# Patent

## Title
Methods for identifying ecdysteroid synthesis inhibitors using the drosophila P450 enzyme shade

## Abstract

The invention relates to methods and materials useful for identifying inhibitors of ecdysteroid biosynthetic enzymes, specifically the Drosophila P450 enzyme, shade. These methods and materials can be used, for example, to identify molecules having insecticidal properties.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119(e) of U.S. Application No. 60/318,006, filed Sep. 7, 2001, and U.S. Application No. 60/317,890, filed Sep. 7, 2001.


FEDERALLY SPONSORED RESEARCH
The U.S. Government may have certain rights in this invention pursuant to Grant No. IBN 9603710 awarded by The National Science Foundation, and Grant No. DK-30118 awarded by The National Institutes of Health.


TECHNICAL FIELD
The invention relates to methods and materials for identifying molecules that have insecticidal properties. In particular, the invention pertains to methods and materials for identifying molecules that inhibit ecdysteroid biosynthetic enzymes. The invention also pertains to transgenic organisms that express exogenous ecdysone biosynthetic enzymes.
BACKGROUND
Pest insects adversely affect agriculture production by decreasing crop yield and/or crop quality. One way to control pest insects involves disrupting metabolic functions that are vital to insect development and growth.
The ecdysteroids ecdysone (“ECD”) and 20-hydroxyecdysone (“20E”) play an important role in insect development. Ecdysteroid pulses regulate larval molts, larval and prepupal transitions, and differentiation of adult tissues during pupation. See e.g., Riddiford, L. M. (1993), Hormones and Drosophila Development, In The Development of Drosophila melanogaster, Vol. II (ed. M. Bate and A. Martinez Arias), pp. 899–939, Cold Spring Harbor, Cold Spring Harbor Press. Ecdysteroids also regulate oogenesis and patterning of the embryonic cuticle. See e.g., Buszczak et al. (1999) Development 126:4581–4589; Bender et al. (1997) Cell 91:777–788; Henrich et al. (1999) Peptide hormones, syteroid hormones and puffs: Mechanisms and models in insect development, In Vitamins and Hormones (ed. Litwack) Vol. 55, pp. 73–125, Academic Press, San Diego.
Insects cannot make the cyclopentanoperhydrophenanthrene structure of ecdysteroids de novo, and build ecdysteroids from dietary steroids (e.g., cholesterol and phytosteroids). Generally, biosynthesis of ECD and 20E from cholesterol involves a series of oxidation, reduction and hydroxylation steps. See e.g., Gilbert et al., Control and Biochemical Nature of the Ecdysteroidogenic Pathway, Ann Rev Entomology (in press). 20E is made from ECD.
While the ecdysteroid biosynthetic pathway and the effects of ECD and 20E on insect development are relatively well characterized, the identity of the genes and proteins involved in ecdysteroid biosynthesis is as yet largely unknown.
SUMMARY
The invention features methods and materials for identifying molecules that target the enzymes involved in ecdysone biosynthesis. The methods and materials of the invention can facilitate the discovery and manufacture of new insecticides.
In one aspect, the invention provides methods for identifying inhibitors of ecdysteroid synthesis. Such methods can include contacting an ecdysteroid biosynthetic enzyme with a candidate inhibitor molecule, and determining whether or not the molecule inhibits the activity of the enzyme. Typically, the enzyme has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
Generally, the determining step uses a product-specific antibody or a substrate-specific antibody. A product-specific antibody can have specific binding affinity for a product such as ketotriol (2,22-dideoxyecdysone), ketodiol (2,22,25-trideoxyecdysone), diketol (3-dehydroketodiol), 2dE (2-deoxyecdysone), 22dE (22-deoxyecdysone), E (ecdysone), 20E (20-hydroxyecdysone), C (cholesterol), 25C (25-hydroxycholesterol), 7dC (7-dehydrocholesterol), 7d25C (7-dehydro-25-hydroxycholesterol), sitosterol, fucosterol, and stigmasterol. A substrate-specific antibody can have specific binding affinity for a substrate such as 22dE, 2dE, E, 20E, 26E (26-hydroxyecdysone), 20,26E (20, 26-dihydroxyecdysone), 7dC, 7d25C, “delta”-4-diketol, diketol, ketodiol, ketotriol, fucosterol, fucosterol epoxide stigmasterol epoxide and 5- and/or 11-hydroxyecdysteroids. Typically, the product-specific antibody or the substrate-specific antibody is attached to a solid substrate, and the solid substrate can be a microtiter plate.
