Methods and materials for identifying novel pesticide agents

This application claims the benefit of U.S. Provisional Application Ser. No. 60/153,116, filed Sep. 7, 1999.

Aminopeptidase N (APN) is an exopeptidase that hydrolyses neutral amino acids from the amino (N)-termini of different proteins. In different cell types, APN is expressed as a soluble cytoplasmic enzyme and a membrane-bound ectoenzyme. This enzyme is found on the surface of diverse cell types including lung, kidney, intestine and brain cells of many animals (Kenny et al., 1987). The ectoenzyme form is attached to epithelial cells of intestinal brush borders and respiratory tracts of vertebrates by a hydrophobic N-terminal stalk (Kenny et al., 1987 and Takasaki et al., 1991). In insects, however, ectoenzyme attachment is via a glycosyl-phosphatidylinositol (GPI) anchor (Tomita et al., 1994; Garczynski and Adang, 1995; Luo et al., 1996a; Luo et al., 1996b; Luo et al., 1997a; and Luo et al., 1 997b). GPI-anchored proteins are relatively mobile on the membrane surface and can be clustered in microdomains with other proteins and specific lipids. The base of the GPI-anchor interacts with the intracellular environment and has been implicated in physiological functions, intracellular sorting and transmembrane signaling (McConville and Ferguson, 1993).

In intestinal epithelial cells, APN is important for the final hydrolysis step of ingested proteins. APN also has several important physiological roles in other tissues. For example, APN is implicated in tumor cell invasion and inhibition of aminopeptidase activity can suppress tumor cell spread (Fujii et al., 1995). In brain cells, APN serves a role in the breakdown and inactivation of peptide neurotransmitters (Kenny et al., 1987). In bovine renal brush border membrane vesicles (BBMV), partially purified APN was found to be associated with a Na + -dependent amino acid co-transporter (Plakidou-dymock et al., 1993).

APN molecules function as adventitious receptors for viruses. Human, feline, canine, and porcine coronaviruses utilize APN as their cellular receptors (Delmas et al., 1992; Yeager et al., 1992; and Tresnan et al., 1996). Cells refractory to coronaviruses from a particular animal species can be made susceptible by expression of an APN cDNA from that species (Benbacer et al., 1997). Human APN was shown to mediate human cytomegalovirus infection by increasing virus binding (McLaughlin and Aderem, 1995). Human, porcine and feline APNs have been cloned and expressed in different cell lines (Delmas et al.,. 1992; Yeager et al., 1992; Kolb et al., 1996; and Tresnan et al., 1996). Each of these vertebrate APNs were expressed on the cell surface as the N-terminal stalked form and bound a coronavirus.

Isoforms of APN located in the epithelial cells of insect midguts bind specifically to Bacillus thuringiensis Cry1 δ-endotoxins. Toxin-binding APNs are reported for several lepidopteran species (see, e.g., Knight et al., 1994; Sangadala et al., 1994; Gill et al., 1995; Valaitis et al., 1995; Luo et al., 1996; and Yaoi et al., 1997). For example, Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C or Cry1E toxins bind to a purified 115 kDa APN from Manduca sexta (Masson et al., 1995). Also partially purified preparations of APN catalyze toxin-induced pore formation in membrane vesicles (Sangadala et al., 1994) and planar lipid bilayers (Schwartz et al., 1997).

Several APN isoforms have been purified and cloned from different insect species (see, e.g., Knight et al., 1995; Gill et al., 1995; Valaitis et a, 1995; Luo et al., 1996; Yaoi et al., 1997; Denolf et al., 1997; and Hua et al., 1998). However, there has been limited success in expressing insect APN cDNA in insect cells. The only example to date involved the expression of Plutella xylostella 105 kDa APN in Sf9 cells using a baculovirus vector (Denolf et al., 1997). While the transformed cells of this study produced APN localized to the cell membrane, the APN was unable to bind to B. thuringiensis Cry1A toxins. Further, Denolf et al were unsuccessful in expressing two 120 kDa APNs from Manduca sexta using the same vector.

The complete structural and functional characterization of insect APN will require the successful expression of insect APN in insect cells. Successful expression of insect APN in insect cells as described in Luo et al. (1999) would also facilitate study of APN-toxin interactions, as well as provide a screening system for obtaining novel pesticide agents.

The subject invention pertains to cells expressing a polynucleotide encoding an Aminopeptide N (APN), and methods of using the same to identify pesticide agents. One aspect of the invention pertains to an isolated polynucleotide which encodes a full length APN from Manduca sexta (M sexta) (SEQ ID NO: 1). Another aspect pertains to a fragment of said fill length polynucleotide which is sufficient to encode a functional protein.

In another aspect, the subject invention pertains to a cell or cells transfected with a polynucleotide encoding an APN or fragments thereof, such that a functional polynucleotide is expressed. Preferably, the polynucleotide is expressed forming a protein which is localized at the cell membrane of said cell or cells. A further aspect pertains to descendent generations of said cells which express APN that is localized on the cell membrane.

In a further aspect, the subject invention is directed to a method of identifying pesticide agents comprising obtaining a cell or cells transfected with a polynucleotide encoding an APN or fragments thereof, such that said polynucleotide is expressed to produce a protein localized at the cell membrane of said cell or cells, and screening one or more pesticide agents for their ability to produce an observable effect on said cell or cells.

In yet another aspect, the subject invention is drawn to novel pesticide agents obtained according to the subject methods.

In a still further aspect, the subject invention is drawn to an expression vector comprising a polynucleotide encoding APN or a functional fragment thereof.

An alternative aspect of the subject invention pertains to a method of identifying novel aminopeptidase inhibitors comprising obtaining cells having APN localized on the cell membranes thereof; and screening one or more compounds of interest for their ability to inhibit aminopeptidase activity.

FIG. 1 represents construction of the expression vector pHSP120.

FIGS. 2A-2E represents the amino acid/polynucleotide sequence of the APN1a from M. sexta (SEQ ID NO: 1 and SEQ ID NO: 2, respectively).

FIG. 3 represents a comparison of the cloned Manduca Sexta 120 kDa APN1a ( M. sexta 1a)(SEQ ID NO: 1) with M. sexta 120 kDa APN2 ( M. sexta 2)[Denolf et al. 1997](SEQ ID NO: 9), Plutella xylostella APN ( P. xylostella )[Denolf et al. 1997](SEQ ID NO: 10), M. sexta 120 kDa APN1 ( M. sexta 1)[Knight et al. 1995](SEQ ID NO: 11), Bombyx mori APN (B. mori)[Hua et al. 1998](SEQ ID NO: 12), and Heliothis virescens APN (H. virescens) [Gill et al. 1995](SEQ ID NO: 13).

FIG. 4 represents immunoblot analysis of M sexta 120 kDa APN expressed in Sf21 cells and 115 kDa APN purified from M sexta midguts.

FIG. 5 represents autoradiograph of in vitro transcription and translation using various plasmid DNAs.

FIGS. 6A-6B represents optimization of expression of M sexta 120 kDa APN in Sf21

FIGS. 7A-7B represents immunoblot analysis of M sexta 120 kDa APN using anti-CRD antiserium.

FIGS. 8A-8D represents immunofluorescence localization of M sexta 120 kDa APN1a in Sf21 cells by confocal microscopy.

FIG. 9 shows APN activity of Sf21 cells transfected with pHSP120 (columns A and B) or pHSP-HR5 (columns C and D).

FIGS. 10A-10D represents immunofluorescence analyses of the binding of Cry1Ac toxin and anti-APN antibody to Sf21 cell surface by confocal microscopy.

FIGS. 11A-11D represents immunofluorescence analyses of the binding of Cry1Ba toxin 55 kDa form) and anti-APN antibody to Sf21 cell surface by confocal microscopy.

FIGS. 12A-12D represents immunofluorescence analyses of the binding of Cry1Ba toxin (65 kDa form) and anti-APN antibody to Sf21 cell surface by confocal microscopy.

FIG. 13 represents immunoblot analysis of Cry1Ac, Cry1Ba and Cry3 toxin-affinity column purified M sexta 120 kDa APN from Sf21 cells and from M sexta midguts.

FIG. 14 represents cytotoxicity of Cry1Ba (55 kDa form) to Sf21 cells transfected with pHSP120 (open circle) and pHSP-HR5 (closed circle).

SEQ ID NO: 1) is the amino acid sequence for the APN1a from M. sexta.

SEQ ID NO: 2) is the nucleotide sequence for the APN1a from M. sexta.

SEQ ID NO: 3) is the nucleotide sequence for PCR primer MS1.

SEQ ID NO: 4) is the nucleotide sequence for PCR primer MS1R.

SEQ ID NO: 5) is the nucleotide sequence for PCR primer MS5R.

SEQ ID NO: 6) is the nucleotide sequence for PCR primer MS4.

SEQ ID NO: 7) is the nucleotide sequence for vector primer T3.

SEQ ID NO: 8) is the nucleotide sequence for vector primer T7.

SEQ ID NO: 9) is the nucleotide sequence for the M. sexta 120 Kda APN2 (Denolf et al. 1997).

SEQ ID NO: 10) is the nucleotide sequence for the P. xylostella APN (Denolf et al. 1997).

SEQ ID NO: 11) is the nucleotide sequence for the M. sexta 120 kDa APN1 (Knight et al. 1995).

SEQ ID NO: 12) is the nucleotide sequence for the B. mori APN (Hua et al. 1998).

SEQ ID NO: 13) is the nucleotide sequence for the H. virescens APN (Gill et al. 1995).

SEQ ID NO: 14) is the nucleotide sequence for the PCR primer 5′-pAHR5.

SEQ ID NO: 15) is the nucleotide sequence for PCR primer 3′-pAHR5.

As noted above, the subject invention relates to a cell or cells transfected with a polynucleotide encoding an APN protein, or fragment thereof, and methods using the subject cells for identifying novel pesticide agents. The subject invention provides, for the first time, insect cells that express a B.t. toxin binding aminopeptidase localized at their cell membrane. Further, the subject invention is the first demonstration of cultured cells which express a protein capable of binding to a toxin. Further, binding to the toxin is capable of producing an observable effect on such cells, including effecting the death of such cells.

