys Asn Ile Asn Thr Trp Gly Thr Lys Pro Lys Leu Asn Asp 835 840 845Leu Ser Thr Tyr Ile Lys Tyr Thr Lys Asp Lys Ser Thr Val Trp Val 850 855 860Ser Thr Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser Gly Ala Pro865 870 875 880Leu His Glu Ile Asn Met Asp Leu Tyr Glu Phe Thr Ile Asp Gly Gln 885 890 895Lys Leu Asn Pro Leu Pro Arg Gly Arg Ser Lys Asp Arg Val Pro Ser 900 905 910Leu Leu Leu Asp Thr Pro Glu Ile Glu Thr Ala Ser Ile Ile Ala Leu 915 920 925Asn His Gly Pro Val Asn Asp Ala Glu Val Ser Phe Leu Thr Thr Ile 930 935 940Pro Leu Lys Asn Val Lys Pro Tyr Lys Arg945 950 13 2522 PRT Photorhabdus luminescens 13Met Ile Leu Lys Gly Ile Asn Met Asn Ser Pro Val Lys Glu Ile Pro 1 5 10 15Asp Val Leu Lys Ile Gln Cys Gly Phe Gln Cys Leu Thr Asp Ile Ser 20 25 30His Ser Ser Phe Asn Glu Phe His Gln Gln Val Ser Glu His Leu Ser 35 40 45Trp Ser Glu Ala His Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys 50 55 60Asp Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Thr Asn Pro Gln 65 70 75 80Leu Gln Asn Ala Val His Leu Ala Ile Val Ala Pro Asn Ala Glu Leu 85 90 95Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala 100 105 110Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu 115 120 125Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr Arg 130 135 140Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln145 150 155 160Asn Met Asp Thr Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu 165 170 175Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Asp Asn Tyr Thr Gln Val 180 185 190Met Glu Met Leu Ser Ala Phe Arg Pro Ser Gly Ala Thr Pro Tyr His 195 200 205Asp Ala Tyr Glu Asn Val Arg Lys Val Ile Gln Leu Gln Asp Pro Gly 210 215 220Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln225 230 235 240Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn 245 250 255Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys 260 265 270Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr 275 280 285Leu Arg Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile 290 295 300Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu305 310 315 320Ile Thr Pro Ile Val Asn Ser Asn Asp Gly Thr Val Lys Val Tyr Arg 325 330 335Ile Thr Arg Glu Tyr Thr Thr Asn Ala Asn Gln Val Asp Val Glu Leu 340 345 350Phe Pro Tyr Gly Gly Glu Asn Tyr Gln Leu Asn Tyr Lys Phe Lys Asp 355 360 365Ser Arg Gln Asp Val Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg 370 375 380Glu Leu Ile Arg Ile Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser385 390 395 400Glu His Ile Thr Leu Ser Thr Thr Asp Ile Ser Gln Pro Phe Glu Ile 405 410 415Gly Leu Thr Arg Val Tyr Pro Ser Ser Ser Trp Ala Tyr Ala Ala Ala 420 425 430Lys Phe Thr Ile Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu 435 440 445Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile 450 455 460Leu Glu Ser Ile Val Arg Ser Val Asn Gln Gln Leu Asp Ile Asn Ala465 470 475 480Glu Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr 485 490 495Ala Ile Asn Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Leu Ile Ser 500 505 510Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn 515 520 525Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile 530 535 540Asp Leu Asn Pro Gly Ser Thr Gly Asp Trp Arg Lys Ser Val Leu Lys545 550 555 560Arg Ala Phe Asn Ile Asp Asp Ile Ser Leu Tyr Arg Leu Leu Lys Ile 565 570 575Thr Asn His Asn Asn Gln Asp Gly Lys Ile Lys Asn Asn Leu Asn Asn 580 585 590Leu Ser Asp Leu Tyr Ile Gly Lys Leu Leu Ala Glu Ile His Gln Leu 595 600 