In another aspect, the invention provides methods for determining whether or not a molecule has insecticidal properties. Such methods can include contacting an insect with a molecule that inhibits an ecdysteroid biosynthetic enzyme, and determining whether or not the molecule decreases the viability of the insect. Typically, the enzyme has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
In yet another aspect, the invention provides methods for making inhibitors of ecdysteroid synthesis. Such methods can include contacting an ecdysteroid biosynthetic enzyme with at least one candidate inhibitor molecule, determining whether or not the molecule inhibits the activity of the enzyme, and synthesizing a plurality of the molecules that inhibit the activity of the enzyme. Typically, the enzyme has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
In another aspect of the invention, there are provided methods for making an insecticide. Such methods can include contacting an insect with a molecule that inhibits the activity of an ecdysteroid biosynthetic enzyme, determining whether or not the molecule decreases the viability of the insect, and synthesizing a plurality of the molecules that decrease the viability of the insect. Typically, the enzyme has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
In still another aspect, the invention provides a kit for testing whether or not a molecule affects the activity of an ecdysteroid biosynthetic enzyme. A kit of the invention can include, separately or in association, one or more elements selected from the group consisting of the enzyme, a nucleic acid construct encoding the enzyme, and a host cell containing the enzyme, and one or more elements selected from the group consisting of a substrate of the enzyme, a product of the enzyme, a tracer, an antibody specific for the substrate, and an antibody specific for the product. Typically, the enzyme has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
In another aspect, the invention provides a transgenic plant that expresses an exogenous enzyme having the amino acid sequence shown in SEQ ID NO:3 or an exogenous enzyme having an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5. Representative plants can be selected from a genus such as Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna and Zea. 
In another aspect, the invention provides an isolated polypeptide that includes an amino acid sequence substantially identical to SEQ ID NO: 1. Such a polypeptide can catalyze the conversion of 2,22-dideoxyecdysone to 2-deoxyecdysone. Generally, a Drosophila embryo lacking the activity of the polypeptide is non-viable, and a Drosophila organism having a reduced level of activity of the polypeptide produces reduced levels of ecdysteroids ecdysone (ECD) and 20-hydroxyecdysone (20E) relative to a wild-type Drosophila organism. Typically, such a polypeptide can functionally complement a Drosophila homozygous dib mutant. The invention further provides a host cell containing such a polypeptide. In addition, the invention provides an isolated nucleic acid consisting essentially of a nucleic acid having the sequence shown in SEQ ID NO:6, or the complement thereof. The invention additionally provides a nucleic acid construct comprising a nucleic acid having the sequence shown in SEQ ID NO:6, or the complement thereof.
In another aspect, the invention provides an isolated polypeptide that includes an amino acid sequence substantially identical to SEQ ID NO:2. Generally, a Drosophila embryo lacking the activity of the polypeptide is non-viable, and a Drosophila organism having a reduced level of activity of the polypeptide produces reduced levels of ecdysteroids ecdysone (ECD) and 20-hydroxyecdysone (20E) relative to a wild-type Drosophila organism. Typically, such a polypeptide can functionally complement a Drosophila homozygous phm mutant. The invention further provides a host cell containing such a polypeptide. In addition, the invention provides an isolated nucleic acid consisting essentially of a nucleic acid having the sequence shown in SEQ ID NO:7, or the complement thereof. The invention additionally provides a nucleic acid construct comprising a nucleic acid having the sequence shown in SEQ ID NO:7, or the complement thereof.
In yet another aspect, the invention provides an isolated polypeptide that includes the amino acid sequence shown in SEQ ID NO:3. Generally, a Drosophila embryo lacking the activity of the polypeptide is non-viable, and a Drosophila organism having a reduced level of activity of the polypeptide produces reduced levels of ecdysteroids ecdysone (ECD) and 20-hydroxyecdysone (20E) relative to a wild-type Drosophila organism. Typically, such a polypeptide can functionally complement a Drosophila homozygous spo mutant. The invention further provides a host cell containing such a polypeptide. In addition, the invention provides an isolated nucleic acid consisting essentially of a nucleic acid having the sequence shown in SEQ ID NO:8, or the complement thereof. The invention further provides a nucleic acid construct comprising a nucleic acid having the sequence shown in SEQ ID NO:8, or the complement thereof.