In one embodiment, the subject invention is drawn to a polynucleotide that encodes an APN from M. sexta . In a preferred embodiment, the polynucleotide of the subject invention comprises a nucleotide sequence as shown in FIGS. 2A-2E (SEQ ID NO: 2).

In another embodiment, the subject invention is drawn to a cell or cells transfected with a polynucleotide molecule that comprises a nucleotide sequence encoding an APN protein or fragment thereof, wherein said APN protein or fragment thereof is localized at the cell membrane of said cell or cells. Further, the APN protein or fragment thereof is preferably, but not necessarily, anchored to said cell membrane by at least one glycosyl-phosphatidyl inositol anchor. In a preferred embodiment, said APN protein or fragment thereof which is localized at the cell membrane is capable of binding a toxin. In a more preferred embodiment, said APN protein or fragment thereof mediates an observable toxicity to said cell or cells, including death upon contacting a toxin.

The term “transfection” as used herein means an introduction of a foreign DNA or RNA into a cell by mechanical inoculation, electroporation, agroinfection, particle bombardment, microinjection, or by other known methods.

The term “transformation” as used herein means a stable incorporation of a foreign DNA or RNA into the cell which results in a permanent, heritable alteration in the cell. Accordingly, the skilled artisan would understand that transfection of a cell may result in the transformation of that cell.

As described in the background of the invention, many Bt toxins have been isolated and sequenced. Polynucleotides encoding any known Bt toxins or those yet to be discovered and active fragments thereof (see, for example, U.S. Pat. No. 5,710,020) can be used in accord with the teachings herein. These include, but are not limited to, polynucleotides encoding Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry3A. See Crickmore et al. (1998) for a description of other Bt toxins.

As used hereinafter, “APN” includes full-length APN and fragments of APN operable for the uses disclosed herein.

In order to provide an understanding of a number of terms used in the specification and claims herein, the following definitions are provided.

An isolated nucleic acid or polynucleotide is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding or noncoding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, and (ii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.

A nucleotide sequence is operably linked when it is placed into a functional relationship with another nucleotide sequence. For instance, a promoter is operably linked. to a coding sequence if the promoter effects its transcription or expression. Generally, operably linked means that the sequences being linked are continguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.

Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate other APN-encoding polynucleotides. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single or double stranded nucleic acids or be chemically synthesized. Polynucleotide probes may be labelled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.

The polynucleotides may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers (1981) Tetra. Letts ., 22:1859-1862 or the triester method according to Matteuci et al. 91981) J. Am. Chem. Soc ., 103:3185, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system (i.e. vector) recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typically selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g., ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.

It will be recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which code for the APN are included in this invention, including DNA sequences as given in SEQ ED NO: 2 having an ATG preceding the coding region for the mature protein.

Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences which will not significantly change activity of the amino acid sequences of the peptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of the regulated promoter region. The skilled artisan will understand that the sequence of the exemplified APN sequence can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode functional equivalents to the sequences given in SEQ ID NO: 2, or an amino acid sequence of greater than 90% identity thereto and having equivalent biological activity. DNA sequences having at least 90, or at least 95% identity to the recited DNA sequences of SEQ ID NO: 2 and encoding functioning APN are considered equivalent to the sequences of SEQ ID NO: 2 and are included in the definition of an APN encoding sequence. Following the teachings herein and using knowledge and techniques well known in the art, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein without the expense of undue experimentation. sequence. Following the teachings herein and using knowledge and techniques well known in the art, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein without the expense of undue experimentation.

As used herein percent sequence identity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al (1990) J. Mol. Biol . 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score =100, wordlength =12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See http://www.ncbi.nih.gov.

Various degrees of stringency of hybridization can be employed. The more stringent the conditions, the greater the complementarity that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes , Stockton Press, New York, N.Y., pp. 169-170.

As used herein “moderate to high stringency” conditions for hybridization refers to conditions that achieve the same, or about the same, degree of specificity of hybridization as the conditions “as described herein.” Examples of moderate to high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed using standard methods (Maniatis et al.). In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to sequences exemplified herein. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula from Beltz et al. (1983).

Tm=81.5° C.+16.6 Log [Na+]+0.41(%G+C)−0.61 (%formamide) 600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at 10-20° C.

below the melting temperature (Tm) of the hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula from Suggs et al. (1981):

Tm (° C.)=2 (number T/A base pairs)+4(number G/C base pairs)

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%

SDS (moderate stringency wash)

In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment of greater than about 70 or so bases in length, the following can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or2×SSPE, 42° C.

Moderate: 0.2×or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, polynucleotide sequences of the subject invention include mutations (both single and multiple), deletions, and insertions in the described sequences, and combinations thereof, wherein said mutations, insertions, and deletions permit formation of stable hybrids with a target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence using standard methods known in the art. Other methods may become known in the future.

The mutational, insertional, and deletional variants of the polynucleotide sequences of the invention can be used in the same manner as the exemplified polynucleotide sequences so long as the variants have substantial sequence similarity with the original sequence. As used herein, substantial sequence similarity refers to the extent of nucleotide similarity that is sufficient to enable the variant polynucleotide to function in the same capacity as the original sequence. Preferably, this similarity is greater than 50%; more preferably, this similarity is greater than 75%; and most preferably, this similarity is greater than 90%. The degree of similarity needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations that are designed to improve the function of the sequence or otherwise provide a methodological advantage.

In a further embodiment, the subject invention provides expression vectors comprising one or more polynucleotides comprising nucleotide sequences encoding APN and capable of expressing APN in a suitable host cell. In the vectors of the subject invention, the polynucleotide encoding APN is operably linked to suitable transcriptional and/or translational regulatory elements to effect expression of the APN in a suitable host cell. The regulatory elements may be derived from mammalian, microbial, viral or insect genes and include, for example, promoters, enhancers, transcription and translation initiation sequences, termination sequences, origins of replication, and leader and transport sequences. Suitable regulatory elements are selected for optimal expression in a desired host cell.

Possible regulatory sequences can include, but are not limited to, any promoter already shown to be constitutive for expression, such as those of viral origin (e.g., IEI promoter from Baculoviruses) or so-called “housekeeping” genes (ubiquitin, actin, tubulin) with their corresponding termination/poly A +sequences. In addition, the gene can be placed under the regulation of inducible promoters and their termination sequences so that gene expression is induced by light (rbcS-3A, cab-1), heat (hsp gene promoters) or wounding (mannopine, HGPGs). Other suitable promoters include the metallothionein promoter, dexamethasone promoter, alcohol dehydrogenase promoter, and the baculovirus promoters, i.e., the early promoter (e.g., IE-1 and et1), the late promoters (e.g., vp39 and p6.9), the very late promoters (e.g., po1h and p10) and the hybrid promoter (e.g., vp39/po1h).

It is clear to one skilled in the art that a promoter may be used either in native or truncated form, and may be paired with its own or a heterologous termination/polyA +sequence. In a preferred embodiment, the subject vectors are regulated by D. melanogaster HSP70 promoter.

Expression vectors can be constructed by well known molecular biological methods as described for example in Sambrook et al. (1989), or any of a myriad of laboratory manuals on recombinant DNA technology that are widely available. Expression vectors into which the polynucleotides of the present invention can be cloned under the control of a suitable promoter are also commercially available. Recombinant viral vectors, including retroviral, baculoviral, parvoviral and densoviral vectors can be used but are not particularly preferred. In host cells containing vectors having an inducible promoter controlling the expression of the nucleic acid encoding APN, expression is induced by methods known in the art and suitable for the selected promoter. For example, expression of nucleic acids under the control of the metallothionein promoter is induced by adding cadmium chloride or copper sulfate to the growth media of host cells.

In a specific embodiment, the subject invention provides a host cell containing a vector comprising nucleotide sequences encoding APN under the control of a promoter. The host cell may be procaryotic or eukaryotic, including bacterial, yeast, insect and mammalian cells. Insect and mammalian cells are preferred. Particularly preferred host cells include insect cell lines, such as, for example, Spodoptera frugiperda (Sf9 and Sf21) and Trichoplusia ni (Tn cells), Estigma acrae (Ea4 cells), Drosophila melanogaster (Dm cells), Choristoneura fumiferama (Cf-y cells), Mamestra brassicae (MaBr-3 cells), Bombyx mori (MnN-4 cells), Helicoverpa zea (Hzlb3 cells), and Lymantria dispar (Ld652Y cells), among others. The host cells. may be transformed, transfected or infected with the expression vectors of the present invention by methods well-known to those of ordinary skill in the art. Transfection may be accomplished by known methods, such as liposome mediated transfection, calcium phosphate mediated transfection, microinjection and electroporation.

Cells of the subject invention may be transfected with a polynucleotide comprising a nucleotide sequence of FIGS. 2A-2E (SEQ ID NO: 2), or fragment thereof. One skilled in the art would readily appreciate that polynucleotides encoding other APNs may be substituted for FIGS. 2A-2E (SEQ ID NO: 2). Examples of toxin binding APNs have been reported for several species (see, e.g., Knight et al., 1994; Sangadala et al., 1994; Gill et al., 1995; Luo et al., 1996; Yaoi et al., 1997; Denolf et al., 1997; and Huo et al., 1998 incorporated herein by this reference). Equipped with the teachings herein, the skilled artisan would be able to transfect insect cells with these, as well as future isolated APN-encoding polynucleotides, to produce APN expressing cells.

The skilled artisan will note that polynucleotides preferred for practicing the subject invention encode proteins capable of expression in cells, localization to cell membrane, and toxin binding. Accordingly, fragments of APN sequences as well as functional mutants may equally be used in practicing the subject invention. Such fragments and mutants will be readily obtainable following the teachings herein coupled with the state of the art. For example, using specifically exemplified polynucleotides as probes, useful polynucleotides can be obtained under conditions of appropriate stringency. Standard hybridization conditions include hybridization with nonspecific DNA, such as salmon DNA, at 50° C. and washing at 45° C. To obtain polynucleotides having the lowest detectable homology with exemplified APNs, hybridization is conducted under conditions of low standard stringency (30-37° C. and 4-6×SSC). More closely related APN-like polynucleotides can be obtained under moderate standard stringency conditions (40-50° C. in 1×SSC).