605Thr Ile Asp Glu Leu Asp Leu Leu Leu Val Ala Val Gly Glu Gly Glu 610 615 620Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Ala Leu Ile Arg625 630 635 640Lys Leu Asn Thr Ile Thr Val Trp Leu Gln Thr Gln Lys Trp Ser Ala 645 650 655Phe Gln Leu Phe Val Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr 660 665 670Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly 675 680 685Phe Asp Lys Asp Lys Ala Asn Leu Leu His Val Met Ala Pro Tyr Ile 690 695 700Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu705 710 715 720Leu Trp Ala Asp Lys Leu Lys Pro Gly Asp Gly Ala Met Thr Ala Glu 725 730 735Lys Phe Trp Asp Trp Leu Asn Thr Gln Tyr Thr Pro Asp Ser Ser Glu 740 745 750Val Leu Ala Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala 755 760 765Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe 770 775 780Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ser Ser Thr Glu Ala785 790 795 800Val Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala 805 810 815Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala 820 825 830Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn 835 840 845Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Thr Gln Ala Gln Asn His 850 855 860Gln His Leu Pro Pro Val Thr Gln Lys Asn Ala Phe Ser Cys Trp Thr865 870 875 880Ser Ile Asp Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn 885 890 895Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln 900 905 910Leu Asn Gln Lys Ile Pro Thr Tyr Ala Gln Trp Glu Ser Ala Gly Glu 915 920 925Ile Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asp Ile Leu His Ala 930 935 940Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg945 950 955 960Gln Val Ala Lys Pro Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr 965 970 975Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr 980 985 990Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Thr 995 1000 1005Leu Glu Asn Val Glu Glu Asn Ala His Ser Gly Val Ile Ser Arg Gln 1010 1015 1020Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala1025 1030 1035 1040Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr 1045 1050 1055Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val 1060 1065 1070Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser 1075 1080 1085Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala 1090 1095 1100Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly1105 1110 1115 1120Leu Ser Glu Thr Asp Thr Gly Glu Tyr Tyr Trp Arg Ser Val Asp His 1125 1130 1135Ser Lys Phe Ser Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp 1140 1145 1150His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Arg Ser Thr Ile Arg Pro 1155 1160 1165Val Met Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu 1170 1175 1180Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr1185 1190 1195 1200Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr 1205 1210 1215Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Glu Lys Ile Ser Lys Leu 1220 1225 1230Glu Leu Ala Lys Asn Lys Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln 1235 1240 1245Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu 1250 1255 1260Asp Ser Tyr Lys Thr Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp1265 1270 1275 1280Met Glu Tyr Lys Asp Met Thr Asp Gly Gln Tyr Lys Ser Tyr Arg Asp 1285 1290 1295Asn Ser Tyr Lys Gln Phe Asp Thr Asn Ser Val Arg Arg Val Asn Asn 1300 1305 1310Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Asn Ser Arg Lys 1315 1320 1325Gly