In another aspect, the invention provides an isolated polypeptide that includes an amino acid sequence substantially identical to SEQ ID NO:4. Generally, a Drosophila embryo lacking the activity of the polypeptide is non-viable, and a Drosophila organism having a reduced level of activity of the polypeptide produces reduced levels of ecdysteroids ecdysone (ECD) and 20-hydroxyecdysone (20E) relative to a wild-type Drosophila organism. Typically, such a polypeptide can functionally complement a Drosophila homozygous shd mutant. The invention further provides a host cell containing such a polypeptide. In addition, the invention provides an isolated nucleic acid consisting essentially of a nucleic acid having the sequence shown in SEQ ID NO:9, or the complement thereof. The invention additionally provides a nucleic acid construct comprising a nucleic acid having the sequence shown in SEQ ID NO:9, or the complement thereof.
In still another aspect, the invention provides an isolated polypeptide that includes an amino acid sequence substantially identical to SEQ ID NO:5. Such a polypeptide can catalyze the conversion of 2,22-dideoxyecdysone to 22-deoxyecdysone and/or 2-deoxyecdysone to ecdysone. Generally, a Drosophila embryo lacking the activity of the polypeptide is non-viable, and a Drosophila organism having a reduced level of activity of the polypeptide produces reduced levels of ecdysteroids ecdysone (ECD) and 20-hydroxyecdysone (20E) relative to a wild-type Drosophila organism. Typically, such a polypeptide can functionally complement a Drosophila homozygous sad mutant. The invention further provides a host cell containing such a polypeptide. In addition, the invention provides an isolated nucleic acid consisting essentially of a nucleic acid having the sequence shown in SEQ ID NO:10, or the complement thereof. The invention additionally provides a nucleic acid construct comprising a nucleic acid having the sequence shown in SEQ ID NO: 10, or the complement thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.



DESCRIPTION OF DRAWINGS
FIG. 1 shows an alignment of novel P450 polypeptide sequences. DIB, SEQ ID NO:14; SPO, SEQ ID NO:16; PHM, SEQ ID NO:15; SHADE, SEQ ID NO:17; SHADOW, SEQ ID NO:18.
FIG. 2 shows the results of HPLC analysis of methanol extracts of the cells transfected with dib cDNA.
FIG. 3 shows the results of HPLC analysis of methanol extracts of the cells transfected with shadow cDNA.



DETAILED DESCRIPTION
The invention provides methods and materials for identifying inhibitors of enzymes involved in ecdysteroid biosynthesis. The invention is based, in part, on the discovery of novel ecdysteroid biosynthetic enzymes. Such enzymes can be used in screens to identify insecticidal molecules that target the ecdysone biosynthetic pathway. The invention also provides transgenic organisms that express exogenous ecdysteroid biosynthetic enzymes.
Novel Ecdysteroid Biosynthetic Enzymes
The products of the Drosophila disembodied (dib), spook (spo), shade (shd), shadow (sad), and phantom (phm) genes appear to be involved in a common developmental pathway. Mutations in each of these genes cause a very similar embryonic lethal phenotype: failure of cuticle differentiation; failure of head involution; abnormal gut morphogenesis and dorsal closure. The common defect in cuticle development suggests that dib, spo, shd, sad, and phm might encode enzymes involved, directly or indirectly, in ecdysteroid biosynthesis.
The products of dib, spo, shd, sad, and phm appear to be required for the synthesis of ECD and/or 20E. Mutations in dib, spo, shd, sad, and phm decrease or eliminate 20E-dependant transcription of the IMP-E1 gene in the embryonic epidermis. See Example 3. Biochemical analyses confirm that ECD and 20E levels are reduced at least 4-fold in heterozygous dib, spo, shd, sad, and phm mutants relative to wild type flies. See Example 4, Tables 1 & 2 (showing the effect of dib and spook mutations on ECD and 20E levels).
Enzymes that exhibit biochemical properties characteristic of cytochrome P450 enzymes reportedly catalyze at least four of the steps in the ECD and 20E biosynthetic pathways. See Grieneisen et al. (1993) Insect Biochem. Mol. Biol. 23:13–23. The dib, spo, shd, sad, and phm genes encode P450 enzymes. P450 sequences are located in the vicinity of the map locations of the dib, spo, shd, sad, and phm loci. Genomic DNA from wild type and one or more mutant strains corresponding to each of these P450 genes was sequenced. At least one mutant allele was identified for each P450 gene. An alignment of the dib, spo, shd, sad, and phm P450 gene products is presented in FIG. 1.