In a further embodiment, the subject invention is directed to a method of identifying novel pesticide agents comprising the steps of obtaining cells transfected with one or more of the polynucleotides encoding APNs, whereby said polynucleotides are expressed to produce at least one protein that is localized at the cell membrane of said cells; and screening one or more pesticide agents for their ability to produce an observable effect on said cells. The observable effect may be related to a change in metabolism or morphology. The effect may be cytotoxic which may manifest itself, for example, as reduced thymidine uptake, slower increase in optical density of the culture, reduced exclusion of vital dyes (e.g., trypan blue), increased release of viability markers such as chromium and rubidium and the like. The differential response between the pesticide-treated cells and the cells absent the pesticide may be qualitatively or quantitatively noted. Further, the strength of the pesticide can be assessed by noting the strength of the response. While the subject invention is useful for screening a variety of pesticide agents, one skilled in the art will appreciate that the subject methods are particularly useful in identifying novel natural or mutated B. t. toxins.

In a further embodiment, the subject invention is directed to novel pesticide agents obtained by the subject screening methods.

In yet another embodiment, the subject invention is drawn to a method of identifying novel APN inhibitors comprising obtaining cells as described herein, and screening compounds of interest for their ability to inhibit aminopeptidase activity.

The teachings of all patents and publications cited throughout this specification are incorporated by reference in their entirety to the extent not inconsistent with the teachings herein.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

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Gill, S., Cowles, E. A. and Francis, V. (1995) “Identification, isolation, and cloning of a Bacillus thuringiensis Cry1Ac toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens,” J. Biol. Chem . 270:27277-27282. Hua, G., Tsukamoto, K., Rasilo, M. and Ikezawa, H. (1998) “Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut: a putative receptor for Bacillus thuringiensis Cry1A toxin,” Gene 214:177-185.

Kennard, M. L., Shimizu, K. Y., Gabathuler, R., Rothenberger, S., Therlmann, D. and Jefferies, W. A. (1997) “Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells,” Biotech. Bioeng . 55:41-53.

Kenny, A. J., Stephenson, S. L. and Turner, A. J. (1987) “Cell surface peptidases,” In Kenny, A. J. and Turner, A. J. (eds.) Mammalian ectoenzymes , Elsevier, Amsterdam, pp. 169-210.

Knight, P. J. K., Crickmore, N. and Ellar, D. J. (1994) “The receptor for Bacillus thuringiensis CryIA(c) delta-endotoxin in the brush border membrane is aminopeptidase N,” Mol. Microbiol . 11:429-436.

Knight, P. J. K., Knowles, B. H. and Ellar, D. J. (1995) “Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin,” J. Biol. Chem . 270:17765-17770.

Kolb, A. F., Maile, J., Heister, A. and Siddell, S. G. (1996) “Characterization of functional domains in the human coronavirus HCV 229E receptor,” J. Gen. Viro . 77:2515-2521.

Kozak, M. (1987) “An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs,” Nucl. Acids Res . 15:8125-8132.

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Look, A. T., Ashmun, R. A., Shapiro, L. H. and Peiper, S. C. (1989) “Human myeloid plasma membrane glycoprotein CD13 (gp 150) is identical to aminopeptidase N,” J. Clin. Invest . 83:1299-1307.

Lu, Y. and Adang, M. J. (1996) “Conversion of Bacillus thuringiensis CryIAc-binding aminopeptidase to a soluble form by endogenous phosphatidylinositol phospholipase C,” Insect Biochem. Molec. Biol . 226:33-40.

Luo, K., Lu, Y. J. and Adang, M. J. (1996) “A 106 kDa form of aminopeptidase is a receptor for Bacillus thuringiensis CryIC delta-endotoxin in the brush border membrane of Manduca sexta,” Insect Biochem. Mol. Biol . 26:783-791.

Luo, K., Sangadala, S., Masson, L., Mazza, A., Brousseau, R. and Adang, M. J. (1997a) “The Heliothis virescens 170-kDa aminopeptidase functions as ‘Receptor A’ by mediating specific Bacillus thuringiensis Cry1A d-endotoxin binding and pore formation,” Insect Biochem. Mol. Biol.

Luo, K., Tabashnik, B. E. and Adang, M. J. (1997b) “Binding of Bacillus thuringiensis Cry1Ac toxin to aminopeptidase in susceptible and resistant diamondback moths ( Plutella xylostella ),” Appl. Environ. Microbiol . 63:1024-1027.

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Masson, L., Lu, Y., Mazza, A., Brosseau, R. and Adang, M. J. (1995) “The CryIA(c) receptor purified from Manduca sexta displays multiple specificities,” J. Biol. Chem . 270:20309-20315.

McConville, M. J. and Ferguson, M. A. J. (1993) “The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes,” Biochemical Journal 294:305-324.

McLaughlin, S. and Aderem, A. (1995) “The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions,” TIBS 20:272-276.

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Richardson, M. A., Smith, D. R. J., Kemp, D. H. and Tellam, R. L. (1993) “Native and baculovirus-expressed forms of the immunoprotective protein BM86 from boophilus microplus are anchored to the cell membrane by a glycosylphosphatidyl inositol linkage,” Insect Mol. Biol . 1:139-147.

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Sangadala, S., Walters, F., English, L. H. and Adang, M. J. (1994) “A mixture of Manduca sexta aminopeptidase and alkaline phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86 Rb + -K + leakage in Vitro,” J. Biol. Chem . 269:10088-10092.