Tyr Asp Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp 1330 1335 1340Ile Pro Thr Ile Ser Tyr Lys Ala Thr Ser Ser Asp Leu Lys Ile Tyr1345 1350 1355 1360Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Gln 1365 1370 1375Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys 1380 1385 1390Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn 1395 1400 1405Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Asn Gly Asn Val Ser Gly 1410 1415 1420Leu Ser Gln Gly Arg Leu Leu Phe His Arg Asp Thr Asn Tyr Ser Ser1425 1430 1435 1440Lys Val Glu Ala Trp Ile Pro Gly Ala Gly Arg Ser Leu Thr Asn Pro 1445 1450 1455Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro 1460 1465 1470Asn Asp Leu Lys Gln Tyr Val Tyr Met Thr Asp Ser Lys Gly Thr Ala 1475 1480 1485Thr Asp Val Ser Gly Pro Val Asp Ile Asn Thr Ala Ile Ser Pro Ala 1490 1495 1500Lys Val Gln Val Thr Val Lys Ala Gly Ser Lys Glu Gln Thr Phe Thr1505 1510 1515 1520Ala Asp Lys Asn Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met 1525 1530 1535Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Ser Leu Asn Phe 1540 1545 1550Thr Asn Asn Ser Ala Ser Ile Asp Ile Thr Phe Thr Ala Phe Ala Glu 1555 1560 1565Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Ile Thr Arg 1570 1575 1580Lys Val Ser Thr Asp Asn Ser Leu Thr Leu Arg His Asn Glu Asn Gly1585 1590 1595 1600Ala Gln Tyr Met Gln Trp Gly Val Tyr Arg Ile Arg Leu Asn Thr Leu 1605 1610 1615Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile 1620 1625 1630Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly 1635 1640 1645Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Pro Ser Thr His Gly 1650 1655 1660Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn1665 1670 1675 1680Ser His Ile Ile Tyr Ser Gly Gln Leu Lys Asp Thr Asn Ile Ser Thr 1685 1690 1695Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr Ser 1700 1705 1710Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp 1715 1720 1725Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn 1730 1735 1740Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn1745 1750 1755 1760Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe 1765 1770 1775Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu 1780 1785 1790His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp 1795 1800 1805Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp 1810 1815 1820Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu1825 1830 1835 1840Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr 1845 1850 1855Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly 1860 1865 1870Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys 1875 1880 1885Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu 1890 1895 1900Pro Leu Ser Thr Thr Trp Asn Asp Pro Arg Leu Asp Lys Ala Ala Asp1905 1910 1915 1920Ile Thr Thr Gln Ser Ala His Ser Ser Ser Ile Val Ala Leu Arg Gln 1925 1930 1935Ser Thr Pro Ala Leu Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp 1940 1945 1950Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr 1955 1960 1965Leu Ala Gln Arg Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp Gly 1970 1975 1980Gln Pro Leu Tyr Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala1985 1990 1995 2000Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro 2005 2010 2015Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala 2020 2025 2030Arg Ser Met Val Ser Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn 2035 2040 