Cytochrome P450 enzymes are a highly diverse superfamily of heme-containing proteins. These proteins display an unusual reduced carbon monoxide difference spectrum that has an absorbance peak at 450 nm (hence Pigment at 450 m or “P450”). The characteristic spectrum is caused by a thiolate anion acting as the 5th ligand to the heme. P450 enzymes most commonly catalyze hydroxylation reactions, often of a lipophilic substrate. P450 proteins perform a wide spectrum of other reactions including N-oxidation, sulfoxidation, epoxidation, N-, S-, and O-dealkylation, peroxidation, deamination, desulfuration and dehalogenation.
More than 1500 P450 cytochrome sequences are known. Among P450 enzymes, the C-terminal half is more highly conserved than the N-terminal half. One P450 signature motif is the heme ligand, usually represented as FXXGXXXCXG (where “X” is any amino acid) (SEQ ID ID NO:12) where the Te residue is pat of the oxygen binding site. The K-helix has an invariant ENXR (SEQ ID NO:13) sequence that tolerates no substitutions.
The proteins encoded by dib, spo, shd, sad, and phm are less than 40% identical to P450 enzymes listed in public databases, and are, by the rules of the P450 Nomenclature Committee (see drnelson.utmem.edu/cytochromeP450.html on the World Wide Web founding members of novel P450 families. In accord with P450 Nomenclature Committee rules, the enzyme encoded by dib is designated CYP302a1, phm encodes CYP306a1, spo encodes CYP307a1, shd encodes CYP314a1, and sad encodes CYP315a1. The cDNA sequences for dib, spo, shd, sad, and phm and the amino acid sequences of the corresponding encoded proteins are shown in Table 3.
Nucleic acid molecules encoding each of the novel P450 enzymes were obtained from Drosophila embryonic cDNA libraries, cloned into expression vectors, and introduced into Drosophila S2 cells. Transfected cells were incubated with particular substrates for 2 to 24 hours and then harvested and extracted with methanol. Methanol extracts were analyzed by HPLC, and reaction products were identified based on their mobility characteristics relative to known standards. CYP302a1, encoded by dib, can catalyze the conversion of 2,22-dideoxyecdysone to 2-deoxyecdysone. See FIG. 2. CYP315a1, encoded by sad, can convert 2,22-dideoxyecdysone to 22-deoxyecdysone. See FIG. 3. CYP315a1 can also convert 2-deoxyecdysone to ecdysone. The spo, shd and phm genes appear to encode enzymes that catalyze earlier steps in the ecdysteroid biosynthetic pathway.
Methods and Materials for Identifying Inhibitors of Ecdysteroid Synthesis
In one aspect, the invention provides methods for identifying inhibitors of ecdysteroid synthesis. The methods involve contacting an ecdysteroid biosynthetic enzyme with a candidate inhibitor molecule and determining whether or not the molecule decreases the activity of the enzyme.
Ecdysteroid biosynthetic enzymes suitable for use in the invention include the CYP302a1 protein (SEQ ID NO:1), the CYP306a1 protein (SEQ ID NO:2), the CYP307a1 protein (SEQ ID NO:3), the CYP314a1 protein (SEQ ID NO:4), and the CYP315a1 protein (SEQ ID NO:5). In addition, ecdysteroid biosynthetic enzymes suitable for use in the invention include enzymes substantially identical to the CYP302a1 protein (SEQ ID NO: 1), the CYP306a1 protein (SEQ ID NO:2), the CYP314a1 protein (SEQ ID NO:4), and the CYP315a1 protein (SEQ ID NO:5). With respect to the CYP302a1 protein, “substantially identical” refers to proteins having at least 95% sequence identity to SEQ ID NO: 1. With respect to the CYP306a1 protein, “substantially identical” refers to proteins having at least 85% sequence identity to SEQ ID NO:2. With respect to the CYP314a1 protein, “substantially identical” refers to proteins having at least 98% sequence identity to SEQ ID NO:4. With respect to the CYP315a1 protein, “substantially identical” refers to proteins having at least 95% sequence identity to SEQ ID NO:5.