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15

1

995

Manduca sexta

1

Met Val Asn Leu Gly Phe Thr Ile Phe Leu Gly Val Ala Leu Leu Gln

1 5 10 15

Gly Val Leu Thr Leu Ser Pro Ile Pro Val Pro Glu Glu Glu Trp Ala

20 25 30

Glu Phe Ser Arg Met Leu Arg Asp Pro Ser Tyr Arg Leu Pro Thr Thr

35 40 45

Thr Arg Pro Arg His Tyr Ala Val Thr Leu Thr Pro Tyr Phe Asp Val

50 55 60

Val Pro Ala Gly Val Ser Ser Leu Thr Thr Phe Ser Phe Asp Gly Glu

65 70 75 80

Val Thr Ile Tyr Ile Ser Pro Thr Gln Ala Asn Val Asn Glu Ile Val

85 90 95

Leu His Cys Asn Asp Leu Thr Ile Gln Ser Leu Arg Val Thr Tyr Val

100 105 110

Ser Gly Asn Ser Glu Val Asp Ile Thr Ala Thr Gly Gln Thr Phe Thr

115 120 125

Cys Glu Met Pro Tyr Ser Phe Leu Arg Ile Arg Thr Ser Thr Pro Leu

130 135 140

Val Met Asn Gln Glu Tyr Ile Ile Arg Ser Thr Phe Arg Gly Asn Leu

145 150 155 160

Gln Thr Asn Met Arg Gly Phe Tyr Arg Ser Trp Tyr Val Asp Arg Thr

165 170 175

Gly Lys Arg Trp Met Ala Thr Thr Gln Leu Gln Pro Gly His Ala Arg

180 185 190

Gln Ala Phe Pro Cys Tyr Asp Glu Pro Gly Phe Lys Ala Thr Phe Asp

195 200 205

Ile Thr Met Asn Arg Glu Ala Asp Phe Ser Pro Thr Ile Ser Asn Met

210 215 220

Pro Ile Arg Ala Thr Thr Thr Leu Thr Asn Gly Arg Ile Ser Glu Thr

225 230 235 240

Phe Phe Thr Thr Pro Leu Thr Ser Thr Tyr Leu Leu Ala Phe Ile Val

245 250 255

Ser His Tyr Gln Val Ile Ser Asn Asn Asn Asn Ala Ala Arg Pro Phe

260 265 270

Arg Ile Tyr Ala Arg Asn Asn Val Gly Ser Gln Gly Asp Trp Ser Leu

275 280 285

Glu Met Gly Glu Lys Leu Leu Leu Ala Met Glu Asn Tyr Thr Ala Ile

290 295 300

Pro Tyr Tyr Thr Met Ala Gln Asn Ile Asp Met Lys Gln Ala Ala Ile

305 310 315 320

Pro Asp Phe Ser Ala Gly Ala Met Glu Asn Trp Gly Leu Leu Thr Tyr

325 330 335

Arg Glu Ala Leu Ile Leu Tyr Asp Pro Leu Asn Ser Asn His His Tyr

340 345 350

Arg Gln Arg Val Ala Asn Ile Val Ser His Glu Ile Ala His Met Trp

355 360 365

Phe Gly Asn Leu Val Thr Cys Ala Trp Trp Asp Asn Leu Trp Leu Asn

370 375 380

Glu Gly Phe Ala Arg Phe Tyr Gln Tyr Tyr Leu Thr Ala Thr Val Asp

385 390 395 400

Pro Glu Leu Gly Tyr Glu Ile Arg Phe Ile Pro Glu Gln Leu Gln Val

405 410 415

Ala Met Phe Ser Asp Ser Val Asp Ser Ala His Ala Leu Thr Asp Thr

420 425 430

Ser Val Asn Asp Pro Val Ala Val Ser Ala His Phe Ser Thr Ile Thr

435 440 445

Tyr Ala Arg Gly Ala Ala Ile Leu Arg Met Thr Gln His Leu Leu Ser

450 455 460

Tyr Asp Thr Phe Val Lys Gly Leu Arg Gln Tyr Leu Arg Ala Arg Gln

465 470 475 480

Phe Asp Val Ala Glu Pro Tyr His Leu Phe Ser Ala Leu Asp Ala Ala

485 490 495

Ala Ala Glu Asp Asn Ala Leu Ala Ala Tyr Thr Gly Ile Thr Ile Asp

500 505 510

Ala Tyr Phe Arg Thr Trp Ser Glu Lys Ala Gly His Pro Leu Leu Ser

515 520 525

Val Thr Val Asp His Glu Thr Gly Arg Met Thr Leu Val Gln Ala Arg

530 535 540

Trp Glu Arg Asn Thr Gly Val Ser Arg Phe Pro Gly Leu Trp His Ile

545 550 555 560

Pro Ile Thr Trp Thr Arg Ala Gly Ala Pro Asp Phe Glu Asn Leu Lys

565 570 575

Pro Ser Gln Val Met Thr Gly Gln Ser Leu Val Ile Asp Arg Gly Thr

580 585 590

Arg Gly Gln Glu Trp Val Ile Phe Asn Lys Gln Val Ser Gly Phe Tyr

595 600 605

Arg Val Asn Tyr Asp Asn Thr Thr Trp Gly Leu Ile Thr Arg Ala Leu

610 615 620

Arg Ser Ala Asn Arg Thr Val Ile His Glu Leu Ser Arg Ser Gln Ile

625 630 635 640

Val Asp Asp Val Phe Gln Leu Ala Arg Ser Gly Val Met Ser Tyr Gln

645 650 655

Arg Ala Leu Asn Ile Leu Ser Tyr Leu Arg Phe Glu Asp Ala Tyr Ala

660 665 670

Pro Trp Leu Ser Ala Ile Ser Gly Phe Asn Trp Val Ile Arg Arg Phe

675 680 685

Ala His Asp Ala Ala Asn Leu Gln Thr Leu Gln Asn Gln Ile Ile Gly

690 695 700

Leu Ser Glu Ala Val Val Ala Arg Leu Gly Phe Thr Glu Val Ser Gly

705 710 715 720

Gly Thr Tyr Met Thr Asp Leu Gln Arg Leu His Val Met Gln Phe Leu

725 730 735

Cys Asn Val Gly His Gln Gln Cys Ile Asp Thr Gly Arg Gln Asn Phe

740 745 750

Leu Asn Trp Arg Asn Gly Ser Phe Ile Pro Ala Asn Met Arg Pro Trp

755 760 765

Val Tyr Cys Thr Gly Leu Arg Tyr Gly Ser Ala Glu Asp Phe Asn Tyr

770 775 780

Phe Trp Asn Arg Tyr Ile Val Glu Asp Leu Ser Asn Glu Lys Val Val

785 790 795 800

Met Leu Glu Ala Ala Gly Cys Thr Arg Asp Gln Ala Ser Leu Glu Lys

805 810 815

Phe Leu Asn Ala Ile Val Ser Gly Asn Asp Asp Val Arg Pro Gln Asp

820 825 830

His Ser Ser Ala Leu Ser Ser Ala Ile Thr Ser Asn Asp Val Asn Thr

835 840 845

Met Arg Ala Phe Asp Trp Leu Thr Lys Asn Val Asp Gln Ile Thr Arg

850 855 860

Thr Leu Gly Ser Ile Thr Ser Pro Leu Asn Thr Ile Thr Ser Arg Leu

865 870 875 880

Leu Thr Glu Ala Gln Met Thr Gln Val Gln Thr Trp Leu Asp Ala Asn

885 890 895

Arg Asn Thr Ile Gly Ala Ala Tyr Asn Thr Gly Val Asn Gly Ile Ala

900 905 910

Thr Ser Arg Ala Asn Leu Gln Trp Ser Ala Asn Arg Met Ser Glu Phe

915 920 925

Leu Arg Phe Phe Glu Thr Gly Phe Val Asp Asp Val Pro Ser Glu Ala

930 935 940

Thr Thr Val Ala Pro Pro Ala Glu Thr Thr Val Thr Pro Ser Thr Phe

945 950 955 960

Pro Pro Thr Glu Ala Pro Ala Thr Thr Pro Ala Pro Gly Ser Gly Asn

965 970 975

Ile Ala Ala Leu Ser Val Val Ser Leu Leu Val Thr Leu Ala Ile Asn

980 985 990

Met Val Ala

995

2

2988

Manduca sexta

2

atggtgaatc tcgggtttac cattttcttg ggggtcgccc ttctccaggg cgttcttact 60

ttgagcccca tacccgtccc agaagaagaa tgggccgaat tctccagaat gctgcgggac 120

ccgagctacc gcctgcctac taccacccgg ccaagacatt acgctgtgac cctgactcca 180

tactttgacg tggtacctgc tggtgtcagc agccttacca ccttcagctt tgacggcgag 240

gtcaccatct acatatcgcc cactcaagct aatgttaatg agatcgtcct ccactgcaat 300

gacttgacga tacagagtct gagggtaaca tatgttagtg gtaatagtga ggtggatatc 360

acggcaactg gacaaacttt tacgtgtgag atgccctaca gttttctcag aataaggacc 420

tctacgcctc tagtgatgaa ccaagagtat attatcagga gtacctttag aggcaacttg 480

cagactaaca tgagagggtt ctacagaagt tggtacgtcg atagaaccgg aaagagatgg 540

atggctacca ctcaacttca acccggacat gcgcgtcaag cgttcccttg ctacgatgag 600

cctggtttca aggccacctt cgacattact atgaacagag aagccgactt tagcccgacc 660

atatctaata tgcctattag ggccactacc acgctcacga atggacgtat ttccgaaaca 720

tttttcacca ctcccttgac atccacctat ctccttgcct tcatagtctc tcactatcag 780

gtcatttcta acaacaacaa tgcagcacgc ccttttagaa tctatgcacg taataatgta 840

gggagccagg gtgactggtc tcttgaaatg ggtgagaaac tgctattagc tatggagaat 900

tatactgcaa taccttatta cacgatggca caaaacattg atatgaaaca agccgccatt 960

cccgacttct ctgctggtgc tatggaaaac tggggtctct tgacatacag ggaagccctc 1020

atcttatacg accccctcaa ttcgaaccat cactaccgtc agcgcgtagc gaacattgtc 1080

tcccacgaga tcgctcacat gtggttcggt aaccttgtca catgcgcatg gtgggataac 1140

ctttggctga acgaaggttt tgcgcggttc taccaatact accttactgc aacggtcgac 1200

ccagagctcg gttatgaaat tcgtttcatc ccagagcagc ttcaagtggc gatgttctct 1260

gactccgtag acagcgccca cgctcttact gacaccagtg ttaatgatcc tgttgctgtc 1320

agcgctcact tctcaacaat cacttacgcc aggggagccg ccatcctcag aatgacacag 1380