2045Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn 2050 2055 2060Gln Ala Ala Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr2065 2070 2075 2080Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala 2085 2090 2095Gly Ala Gln Ser Arg Phe Asp Ser Tyr Ser Lys Leu His Asp Glu Asn 2100 2105 2110Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala 2115 2120 2125Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala 2130 2135 2140Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp2145 2150 2155 2160Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn 2165 2170 2175Val Met Asn Thr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg 2180 2185 2190Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu 2195 2200 2205Leu Lys Gln Leu Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu 2210 2215 2220Ala Ala Val Leu Gln Lys Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr2225 2230 2235 2240Gln Ala Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu 2245 2250 2255Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr 2260 2265 2270Asp Leu Ala Ile Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp 2275 2280 2285Glu Ile Ser Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln 2290 2295 2300Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu2305 2310 2315 2320Ala Gln Met Glu Asp Ala His Leu Arg Arg Asp Lys Arg Ala Leu Glu 2325 2330 2335Val Glu Arg Thr Val Ser Leu Ala Glu Ile Tyr Ala Gly Leu Pro Gln 2340 2345 2350Asp Lys Gly Pro Phe Ser Leu Thr Gln Glu Ile Glu Lys Leu Val Asn 2355 2360 2365Ala Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly 2370 2375 2380Ala Gly Thr Asp Thr Lys Thr Ser Leu Gln Ala Ser Ile Ser Leu Ala2385 2390 2395 2400Asp Leu Lys Ile Arg Glu Asp Tyr Pro Glu Ser Ile Gly Lys Ile Arg 2405 2410 2415Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr 2420 2425 2430Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala 2435 2440 2445Asn Gly Cys Ala Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser Gly 2450 2455 2460Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly2465 2470 2475 2480Ile Ala Ile Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser 2485 2490 2495Thr Pro Ala Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp 2500 2505 2510Ile Ile Leu His Ile Arg Tyr Thr Ile Lys 2515 2520 14 1481 PRT Photorhabdus luminescens 14Met Gln Asn Ser Gln Thr Phe Ser Met Thr Glu Leu Ser Leu Pro Lys 1 5 10 15Gly Gly Gly Ala Ile Thr Gly Met Gly Glu Ala Leu Thr Pro Ala Gly 20 25 30Pro Asp Gly Met Ala Ala Leu Ser Leu Pro Leu Pro Ile Ser Ala Gly 35 40 45Arg Gly Tyr Ala Pro Ser Leu Thr Leu Asn Tyr Asn Ser Gly Thr Gly 50 55 60Asn Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val Met Thr Ile Arg 65 70 75 80Arg Arg Thr Ser Thr Gly Val Pro Asn Tyr Asp Glu Thr Asp Thr Phe 85 90 95Leu Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu Asn Glu Ala Gly 100 105 110Gln Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly Ile Asn Leu Gly 115 120 125Met Thr Phe Thr Val Thr Gly Tyr Arg Ser Arg Leu Glu Ser His Phe 130 135 140Ser Arg Leu Glu Tyr Trp Gln Pro Gln Thr Thr Gly Ala Thr Asp Phe145 150 155 160Trp Leu Ile Tyr Ser Pro Asp Gly Gln Ala His Leu Leu Gly Lys Asn 165 170 175Pro Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn Gln Thr Ala Gln 180 185 190Trp Leu Leu Glu Ala Ser Val Ser Ser His Gly Glu Gln Ile Tyr Tyr 195 200 205Gln Tyr Arg Ala Glu Asp Glu Thr Asp Cys Glu Thr Asp Glu Leu Thr 210 215 220Ala His Pro Asn Thr Thr Val Gln Arg Tyr Leu Gln Val Val