Suitable polypeptides that are substantially identical to the polypeptides having the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5 can have one or more amino acid substitutions, insertions or deletions relative to one of the above-identified P450 enzymes. Thus, suitable homologs can correspond to a part (e.g., a characteristic amino acid sequence element or functional domain) of one of the above-identified P450 enzymes. For use in the methods of the invention, the above-described ecdysteroid biosynthetic enzymes should possess functional activity to determine whether or not the enzyme is inhibited by a candidate inhibitor molecule. Non-functional ecdysteroid biosynthetic enzymes, however, also have utility. Non-functional ecdysteroid biosynthetic enzymes can be used, for example, to purify a substrate. A non-functional ecdysteroid biosynthetic enzyme also can be used as a candidate inhibitor molecule since such a molecule can compete with the wild-type enzyme for substrate binding but will not generate a product.
Suitable homologs typically have amino acid sequence elements characteristic of P450 enzymes. One characteristic P450 amino acid sequence element is the heme ligand, usually represented as FXXGXXXCXG (SEQ ID NO:11), which can tolerate exceptions at the three non-cysteine positions. The heme-binding region is typically about 50 amino acids from the C-terminal of the protein. Another characteristic P450 amino acid sequence element is the 1helix, which has a conserved motif A(A,G)X(E,D)T (SEQ ID NO: 12). The K-helix has an invariant EXXR (SEQ ID NO: 13) sequence that tolerates no substitutions. Other characteristic P450 sequence elements are described in Nelson, Methods in Molecular Biology. Vol. 107. Cytochrome P450 Protocols. eds. Phillips & Shephard. Humana Press, Totowa, N. J., pp. 15–24, 1998.
Suitable homologs of the above-identified P450 enzymes can be synthesized on the basis of amino acid sequence elements characteristic of the above-identified P450 enzymes. Suitable homologs can also be identified by homologous sequence analysis from a database of nucleotide or polypeptide sequences. For example, homologous sequence analysis can involve BLAST or PSI-BLAST analysis of databases using the amino acid sequences of the above-identified P450 enzymes. Potentially useful homologs also can be identified by manual inspection of candidates that appear to have amino acid sequence elements characteristic of the above-identified P450 enzymes.
A percent identity for any “target” nucleic acid or amino acid sequence relative to another “subject” nucleic acid or amino acid sequence (e.g.,, the nucleic acid or amino acid sequence of any of above-identified P450 enzymes) can be determined by using the BLAST 2 Sequences (B12seq) program from the stand-alone BLASTZ application (version 2.0.14) containing BLASTN and BLASTP. BLASTZ version 2.0.14 can be obtained at fr.com or at ncbi.nlm.nih.gov on the World Wide Web. Instructions explaining how to use BLASTZ, and specifically the B12seq program, can be found in the ‘readme’ file accompanying BLASTZ. The programs also are described in detail by Karlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264; Karlin et al, 1990, Proc. Natl. Acad. Sci., 90:5873; and Altschul et al, 1997, Nuci. Acids Res., 25:3389.
B12 seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and all other options are left at their default setting. The following command can be used to generate an output file containing a comparison between two nucleic acid sequences: C:\B12seq-i c:\seq1.txt-j c:\seq2.txt -p blastn-o c:\output.txt-q-1-r 2. To compare two amino acid sequences, the options of B12 seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. The following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12 seq-i c:\seq1.txt-j c:\seq2.txt -p blastp-o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
The percent identity is determined by dividing the number of matches by the length of the subject sequence followed by multiplying the resulting value by 100. For example, if a target sequence is compared to a subject sequence having a length of 1000 and the number of matches is 900, then the sequence has a percent identity of 90% (i.e., 900÷1000×100=90%) relative to the subject sequence.
Whether a homolog of one of the above-identified P450 enzymes is suitable for the invention can be readily determined by functional complementation. Functional complementation involves introducing a gene encoding a homolog into Drosophila homozygous for the dib, spo, shd, sad, or phm mutations indicated in FIG. 1. Homologs that increase the ecdysone titer in a statistically significant way relative to the appropriate homozygous mutant are useful for the invention. Ecdysone titers can be measured as described in Example 4.
Additional amino acid sequences (e.g., FLAGTM (U.S. Pat. No. 4,851,341); 6×HIS; c-myc; Protein C; VSV-G; Hemagglutinin; biotin; and GFP) can be attached to any one of the above-identified P450 enzymes or homologs to assist in, for example, purification of one or more of the above-identified P450 enzymes or homologs thereof.