catttgttga gctatgacac cttcgtcaaa ggtcttaggc agtatctgcg tgctcgacaa 1440

tttgacgtcg ccgaacccta ccacctgttc tccgctttgg atgctgcggc tgctgaagac 1500

aatgctctcg ctgcctacac aggcatcact attgacgctt acttcaggac ttggtcagag 1560

aaggcgggac atccccttct ctcagttact gttgatcatg aaaccggccg tatgactctc 1620

gttcaggcaa gatgggagcg caataccggt gtgtctcgat tcccgggctt atggcatatc 1680

cctatcacat ggacaagggc tggagcccca gacttcgaaa acctgaagcc ctcgcaagtt 1740

atgactggac agtctttagt cattgaccgt ggtaccagag gacaagagtg ggtcatcttc 1800

aacaagcaag tatcaggttt ctaccgtgtc aactacgata ataccacctg gggtctcatc 1860

acaagggctc tgaggtctgc gaacaggaca gttattcacg aattgagtcg ctctcagata 1920

gtagacgatg tcttccaact ggctagatcc ggcgtgatgt cataccaacg agcacttaac 1980

attctgtcct acttgagatt cgaagacgcg tacgcaccgt ggttgtccgc catcagcggg 2040

ttcaactggg tcatcaggag attcgcccat gacgccgcca atttacaaac tttacagaac 2100

caaatcatcg gactgagcga agctgtggtg gctcggcttg gcttcaccga agtatccggt 2160

ggtacttata tgaccgacct ccagaggttg catgtaatgc agtttctctg caatgtggga 2220

catcagcagt gcattgacac tggaagacag aacttcttga actggaggaa cggtagcttt 2280

atcccagcta acatgcgtcc atgggtgtac tgcactggtc ttcgttacgg ctctgctgag 2340

gacttcaatt acttctggaa tcgttacatc gtagaagatc tgtctaatga aaaggttgtg 2400

atgctcgaag cggccggttg cacgcgtgac caggccagct tggagaagtt cttgaacgct 2460

atcgtttctg gcaatgatga cgtcagacca caggatcatt cgagtgccct gagctcagct 2520

atcacatcca acgacgtcaa caccatgaga gcgttcgact ggttgaccaa gaatgtagat 2580

caaattacac gaactcttgg tagtatcacc tcgccgctga acaccatcac gagccgtctc 2640

ttgaccgagg cacagatgac tcaggtacaa acttggcttg acgcaaaccg taacaccatc 2700

ggcgctgcct acaacactgg cgtgaacggc atcgccacat cgagagctaa tctccagtgg 2760

tcggcgaaca gaatgtctga gttcctgcgc ttcttcgaaa ctgggttcgt cgacgatgtt 2820

cctagtgagg cgactactgt tgcgccccct gccgaaacta cggtgactcc ctctaccttc 2880

cctccgacgg aagcaccggc gactactcca gccccgggct caggaaacat cgccgctttg 2940

agcgttgtca gcctcctcgt cacacttgcc attaacatgg tagcgtaa 2988

3

22

Manduca sexta

3

attttcttgg gggtcgccct tc 22

4

17

Manduca sexta

4

acgctaccat gttaatg 17

5

17

Manduca sexta

5

tgctgtgtca ttctgag 17

6

21

Manduca sexta

6

aggagattcg cccatgacgc c 21

7

20

Manduca sexta

7

aattaaccct cactaaaggg 20

8

22

Manduca sexta

8

gtaatacgac tcactatagg gc 22

9

942

Manduca sexta

9

Met Tyr Ser Leu Ile Phe Leu Ala Leu Ile Gly Ala Ala Phe Gly Val

1 5 10 15

Pro Leu Ser Thr Asn Glu Asp Ser Thr Arg Asn Gln Asn Leu Ala Ala

20 25 30

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

35 40 45

Leu Phe Ile Asp Pro Gly Tyr Thr Glu Ala Phe His Gly Asn Val Ser

50 55 60

Ile Arg Ile Ile Pro Asn Ile Asn Ile Asp Gln Ile Thr Ile His Ala

65 70 75 80

Met Ala Met Arg Ile Asp Ser Ile Arg Val Val Ser Asp Val Asn Pro

85 90 95

Asn Glu Asp Leu Phe Ser Asp Phe Thr Leu Ala Thr Asp Asp Thr His

100 105 110

Leu Leu Thr Ile Arg Leu Thr Arg Asn Ile Thr Ala Leu Gln Pro His

115 120 125

Val Ile His Ile Asp Tyr Val Ala Gln Tyr Ala Asp Asp Met Phe Gly

130 135 140

Val Tyr Val Ser Thr Tyr Glu Glu Asn Gly Arg Thr Val Asn Leu Val

145 150 155 160

Thr Ser Gln Leu Gln Pro Thr Phe Ala Arg Arg Ala Phe Pro Cys Tyr

165 170 175

Asp Glu Pro Ala Leu Lys Ala Val Phe Arg Thr Thr Ile Tyr Ala Pro

180 185 190

Ala Ala Tyr Ala Thr Val Arg Ser Asn Thr Pro Glu Arg Arg Asp Ser

195 200 205

Leu Lys Pro Asn Glu Pro Gly Tyr Val Lys His Glu Phe Glu Asp Thr

210 215 220

Leu Val Met Ser Thr Tyr Leu Ile Ala Tyr Leu Val Ser Asn Phe Asn

225 230 235 240

Tyr Ile Glu Asn Ser Gln Asn Pro Ile Tyr Pro Ile Pro Phe Arg Val

245 250 255

Tyr Ser Arg Pro Gly Thr Gln Asn Thr Ala Glu Phe Ala Leu Glu Phe

260 265 270

Gly Gln Gln Asn Met Ile Ala Leu Glu Glu Tyr Thr Glu Phe Pro Tyr

275 280 285

Ala Phe Pro Lys Ile Asp Lys Ala Ala Val Pro Asp Phe Ala Ala Gly

290 295 300

Ala Met Glu Asn Trp Gly Leu Val Ile Tyr Arg Glu Val Ala Leu Leu

305 310 315 320

Val Arg Glu Gly Val Thr Thr Thr Ser Val Lys Gln Asn Ile Gly Arg

325 330 335

Ile Ile Cys His Glu Asn Thr His Met Trp Phe Gly Asn Glu Val Gly

340 345 350

Pro Met Ser Trp Thr Tyr Thr Trp Leu Asn Glu Gly Phe Ala Asn Phe

355 360 365

Phe Glu Asn Tyr Ala Thr Asp Phe Val Arg Pro Gln Trp Arg Met Met

370 375 380

Asp Gln Phe Val Ile Ala Met Gln Asn Val Phe Pro Val Arg Arg Cys

385 390 395 400

Ser Lys Cys Gln Pro His Asp Ala Pro Gly Leu Tyr Ser Phe Pro Asp

405 410 415

His Arg Tyr Phe Gln Arg Arg Arg Leu Pro Glu Val Trp Phe Arg Tyr

420 425 430

Ser Asp Val Ala Ala Phe His Asp Thr Arg Asp Phe Gln Glu Arg Ser

435 440 445

Gly His Leu His Gln Ser Gln Leu Ser Arg Pro Ala Ala Pro Ser Asp

450 455 460

Leu Tyr Val Ala Leu Gln Gln Ala Leu Asp Glu Ser Ser His Arg Ile

465 470 475 480

Pro Lys Pro Ile Ser Thr Ile Met Thr Glu Trp Ser Thr Gln Gly Gly

485 490 495

Phe Pro Val Leu Thr Val Arg Arg Thr Ala Pro Asn Ala Asp Ser Val

500 505 510

Phe Val Ala Gln Glu Arg Tyr Leu Thr Asp Arg Ser Leu Thr Ser Thr

515 520 525

Asp Arg Trp His Val Pro Val Asn Trp Val Ile Ser Ser Asn Val Asn

530 535 540

Phe Ser Asp Thr Ser Pro Gln Ala Trp Ile Leu Pro Thr Phe Pro Ala

545 550 555 560

Thr Ala Val Asp Val Pro Gly Leu Ser Asn Ala Asp Trp Tyr Ile Phe

565 570 575

Asn Lys Gln Gln Thr Gly Tyr Tyr Arg Val Asn Tyr Asp Val Glu Asn

580 585 590

Trp Val Ala Leu Ala Arg Val Leu Asn Asn Ser His Glu Ile Ile His

595 600 605

Val Leu Asn Arg Ala Gln Ile Val Asp Asp Ala Phe Asn Leu Ala Arg

610 615 620

Asn Gly Arg Leu His Tyr Lys Asn Ala Phe Glu Ile Ser Arg Tyr Leu

625 630 635 640

Glu Met Glu Lys Asp Tyr Ile Pro Trp Ala Ala Ala Asn Pro Ala Phe

645 650 655

Asn Tyr Leu Asp Ile Val Leu Ser Gly Ala Asn Ser Tyr Asn Leu Tyr

660 665 670

Arg Tyr Tyr Leu Leu Asn Leu Thr Ala Pro Met Phe Glu Asp Leu Gly

675 680 685

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

690 695 700

Ile Ile Leu Asp Ile Asn Cys Arg Phe Gly Asn Gln Arg Cys Ile Ser

705 710 715 720

Arg Ala Gln Glu Ile Leu Gln Ala Phe Lys Asn Asn Pro Asn Gln Arg

725 730 735

Pro Asn Pro Asp Ile Gln Thr Leu Val Tyr Cys Ser Ser Leu Arg Ala

740 745 750

Gly Asn Val Glu Asn Phe Asn Phe Leu Trp Asn Met Tyr Leu Gly Thr

755 760 765

Ser Asp Ser Ser Glu Gln Ser Ile Leu Leu Ser Ala Leu Gly Cys Thr

770 775 780

Ser Asn Ala Glu Arg Arg Asn Phe Tyr Leu Asn Gln Ile Ile Asp Asp

785 790 795 800

Asn Ser Ala Val Arg Glu Gln Asp Arg His Ser Ile Ala Val Ser Val

805 810 815

Ile Asn Ser Ser Pro Glu Gly Met Asn Val Ala Leu Asp Phe Val Val

820 825 830

Glu Asn Phe His Arg Ile Gln Pro Arg Val Gln Ala Leu Thr Gly Thr

835 840 845

Thr Asn Ile Leu Asn Thr Phe Ala Arg Arg Leu Thr Thr Ser Ala His

850 855 860

Asn Glu Lys Ile Asp Glu Leu Val Arg Arg His Glu Ser Ile Phe Ser

865 870 875 880