His Tyr225 230 235 240Gly Asn Leu Thr Ala Ser Glu Val Phe Pro Thr Leu Asn Gly Asp Asp 245 250 255Pro Leu Lys Ser Gly Trp Leu Phe Cys Leu Val Phe Asp Tyr Gly Glu 260 265 270Arg Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys Ala Thr Ser Asn 275 280 285Trp Leu Cys Arg Lys Asp Arg Phe Ser Arg Tyr Glu Tyr Gly Phe Ala 290 295 300Leu Arg Thr Arg Arg Leu Cys Arg Gln Ile Leu Met Phe His Arg Leu305 310 315 320Gln Thr Leu Ser Gly Gln Ala Lys Gly Asp Asp Glu Pro Ala Leu Val 325 330 335Ser Arg Leu Ile Leu Asp Tyr Asp Glu Asn Ala Val Val Ser Thr Leu 340 345 350Val Ser Val Arg Arg Val Gly His Glu Gln Asp Gly Thr Thr Ala Val 355 360 365Ala Leu Pro Pro Leu Glu Leu Ala Tyr Gln Pro Phe Glu Pro Glu Gln 370 375 380Lys Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn Phe Asn Thr Ile385 390 395 400Gln Arg Trp Gln Leu Leu Asp Leu Gln Gly Glu Gly Val Pro Gly Ile 405 410 415Leu Tyr Gln Asp Lys Asn Gly Trp Trp Tyr Arg Ser Ala Gln Arg Gln 420 425 430Thr Gly Glu Glu Met Asn Ala Val Thr Trp Gly Lys Met Gln Leu Leu 435 440 445Pro Ile Thr Pro Ala Ile Gln Asp Asn Ala Ser Leu Met Asp Ile Asn 450 455 460Gly Asp Gly Gln Leu Asp Trp Val Ile Thr Gly Pro Gly Leu Arg Gly465 470 475 480Tyr His Ser Gln His Pro Asp Gly Ser Trp Thr Arg Phe Thr Pro Leu 485 490 495His Ala Leu Pro Ile Glu Tyr Thr His Pro Arg Ala Gln Leu Ala Asp 500 505 510Leu Met Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser 515 520 525Val Arg Leu Tyr Ala Asn Asn Arg Asp Gly Phe Thr Glu Gly Arg Asp 530 535 540Val Val Gln Ser Gly Gly Ile Thr Leu Pro Leu Pro Gly Ala Asp Ala545 550 555 560Arg Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser Gly Gln Ala His 565 570 575Leu Val Glu Val Ser Ala Thr Lys Val Thr Cys Trp Pro Asn Leu Gly 580 585 590His Gly Arg Phe Gly Gln Pro Ile Thr Leu Pro Gly Phe Ser Gln Ser 595 600 605Ala Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala Asp Leu Asp Gly 610 615 620Ser Gly Pro Ala Asp Leu Ile Tyr Val His Ala Asp His Leu Asp Ile625 630 635 640Phe Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro Phe Thr Leu Arg 645 650 655Phe Pro Asp Gly Leu Arg Phe Asp Asp Thr Cys Gln Leu Gln Val Ala 660 665 670Asp Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu Ser Val Pro His 675 680 685Met Ala Pro His His Trp Arg Cys Asp Leu Thr Asn Ala Lys Pro Trp 690 695 700Leu Leu Ser Glu Met Asn Asn Asn Met Gly Ala His His Thr Leu His705 710 715 720Tyr Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys Ala Ala Ala Leu 725 730 735Ala Thr Gly Gln Thr Pro Val Cys Tyr Leu Pro Phe Pro Val His Thr 740 745 750Leu Trp Gln Thr Glu Thr Glu Asp Glu Ile Ser Gly Asn Lys Leu Val 755 760 765Thr Thr Leu Arg Tyr Ala His Gly Ala Trp Asp Gly Arg Glu Arg Glu 770 775 780Phe Arg Gly Phe Gly Tyr Val Glu Gln Thr Asp Ser His Gln Leu Ala785 790 795 800Gln Gly Asn Ala Pro Glu Arg Thr Ser Pro Ala Leu Thr Lys Asn Trp 805 810 815Tyr Ala Thr Gly Ile Pro Glu Val Asp Asn Thr Leu Ser Ala Gly Tyr 820 825 830Trp Arg Gly Asp Thr Gln Ala Phe Thr Gly Phe Thr Pro His Phe Thr 835 840 845Leu Trp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro Glu Asp Asp His 850 855 860Asn Leu Tyr Trp Leu Asn Arg Ala Leu Lys Gly Gln Pro Leu Arg Ser865 870 875 880Glu Leu Tyr Gly Leu Asp Gly Ser Ala Gln Gln Lys Ile Pro Tyr Thr 885 890 895Val Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln Asp Asn Thr Thr 900 905 910Leu Ser Pro Val Leu Trp Ala Ser Val Val Glu Ser Arg Ser Tyr His 915 920 925Tyr Glu Arg Ile Ile Ser Asp Pro Gln Cys Asn Gln Asp Ile Thr Leu 930 935 