In the methods of the invention, suitable ecdysteroid biosynthetic enzymes are brought into contact with a candidate inhibitor molecule. Any molecule can be a suitable candidate inhibitor molecule.
Suitable ecdysteroid biosynthetic enzymes and candidate inhibitor molecules are typically brought into contact with one another within a cell. Nucleic acid constructs encoding suitable ecdysteroid biosynthetic enzymes can be introduced into host cells (e.g., Drosophila S2 cells) by a variety of means, including liposome-mediated transfer, calcium phosphate co-precipitation, electroporation and DDAB-mediated transfection. See e.g., Trotter & Wood (1995) Methods Molec. Biol., 39:97; Han (1996) Nucleic Acid Res., 24:4362. Means for expressing recombinant proteins in insect cells are well known in the art. See e.g., O'Reilly et al. (1992) In: Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Company, 216; Vaughn et al. (1977) In Vitro, 13:213. Exemplary transfer vectors for heterologous protein production are based upon Autographa californica nuclear polyhedrosis virus (AcNPV). The AcNPV viral genome is 134 kb in length and is functionally characterized by genes that are expressed early, late and very late during infection. See e.g., Ayres et al. (1994) Virology, 202:586; Friesen & Miller (1986) In The Molecular Biology of Baculoviruses, Springer-Verlag, Berlin, 31. In some embodiments, a host cell expresses only one ecdysteroid biosynthetic enzyme. In other embodiments, a host cell expresses other enzymes in the ecdysteroid biosynthetic pathway.
Enzyme and radio-immunoassays (“EIA” and “RIA”) can be used to determine whether a candidate inhibitor molecule decreases the activity of an ecdysteroid biosynthetic enzyme. In one format (“Format 1”), an antibody has specific binding affinity for the product of the enzyme but not for the substrate. In another format (“Format 2”), an antibody has specific binding affinity for the substrate of the enzyme but not for the product.
The substrate can be the molecule upon which a suitable ecdysteroid biosynthetic enzyme or homolog directly acts, as well as molecules further upstream in the pathway of ecdysone biosynthesis. See e.g., Gilbert et al., Control and Biochemical Nature of the Ecdysteroidogenic Pathway, Ann Rev Entomology (in press). Thus, exemplary substrates include radiolabeled and non-labeled ketotriol (2,22-dideoxyecdysone), ketodiol (2,22,25-trideoxyecdysone), diketol (3-dehydroketodiol), 2dE (2-deoxyecdysone), 22dE (22-deoxyecdysone), E (ecdysone), 20E (20-hydroxyecdysone), C (cholesterol), 25C (25-hydroxycholesterol), 7dC (7-dehydrocholesterol), 7d25C (7-dehydro-25-hydroxycholesterol), sitosterol, fucosterol, and stigmasterol. The product can be the molecule directly produced by a suitable ecdysteroid biosynthetic enzyme or homolog, as well as molecules further downstream in the pathway of ecdysone biosynthesis. Thus, exemplary products include radiolabeled and non-labeled 22dE, 2dE, E, 20E, 26E (26-hydroxyecdysone), 20,26E (20, 26-dihydroxyecdysone), 7dC, 7d25C, “delta”-4-diketol, diketol, ketodiol, ketotriol, fucosterol, fucosterol epoxide stigmasterol epoxide and 5- and/or 11-hydroxyecdysteroids.
Format 1 assays use a product-specific antibody. The antibody is typically immobilized (e.g., on the surface of a 96-well titer plate). Cells or cell extracts containing a suitable ecdysteroid biosynthetic enzyme are incubated in proximity to the antibody along with inhibitor, tracer (i.e., radioactively or enzymatically labeled product), and an appropriate substrate. Control reactions lack inhibitor. Tracer is typically added prior to substrate. Substrate, product and tracer compete for binding to the antibody. The antibody is separated from the unbound components of the reaction and the amount of antibody-bound tracer is measured by scintillation counting for RIA, or by an appropriate enzymatic assay for EIA. If an inhibitor molecule decreases the activity of an ecdysteroid enzyme, there is less displacement of antibody-bound tracer by unlabeled product than in control reactions that lack inhibitor, resulting in higher post-reaction measurements of antibody-bound tracer.