Ala Gly Glu Arg Ala Ser Ile Ala Ala Ile Arg Glu Asn Ile Ala Ala

885 890 895

Ser Ile Ala Trp Ser Asn Ser Asn Ala Gly Ile Val Glu Asn Trp Leu

900 905 910

Lys Glu Asn Tyr Gly Pro Pro Ser Gly Ala Lys Ser Leu Thr Ala Gly

915 920 925

Leu Leu Val Leu Ile Ser Leu Phe Val Ala Ile Phe Asn His

930 935 940

10

946

Plutella xylostella

10

Met Arg Leu Leu Ile Cys Leu Thr Leu Leu Gly Leu Val Cys Gly Asn

1 5 10 15

Pro Val Gln Leu Thr Asp Asn Ser Ile Ala Leu Gln Asn Thr Tyr Asp

20 25 30

Asn Tyr Val Leu Pro Gly Glu Ser Phe Pro Thr Phe Tyr Asp Val Gln

35 40 45

Leu Phe Phe Asp Pro Glu Tyr Glu Ala Ser Phe Asn Gly Thr Val Ala

50 55 60

Ile Arg Val Val Pro Arg Ile Ala Thr Gln Glu Ile Val Leu His Ala

65 70 75 80

Met Glu Met Glu Ile Leu Ser Ile Arg Ala Tyr Ser Asp Leu Pro Ser

85 90 95

Asp Asp Asn Leu Asn Glu Asn Leu Phe Ser Ser Tyr Thr Leu Ala Thr

100 105 110

Asp Asp Thr His Leu Leu Lys Ile Gln Phe Thr Arg Val Leu Asp Ala

115 120 125

Leu Gln Pro Ile Thr Val Glu Ile Ser Tyr Ser Ala Gln Tyr Ala Pro

130 135 140

Asn Met Phe Gly Val Tyr Val Ser Arg Tyr Val Glu Asn Gly Ala Thr

145 150 155 160

Val Ser Leu Val Thr Ser Gln Leu Gln Pro Thr Phe Ala Arg Arg Ala

165 170 175

Phe Pro Cys Tyr Asp Glu Pro Ala Leu Lys Ala Val Phe Arg Thr Thr

180 185 190

Ile Tyr Ala Pro Pro Ala Tyr Asn Val Val Glu Thr Asn Met Pro Leu

195 200 205

Arg Thr Asp Ser Leu Lys Ser Asp Arg Pro Gly Phe Thr Lys His Glu

210 215 220

Phe Gln Asp Thr Leu Val Met Ser Ser Tyr Leu Leu Ala Tyr Leu Val

225 230 235 240

Ser Lys Phe Asp Tyr Ile Ser Asn Glu Asn Asn Pro Thr Tyr Asp Lys

245 250 255

Ser Met Lys Val Phe Ser Arg Pro Gly Thr Gln Asn Thr Ala Glu Phe

260 265 270

Ala Leu Asp Phe Gly Gln Lys Asn Met Val Glu Leu Glu Lys Tyr Thr

275 280 285

Glu Phe Pro Tyr Ala Phe Pro Lys Ile Asp Lys Val Ala Val Pro Asp

290 295 300

Phe Ala Ala Gly Ala Met Glu Asn Trp Gly Leu Val Ile Tyr Arg Glu

305 310 315 320

Ile Ala Leu Leu Val Gln Glu Gly Val Thr Thr Thr Ser Thr Leu Gln

325 330 335

Gly Ile Gly Arg Ile Ile Ser His Glu Asn Thr His Gln Trp Phe Gly

340 345 350

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

355 360 365

Phe Ala Asn Phe Phe Glu Ser Phe Ala Thr Asp Leu Val Leu Pro Glu

370 375 380

Trp Arg Met Met Asp Gln Phe Val Ile Asn Met Gln Asn Val Phe Gln

385 390 395 400

Ser Asp Ala Val Leu Ser Val Asn Pro Ile Thr Phe Glu Val Arg Thr

405 410 415

Pro Ser Gln Ile Leu Gly Thr Phe Asn Ser Val Ala Tyr Gln Lys Ser

420 425 430

Gly Ser Val Ile Arg Met Met Gln His Phe Leu Thr Pro Glu Ile Phe

435 440 445

Arg Lys Ser Leu Ala Leu Tyr Ile Ser Arg Met Ser Arg Lys Ala Ala

450 455 460

Lys Pro Thr Asp Leu Phe Glu Ala Ile Gln Glu Val Val Asp Ala Ser

465 470 475 480

Asp His Ser Ile Arg Trp Arg Leu Ser Ile Ile Met Asn Arg Trp Thr

485 490 495

Gln Gln Gly Gly Phe Pro Val Val Thr Val Arg Arg Ser Ala Pro Ser

500 505 510

Ala Gln Ser Phe Val Ile Thr Gln Arg Arg Phe Leu Thr Asp Ser Thr

515 520 525

Gln Glu Ser Asn Thr Val Trp Asn Val Pro Leu Asn Trp Val Leu Ser

530 535 540

Thr Asp Val Asn Phe Asn Asp Thr Arg Pro Met Ala Trp Leu Pro Pro

545 550 555 560

Gln Leu Ala Ala Glu Ala Val Gln Val Pro Gly Leu Gln Asn Ala Glu

565 570 575

Trp Phe Ile Val Asn Lys Gln Gln Thr Gly Tyr Tyr Arg Val Asn Tyr

580 585 590

Asp Pro Glu Asn Trp Arg Ala Leu Ala Lys Val Leu Asn Asp Thr His

595 600 605

Glu Ile Ile His Leu Leu Asn Arg Ala Gln Leu Ile Asp Asp Ser Phe

610 615 620

Asn Leu Ala Arg Asn Gly Arg Leu Asp Tyr Ser Leu Ala Phe Asp Leu

625 630 635 640

Ser Arg Tyr Leu Val Gln Glu Arg Asp Tyr Ile Pro Trp Ala Ala Ala

645 650 655

Asn Ala Ala Phe Asn Tyr Leu Asn Ser Val Leu Ser Gly Ser Ser Val

660 665 670

His Pro Leu Phe Gln Glu Tyr Leu Leu Phe Leu Thr Ala Pro Leu Tyr

675 680 685

Gln Arg Leu Gly Phe Asn Ala Ala Thr Gly Glu Glu His Val Thr Pro

690 695 700

Phe His Arg Asn Ile Ile Leu Asn Ile Asn Cys Leu His Gly Asn Glu

705 710 715 720

Asp Cys Val Ser Thr Ala Glu Thr Leu Leu Gln Asn Phe Arg Asp Asn

725 730 735

Pro Thr Gln Thr Leu Asn Pro Asp Ile Gln Thr Thr Val Phe Cys Ser

740 745 750

Gly Leu Arg Gly Gly Asp Val Asp Asn Phe Asn Phe Leu Trp Ala Arg

755 760 765

Tyr Thr Ala Thr Gln Asp Ser Ser Glu Gln Ser Ile Leu Leu Asn Ala

770 775 780

Leu Gly Cys Thr Ser Asn Ala Asp Arg Arg Asp Phe Leu Phe Ser Gln

785 790 795 800

Val Ile Ala Ser Asp Ser Gln Val Arg Glu Gln Asp Arg His Ser Val

805 810 815

Leu Val Ser Ala Ile Asn Ser Gly Pro Asp Asn Met Asn Ala Ala Leu

820 825 830

Asp Phe Val Leu Glu Asn Phe Ala Asn Ile Gln Pro Asn Val Gln Gly

835 840 845

Leu Thr Gly Thr Thr Asn Ile Leu Asn Ala Phe Ala Arg Thr Leu Thr

850 855 860

Thr Gln Glu His Ala Asn Lys Ile Asp Glu Phe Ser Asn Lys Tyr Ala

865 870 875 880

Asn Val Phe Thr Ala Gly Glu Met Ala Ser Val Ala Ala Ile Lys Glu

885 890 895

Asn Ile Ala Ala Ser Ile Thr Trp Asn Ser Gln Asn Ala Ala Thr Val

900 905 910

Glu Ala Trp Leu Arg Lys Asn Phe Gly Thr Asp Gly Ala Ser Thr Val

915 920 925

Ser Ala Ser Ile Thr Ile Ile Ile Ser Ala Met Val Ala Ile Tyr Asn

930 935 940

Ile Leu

945

11

990

Manduca sexta

11

Phe Thr Ile Phe Leu Gly Val Ala Leu Leu Gln Gly Val Leu Thr Leu

1 5 10 15

Ser Pro Ile Pro Val Pro Glu Glu Glu Trp Ala Glu Phe Ser Arg Met

20 25 30

Leu Arg Asp Pro Ser Tyr Arg Leu Pro Thr Thr Thr Arg Pro Arg His

35 40 45

Tyr Ala Val Thr Leu Thr Pro Tyr Phe Asp Val Val Pro Ala Gly Val

50 55 60

Ser Gly Leu Thr Thr Phe Ser Phe Asp Gly Glu Val Thr Ile Tyr Ile

65 70 75 80

Ser Pro Thr Gln Ala Asn Val Asn Glu Ile Val Leu His Cys Asn Asp

85 90 95

Leu Thr Ile Gln Ser Leu Arg Val Thr Tyr Val Ser Gly Asn Ser Glu

100 105 110

Val Asp Ile Thr Ala Thr Gly Gln Thr Phe Thr Cys Glu Met Pro Tyr

115 120 125

Ser Phe Leu Arg Ile Arg Thr Ser Thr Pro Leu Val Met Asn Gln Glu

130 135 140

Tyr Ile Ile Arg Ser Thr Phe Arg Gly Asn Leu Gln Thr Asn Met Arg

145 150 155 160

Gly Phe Tyr Arg Ser Trp Tyr Val Asp Arg Thr Gly Lys Arg Trp Met

165 170 175

Ala Thr Thr Gln Phe Gln Pro Gly His Ala Arg Gln Ala Phe Pro Cys

180 185 190

Tyr Asp Glu Pro Gly Phe Lys Ala Thr Phe Asp Ile Thr Met Asn Arg

195 200 205

Glu Ala Asp Phe Ser Pro Thr Ile Ser Asn Met Pro Ile Arg Ala Thr

210 215 220

Thr Thr Leu Thr Asn Gly Arg Ile Ser Glu Thr Phe Phe Thr Thr Pro

225 230 235 240

Leu Thr Ser Thr Tyr Leu Leu Ala Phe Ile Val Ser His Tyr Gln Val

245 250 255

Ile Ser Asn Asn Asn Asn Ala Ala Arg Pro Phe Arg Ile Tyr Ala Arg

260 265 270

Asn Asn Val Gly Ser Gln Gly Asp Trp Ser Leu Glu Met Gly Glu Lys

275 280 285

Leu Leu Leu Ala Met Glu Asn Tyr Thr Ala Ile Pro Tyr Tyr Thr Met

290 295 300

Ala Gln Asn Leu Asp Met Lys Gln Ala Ala Ile Pro Asp Phe Ser Ala

305 310 315 320

Gly Ala Met Glu Asn Trp Gly Leu Leu Thr Tyr Arg Glu Ala Leu Ile

325 330 335