940Ser Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val Ser Val Gln Tyr945 950 955 960Pro Arg Arg Asn Lys Pro Thr Thr Asn Pro Tyr Pro Asp Thr Leu Pro 965 970 975Asp Thr Leu Phe Ala Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg 980 985 990Leu Thr Tyr Gln Gln Ser Ser Trp His His Leu Ile Ala Asn Glu Leu 995 1000 1005Arg Val Leu Gly Leu Pro Asp Gly Thr Arg Ser Asp Ala Phe Thr Tyr 1010 1015 1020Asp Ala Lys His Val Pro Val Asp Gly Leu Asn Leu Glu Ala Leu Cys1025 1030 1035 1040Ala Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg Glu Tyr Leu Asn 1045 1050 1055Gln Gln Arg Thr Phe Tyr Thr Asp Gly Lys Thr Asp Gly Lys Asn Pro 1060 1065 1070Thr Pro Leu Lys Thr Pro Thr Arg Gln Ala Leu Ile Ala Phe Thr Glu 1075 1080 1085Thr Ala Val Leu Thr Glu Ser Leu Leu Ser Ala Phe Asp Gly Gly Ile 1090 1095 1100Thr Pro Asp Glu Leu Pro Gly Leu Leu Thr Gln Ala Gly Tyr Gln Gln1105 1110 1115 1120Glu Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln Val Trp Val Ala 1125 1130 1135Arg Lys Gly Tyr Thr Asp Tyr Gly Thr Glu Val Gln Phe Trp Arg Pro 1140 1145 1150Val Ala Gln Arg Asn Thr Gln Leu Thr Gly Lys Thr Thr Leu Lys Trp 1155 1160 1165Asp Thr His Tyr Cys Val Ile Thr Gln Thr Gln Asp Ala Ala Gly Leu 1170 1175 1180Thr Val Ser Ala Asn Tyr Asp Trp Arg Phe Leu Thr Pro Met Gln Leu1185 1190 1195 1200Thr Asp Ile Asn Asp Asn Val His Ile Ile Thr Leu Asp Ala Leu Gly 1205 1210 1215Arg Pro Val Thr Gln Arg Phe Trp Gly Ile Glu Asn Gly Val Ala Thr 1220 1225 1230Gly Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro Pro Val Asp Val 1235 1240 1245Asn Ala Ala Ile Ala Leu Thr Gly Pro Leu Pro Val Ala Gln Cys Leu 1250 1255 1260Val Tyr Ala Pro Asp Ser Trp Met Pro Leu Phe Gly Gln Glu Thr Phe1265 1270 1275 1280Asn Thr Leu Thr Gln Glu Glu Gln Lys Thr Leu Arg Asp Leu Arg Ile 1285 1290 1295Ile Thr Glu Asp Trp Arg Ile Cys Ala Leu Ala Arg Arg Arg Trp Leu 1300 1305 1310Gln Ser Gln Lys Ala Gly Thr Pro Leu Val Lys Leu Leu Thr Asn Ser 1315 1320 1325Ile Gly Leu Pro Pro His Asn Leu Met Leu Ala Thr Asp Arg Tyr Asp 1330 1335 1340Arg Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala Phe Ser Asp Gly1345 1350 1355 1360Phe Gly Arg Leu Leu Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala 1365 1370 1375Trp Gln Arg Asn Gln Asp Gly Ser Leu Val Thr Lys Met Glu Asp Thr 1380 1385 1390Lys Thr Arg Trp Ala Ile Thr Gly Arg Thr Glu Tyr Asp Asn Lys Gly 1395 1400 1405Gln Ala Ile Arg Thr Tyr Gln Pro Tyr Phe Leu Asn Asp Trp Arg Tyr 1410 1415 1420Val Ser Asp Asp Ser Ala Arg Lys Glu Ala Tyr Ala Asp Thr His Ile1425 1430 1435 1440Tyr Asp Pro Ile Gly Arg Glu Ile Gln Val Ile Thr Ala Lys Gly Trp 1445 1450 1455Leu Arg Gln Asn Gln Tyr Phe Pro Trp Phe Thr Val Ser Glu Asp Glu 1460 1465 1470Asn Asp Leu Ser Ala Asp Ala Leu Val 1475 1480 15 23 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 15cgggatccga tgattttaaa agg 23 16 16 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 16gcgccattga tttgag 16 17 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 17cattagaggt cgaacgtac 19 18 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 18gagcgagctc ttacttaatg gtgtag 26 19 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 19cagcgagctc catgcagaat tcacagac 28 20 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 20ggcaatggca gcgataag 18 21 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 21cattaacgca ggaagagc 18 22 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 22gacctcgagt 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).