Leu Tyr Asp Pro Leu Asn Ser Asn His His Tyr Arg Gln Arg Val Ala

340 345 350

Asn Ile Val Ser His Glu Ile Ala His Met Trp Phe Gly Asn Leu Val

355 360 365

Thr Cys Ala Trp Trp Asp Asn Leu Trp Leu Asn Glu Gly Phe Ala Arg

370 375 380

Phe Ser Gln Tyr Tyr Leu Thr Ala Thr Val Asp Pro Glu Leu Gly Tyr

385 390 395 400

Glu Ile Arg Phe Ile Pro Glu Gln Leu Gln Val Ala Met Phe Ser Asp

405 410 415

Ser Val Asp Ser Ala His Ala Leu Thr Asp Thr Ser Val Asn Asp Pro

420 425 430

Val Ala Val Ser Ala His Phe Ser Thr Ile Thr Tyr Ala Arg Gly Ala

435 440 445

Ala Ile Leu Arg Met Thr Gln His Leu Leu Ser Tyr Asp Thr Phe Val

450 455 460

Lys Gly Leu Arg Gln Tyr Leu Arg Ala Arg Gln Phe Asp Val Ala Glu

465 470 475 480

Pro Tyr His Leu Phe Ser Ala Leu Asp Ala Ala Ala Ala Glu Asp Asn

485 490 495

Ala Leu Ala Ala Tyr Arg Gly Ile Thr Ile Asp Ala Tyr Phe Arg Thr

500 505 510

Trp Ser Glu Lys Ala Gly His Pro Leu Leu Ser Val Thr Val Asp His

515 520 525

Glu Ser Gly Arg Met Thr Leu Val Gln Ala Arg Trp Glu Arg Asn Thr

530 535 540

Gly Val Ser Arg Phe Pro Gly Leu Trp His Ile Pro Ile Thr Trp Thr

545 550 555 560

Arg Ala Gly Ala Pro Asp Phe Glu Asn Leu Lys Pro Ser Gln Val Met

565 570 575

Thr Gly Gln Ser Leu Val Ile Asp Arg Gly Thr Arg Gly Gln Glu Trp

580 585 590

Val Ile Phe Asn Lys Gln Val Ser Gly Phe Tyr Arg Val Asn Tyr Asp

595 600 605

Asn Thr Thr Trp Gly Leu Ile Thr Arg Ala Leu Arg Ser Ala Asn Arg

610 615 620

Thr Val Ile His Glu Leu Ser Arg Ser Gln Ile Val Asp Asp Val Phe

625 630 635 640

Gln Leu Ala Arg Ser Gly Val Met Ser Tyr Gln Arg Ala Leu Asn Ile

645 650 655

Leu Ser Tyr Leu Arg Phe Glu Asp Ala Tyr Ala Pro Trp Leu Ser Ala

660 665 670

Ile Ser Gly Phe Asn Trp Val Ile Arg Arg Phe Ala His Asp Ala Ala

675 680 685

Asn Leu Gln Thr Leu Gln Asn Gln Ile Ile Gly Leu Ser Glu Ala Val

690 695 700

Val Ala Arg Leu Gly Phe Thr Glu Val Ser Gly Gly Thr Tyr Met Thr

705 710 715 720

Asp Leu Gln Arg Leu His Val Met Gln Phe Leu Cys Asn Val Gly His

725 730 735

Gln Gln Cys Ile Asp Ala Gly Arg Gln Asn Phe Leu Asn Trp Arg Asn

740 745 750

Gly Ser Phe Ile Pro Ala Asn Met Arg Pro Trp Val Tyr Cys Thr Gly

755 760 765

Leu Arg Tyr Gly Ser Ala Glu Asp Phe Asn Tyr Phe Trp Asn Arg Tyr

770 775 780

Ile Val Glu Asp Leu Ser Asn Glu Lys Val Val Met Leu Glu Ala Ala

785 790 795 800

Gly Cys Thr Arg Asp Gln Ala Ser Leu Glu Lys Phe Leu Asn Ala Ile

805 810 815

Val Ser Gly Asn Asp Asp Val Arg Pro Gln Asp His Ser Ser Ala Leu

820 825 830

Ser Ser Ala Ile Thr Ser Asn Asp Val Asn Thr Met Arg Ala Phe Asp

835 840 845

Trp Leu Thr Lys Asn Val Asp Gln Ile Thr Arg Thr Leu Gly Ser Ile

850 855 860

Thr Ser Pro Leu Asn Thr Ile Thr Ser Arg Leu Leu Thr Glu Ala Gln

865 870 875 880

Met Thr Gln Val Gln Thr Trp Leu Asp Ala Asn Arg Asn Thr Ile Gly

885 890 895

Ala Ala Tyr Asn Thr Gly Val Asn Gly Ile Ala Thr Ser Arg Ala Asn

900 905 910

Leu Gln Trp Ser Ala Asn Arg Met Ser Glu Phe Leu Arg Phe Phe Glu

915 920 925

Thr Gly Phe Val Asp Asp Val Pro Ser Glu Ala Thr Thr Val Ala Pro

930 935 940

Pro Ala Glu Thr Thr Val Thr Pro Ser Thr Phe Pro Pro Thr Val Ala

945 950 955 960

Pro Ala Thr Thr Pro Ala Pro Gly Ser Gly Asn Ile Ala Ala Leu Ser

965 970 975

Val Val Ser Leu Leu Val Thr Leu Ala Ile Asn Met Val Ala

980 985 990

12

986

Bombyx mori

12

Ala Arg Glu Trp His Leu Ala Gly Phe Thr Ser Ser Trp Ala Tyr Phe

1 5 10 15

Leu Gln Thr Ser Leu Thr Leu Ser Pro Ile Pro Val Pro Glu Asp Glu

20 25 30

Trp Val Glu Phe Ala Arg Met Leu Arg Asp Pro Ala Phe Arg Leu Pro

35 40 45

Thr Thr Thr Arg Pro Arg His Tyr Gln Val Thr Leu Thr Pro Tyr Phe

50 55 60

Asp Val Val Pro Ala Asn Val Asn Pro Phe Thr Phe Asp Gly Glu Val

65 70 75 80

Thr Ile Tyr Thr Ser Pro Thr Val Ala Asn Val Asn Glu Val Val Ile

85 90 95

His Cys Asn Asp Leu Thr Ile Gln Ser Leu Ser Ile Gly Tyr Gln Ser

100 105 110

Gly Thr Asp Val Val Asp Ile Thr Ala Thr Gly Gln Thr Phe Ala Cys

115 120 125

Glu Met Pro Phe Ser Phe Leu Arg Ile Arg Thr Thr Glu Ala Leu Val

130 135 140

Leu Asn Arg Glu Tyr Ile Ile Lys Ser Thr Phe Arg Gly Asn Leu Gln

145 150 155 160

Thr Asn Met Arg Gly Phe Tyr Arg Ser Trp Tyr Val Asp Ser Thr Gly

165 170 175

Arg Arg Trp Met Gly Thr Thr Gln Phe Gln Pro Gly His Ala Arg Gln

180 185 190

Ala Phe Pro Cys Tyr Asp Glu Pro Gly Phe Lys Ala Thr Phe Asp Ile

195 200 205

Thr Met Asn Arg Glu Glu Ser Phe Ser Pro Thr Ile Ser Asn Met Pro

210 215 220

Ile Arg Thr Thr Asn Thr Leu Ala Asn Gly Arg Val Ser Glu Thr Phe

225 230 235 240

Trp Thr Thr Pro Val Thr Ser Thr Tyr Leu Leu Ala Phe Ile Val Ser

245 250 255

His Tyr Thr Val Val Ser Thr Asn Asn Asn Ala Leu Arg Pro Phe Asp

260 265 270

Ile Tyr Ala Arg Asn Asn Val Gly Arg Thr Gly Asp Trp Ser Leu Glu

275 280 285

Ile Gly Glu Lys Leu Leu Glu Ala Met Glu Ala Tyr Thr Gln Ile Pro

290 295 300

Tyr Tyr Thr Met Ala Glu Asn Ile Asn Met Lys Gln Ala Ala Ile Pro

305 310 315 320

Asp Phe Ser Ala Gly Ala Met Glu Asn Trp Gly Leu Leu Thr Tyr Arg

325 330 335

Glu Ala Leu Ile Leu Tyr Asp Pro Leu Asn Ser Asn His Phe Tyr Lys

340 345 350

Gln Arg Val Ala Asn Ile Val Ala His Glu Ile Ala His Met Trp Phe

355 360 365

Gly Asn Leu Val Thr Cys Ala Trp Trp Asp Asn Leu Trp Leu Asn Glu

370 375 380

Gly Phe Ala Arg Phe Tyr Gln Tyr Tyr Leu Thr Ala Ser Val Ala Pro

385 390 395 400

Glu Leu Gly Tyr Glu Thr Arg Phe Ile Val Glu Gln Val Gln Met Ala

405 410 415

Met Phe Ser Asp Ser Val Asp Thr Ala His Ala Leu Thr Asp Leu Asn

420 425 430

Val Asn Asp Pro Thr Thr Val Ser Ala His Phe Ser Thr Ile Thr Tyr

435 440 445

Ala Arg Gly Ala Ala Ile Leu Arg Met Thr Gln His Leu Leu Gly Val

450 455 460

Glu Thr Phe Val Lys Gly Leu Arg Asn Tyr Leu Arg Glu Arg His Ser

465 470 475 480

Met Leu Leu Ser Ser Ser Leu Phe Thr Ala Leu Asp Ala Ala Ala Val

485 490 495

Glu Asp Gly Ala Leu Asn Gly Tyr Gly Gly Ile Thr Ile Asp Thr Tyr

500 505 510

Phe Arg Thr Trp Ser Glu Lys Ala Gly His Pro Leu Leu Thr Val Thr

515 520 525

Ile Lys Pro Glu Asn Trp Gly Asn Asp Cys Thr Gln Glu Arg Trp Glu

530 535 540

Arg Asn Thr Gly Val Ser Gln Phe Pro Ser Leu Trp His Ile Pro Ile

545 550 555 560

Thr Trp Thr Arg Ala Gly Ala Pro Glu Phe Glu Asp Leu Lys Pro Ser

565 570 575

Gln Phe Ile Ser Gln Gln Val Thr Ser Ile Asn Arg Gly Thr Thr Gly

580 585 590

Leu Glu Trp Val Ile Phe Asn Lys Gln Glu Ala Gly Phe Tyr Arg Val

595 600 605

Lys Tyr Asp Asp Thr Asn Trp Ala Leu Leu Thr Arg Ala Leu Arg Ser

610 615 620

Ser Ser Arg Thr Ala Ile His Gln Leu Asn Arg Ala Gln Ile Val Asp

625 630 635 640

Asp Ile Phe Gln Leu Ala Arg Ala Asn Val Met Lys Tyr Asn Arg Ala

645 650 655

Phe Asn Ile Leu Ser Phe Leu Gln Phe Glu Asp Glu Tyr Ala Pro Trp

660 665 670

Leu Ala Ala Ile Ser Gly Phe Asn Phe Leu Ile Arg Arg Leu Ala His

675 680 685

Asp Ser Thr Asn Ala Ala Leu Leu Gln Lys Leu Ile Leu Glu Leu Ser

690 695 700

Pro Ala Val Val Ala Lys Leu Gly Tyr Leu Glu Pro Glu Asn Gly Ser

705 710 715 720

Tyr Met Thr Asp Leu Gln Arg Met Tyr Val Met Glu Phe Leu Cys Asn

725 730 735

Val Gly Pro Glu Cys Asn Asn Phe Gly Thr Gln Ala Phe Arg Arg Trp

740 745 750

Ser Thr Gly Thr Phe Ile Pro Ala Asn Met Arg Pro Trp Val Tyr Cys

755 760 765

Ala Gly Leu Arg His Gly Thr Ala Glu Asp Phe Asn Phe Phe Trp Asn

770 775 780

Arg Tyr Leu Gln Glu Asp Leu Ser Ser Glu Lys Val Val Met Leu Asn

785 790 795 800

Val Ala Gly Cys Thr Thr Asp Gln Ala Ser Leu Asn Arg Phe Leu Asp

805 810 815

Ala Ile Val Ser Gly Asn Asp Asp Ile Arg Pro Gln Asp Tyr Asn Ala

820 825 830

Ala Leu Thr Ser Ala Ile Thr Ser Asn Glu Ile Asn Thr Leu Arg Ala

835 840 845

Phe Gln Trp Leu Arg Asn Asn Val Asp Gln Ala Thr Arg Thr Leu Gly

850 855 860

Ser Val Ser Thr Ile Leu Asn Thr Ile Ile Gly Arg Leu Leu Asn Glu

865 870 875 880

Glu Gln Ile Asn Glu Val Ser Asn Trp Leu Thr Ala Asn Gln Asn Thr

885 890 895

Leu Gly Ala Thr Tyr Ser Thr Ala Leu Arg Ala Ile Glu Thr Thr Arg

900 905 910

Ser Asn Leu Val Trp Ser Gln Gln Arg Ile Ser Glu Phe Thr Asn Tyr

915 920 925

Phe Glu Ser Gly Tyr Val Glu Asp Val Ile Glu Glu Ile Thr Glu Ala

930 935 940

Pro Pro Thr Ala Pro Pro Thr Ala Pro Pro Thr Glu Ala Pro Ala Val

945 950 955 960

Thr Pro Ala Pro Asp Ser Ala Asn Val Ala Ala Leu Ser Phe Ile Thr

965 970 975

Leu Ile Ile Thr Leu Ala Val Asn Leu Ala

980 985

13

1009

Heliothis virescens

13

Met Ala Ala Ile Lys Leu Leu Val Leu Ser Leu Ala Cys Ala Cys Val

1 5 10 15

Ile Ala His Ser Pro Ile Pro Pro Ala Ser Arg Thr Ile Phe Leu Asp

20 25 30

Glu Arg Leu Glu Gly Gly Ala Phe Glu Asn Ile Asp Ala Phe Glu Asn

35 40 45

Ile Glu Leu Ser Asn Val Val Ala Ser Pro Tyr Arg Leu Pro Thr Thr

50 55 60

Thr Val Pro Thr His Tyr Lys Ile Leu Trp Ile Ile Asp Ile His Gln

65 70 75 80

Pro Val Gln Thr Tyr Ser Gly Asn Val Val Ile Thr Leu His Ala Thr

85 90 95

Gln Ala Gln Val Asn Glu Ile Val Ile His Ser Asp His Met Thr Leu

100 105 110

Ser Ser Val Val Leu Arg Gln Gly Asp Thr Val Ile Pro Thr Thr Pro

115 120 125

Thr Ala Gln Pro Glu Tyr His Phe Leu Arg Val Lys Leu Asn Asp Gly

130 135 140

Tyr Leu Ala Tyr Asn Ala Asp Asn Ala Val Leu Tyr Thr Leu Ser Ile

145 150 155 160

Asp Phe Thr Ala Pro Met Arg Asp Asp Met Tyr Gly Ile Tyr Asn Ser

165 170 175

Trp Tyr Arg Asn Leu Pro Asp Asp Ala Asn Val Arg Trp Met Ala Thr

180 185 190

Thr Gln Phe Gln Ala Thr Ala Ala Arg Tyr Ala Phe Pro Cys Tyr Asp

195 200 205

Glu Pro Gly Phe Lys Ala Lys Phe Asp Val Thr Ile Arg Arg Pro Val

210 215 220

Gly Tyr Ser Ser Trp Phe Cys Thr Arg Gln Lys Gly Ser Gly Pro Ser

225 230 235 240

Thr Val Ala Gly Tyr Glu Glu Asp Glu Tyr His Thr Thr Pro Thr Met

245 250 255

Ser Thr Tyr Leu Leu Ala Leu Ile Val Ser Glu Tyr Thr Ser Leu Pro

260 265 270

Ala Thr Asn Ala Ala Gly Glu Ile Leu His Glu Val Ile Ala Arg Pro

275 280 285

Gly Ala Ile Asn Asn Gly Gln Ala Val Tyr Ala Gln Arg Val Gly Gln

290 295 300

Ala Leu Leu Ala Glu Met Ser Asp His Thr Gly Phe Asp Phe Tyr Ala

305 310 315 320

Gln Asp Pro Asn Leu Lys Met Thr Gln Ala Ala Ile Pro Asp Phe Gly

325 330 335

Ala Gly Ala Met Glu Asn Trp Gly Leu Leu Thr Tyr Arg Glu Ala Tyr

340 345 350

Leu Leu Tyr Asp Glu Gln His Thr Asn Ser Tyr Phe Lys Gln Ile Ile

355 360 365

Ala Tyr Ile Leu Ser His Glu Ile Ala His Met Trp Phe Gly Asn Leu

370 375 380

Val Thr Asn Ala Trp Trp Asp Val Leu Trp Leu Asn Glu Gly Phe Ala

385 390 395 400

Arg Tyr Tyr Gln Tyr Phe Leu Thr Ala Trp Val Glu Asp Leu Gly Leu

405 410 415

Ala Thr Arg Phe Ile Asn Glu Gln Val His Ala Ser Leu Leu Ser Asp

420 425 430

Ser Ser Ile Tyr Ala His Pro Leu Thr Asn Pro Gly Val Gly Ser Pro

435 440 445

Ala Ala Val Ser Ala Met Phe Ser Thr Val Thr Tyr Asn Lys Gly Ala

450 455 460

Ser Ile Ile Arg Met Thr Glu His Leu Leu Gly Phe Asp Val His Arg

465 470 475 480

Thr Gly Leu Arg Asn Tyr Leu Lys Asp Leu Ala Tyr Lys Thr Ala Gln

485 490 495

Pro Ile Asp Leu Phe Thr Ala Leu Glu Ser Ala Gly Asn Gln Ala Gly

500 505 510

Ala Leu Ser Ala Tyr Gly Ser Asp Phe Asp Phe Val Lys Tyr Tyr Glu

515 520 525

Ser Trp Thr Glu Gln Pro Gly His Pro Val Leu Asn Val Gln Ile Asn

530 535 540

His Gln Thr Gly Gln Met Thr Ile Thr Gln Arg Arg Phe Asp Ile Asp

545 550 555 560

Thr Gly His Ser Val Gln Asn Arg Asn Tyr Ile Ile Pro Ile Thr Phe

565 570 575

Thr Thr Gly Ala Asn Pro Ser Phe Asp Asn Thr Lys Pro Ser His Ile

580 585 590

Ile Ser Lys Gly Val Thr Val Ile Asp Arg Gly Val Val Gly Asp Tyr

595 600 605

Trp Thr Ile Phe Asn Ile Gln Gln Thr Gly Phe Tyr Arg Val Asn Tyr

610 615 620

Asp Asp Tyr Thr Trp Asn Leu Ile Val Leu Ala Leu Arg Gly Ala Asp

625 630 635 640

Arg Glu Lys Ile His Glu Tyr Asn Arg Ala Gln Ile Val Asn Asp Val

645 650 655

Phe Gln Phe Ala Arg Ser Gly Leu Met Thr Tyr Gln Arg Ala Leu Asn

660 665 670

Ile Leu Ser Phe Leu Glu Phe Glu Thr Glu Tyr Ala Pro Trp Val Ala

675 680 685

Ala Ile Thr Gly Phe Asn Trp Leu Arg Asn Arg Leu Val Gly Lys Pro

690 695 700

Gln Leu Asp Glu Leu Asn Glu Lys Ile Val Gln Trp Ser Ser Lys Val

705 710 715 720

Met Gly Glu Leu Thr Tyr Met Pro Thr Glu Gly Glu Pro Phe Met Arg

725 730 735

Ser Tyr Leu Arg Trp Gln Leu Ala Pro Val Met Cys Asn Leu Asn Val

740 745 750

Pro Ala Cys Arg Ala Gly Ala Arg Ala Ile Phe Glu Asp Leu Arg Val

755 760 765

Phe Gly His Glu Val Pro Val Asp Ser Arg Asn Trp Val Tyr Cys Asn

770 775 780

Ala Leu Arg Asp Gly Gly Ala Gln Glu Phe Asn Phe Leu Tyr Asn Arg

785 790 795 800

Phe Lys Ser His Asn Val Tyr Thr Glu Lys Ile Val Leu Leu Gln Thr

805 810 815

Leu Gly Cys Thr Ser His Val Glu Ser Leu Asn Thr Leu Leu Thr Asp

820 825 830

Ile Val Thr Pro Asn Gln Met Ile Arg Pro Gln Asp Tyr Thr Thr Ala

835 840 845

Phe Asn Thr Ala Val Ser Gly Asn Glu Val Asn Thr Arg Leu Val Trp

850 855 860

Asn Tyr Ile Gln Ala Asn Leu Gln Leu Val Phe Asn Ala Phe Ala Ser

865 870 875 880

Pro Arg Thr Pro Leu Ser Tyr Ile Ala Ala Arg Leu Arg Thr Val Glu

885 890 895

Glu Val Val Glu Tyr Gln Thr Trp Leu Asn Thr Thr Ala Ile Gln Ser

900 905 910

Ala Leu Gly Thr Asn Tyr Asn Ala Ile Tyr Gly Asp Ser Val Ala Thr

915 920 925

Tyr Asn Ser Ile Leu Trp Val Ser Thr Ile Glu Asp Ser Leu Ser Thr

930 935 940

Tyr Leu Thr Asn Gly Asn Asp Val Ile Glu Pro Ser Thr Ser Thr Thr

945 950 955 960

Ser Thr Thr Ala Ala Pro Thr Thr Val Thr Gln Pro Thr Ile Thr Glu

965 970 975

Pro Ser Thr Pro Thr Leu Pro Glu Leu Thr Asp Ser Ala Met Thr Ser

980 985 990

Phe Ala Ser Leu Phe Ile Ile Ser Leu Gly Ala Ile Leu His Leu Ile

995 1000 1005

Leu

14

39

Manduca sexta

14

ggaagatctt ccactgcatg cgtaactagt gcactcaac 39

15

50

Manduca sexta

15

gggatcccgt ccccgcgggg actcgatttg aaaaacaaat gaccatcatc 50