Format 2 assays use a substrate-specific antibody. The antibody is typically immobilized (e.g., on the surface of a 96-well titer plate). Cells or cell extracts containing a suitable ecdysteroid biosynthetic enzyme are incubated with inhibitor and an appropriate substrate. Control reactions lack inhibitor. The reaction mixture is heated to inactivate the enzyme prior to incubation with antibody and tracer (i.e., radioactively or enzymatically labeled substrate). The antibody is separated from the unbound components of the reaction and the amount of antibody-bound tracer is measured by scintillation counting for RIA, or by an appropriate enzymatic assay for EIA. If an inhibitor molecule decreases the activity of an ecdysteroid enzyme, there is more displacement of antibody-bound tracer by unlabeled substrate than in control reactions that lack inhibitor, resulting in lower post-reaction measurements of antibody-bound tracer.
In assays that employ whole cells, the substrate, product and inhibitor are typically freely diffusible within the intact cell preparation. In high throughput assays, all the necessary reagents are incubated together in a 96-well, or larger, format.
In other embodiments of the invention, a suitable ecdysteroid biosynthetic enzyme and a candidate inhibitor molecule are brought into contact with one another in an in vitro biochemical reaction. In these embodiments, enzymes are purified (i.e., extracted from cells and, optionally, separated from various other cellular biomolecules). Means for purifying P450 type enzymes are well known in the art. See e.g., Chen, W. et al, (1996) Arch Biochem Biophys., 335:123–30. Inhibitory effects of purified enzymes can be tested using the same types of assays described above. In some embodiments, a reaction contains only one ecdysteroid biosynthetic enzyme. In other embodiments, a reaction contains one or more additional enzymes in the ecdysteroid biosynthetic pathway.
In another aspect, the invention provides methods for identifying molecules having insecticidal properties. The methods involve contacting an insect with a molecule that inhibits an ecdysteroid biosynthetic enzyme, and determining whether or not the molecule decreases the viability of the insect. If necessary, an ecdysteroid biosynthetic enzyme can be contacted with the candidate molecule to demonstrate inhibition of the enzyme as discussed herein. Insects can be contacted with a candidate inhibitor, for example, by spraying or soaking newly hatched insect larvae with a liquid suspension or powder containing the inhibitor or by feeding newly hatched insect larvae a food adulterated with the candidate inhibitor molecule. A candidate inhibitor decreases the insect viability if larvae contacted with the candidate inhibitor mature to adulthood at a lower frequency than larvae that are not contacted with the candidate inhibitor molecule. For example, if 100 of 1000 larvae contacted with a candidate inhibitor molecule mature to adulthood and 800 of 1000 uncontacted larvae mature to adulthood, the candidate inhibitor decreases insect viability. With respect to an embryo, a non-viable embryo is an embryo that has died.
In another aspect, the invention provides methods for making inhibitors of ecdysteroid synthesis. The method involves contacting a suitable ecdysteroid biosynthetic enzyme with at least one candidate inhibitor molecule, determining whether or not the molecule decreases the activity of the enzyme, and synthesizing a plurality of those molecules that decrease the activity of the enzyme.
In another aspect, the invention provides methods for making an insecticide. The methods involve contacting an insect with the molecule, determining whether or not the molecule decreases the viability of the insect, and synthesizing a plurality of those molecules that decrease the viability of the insect. Chemical inhibitors can be made by a variety of methods known to the skilled artisan. Peptide inhibitors can be made by a variety of methods, including those described in Merrifield (1963) J. Am. Chem. Soc., 85:2149–2154; and U.S. Pat. No. 6,280,595. The latter method can also be used to generate a large library of potential peptide inhibitors.
In another aspect, the invention provides kits for testing whether a molecule affects the activity of an ecdysteroid biosynthetic enzyme. The kits contain one or more of the following: a nucleic acid construct encoding a suitable ecdysone biosynthetic enzyme, a transfected cell containing a suitable ecdysone biosynthetic enzyme, and a suitable ecdysone biosynthetic enzyme. The kits also can contain, separately or in association, one or more of the following: a suitable substrate, a suitable product, a suitable tracer, or a suitable antibody.
Transgenic Organisms
The invention also provides transgenic eukaryotic organisms that express ecdysteroid biosynthetic enzymes substantially identical to the CYP302a1 protein (SEQ ID NO:1), the CYP306a1 protein (SEQ ID NO:2), CYP307a1 protein (SEQ ID NO:3), the CYP314a1 protein (SEQ ID NO:4), and the CYP315a1 protein (SEQ ID NO:5). A transgenic eukaryote of the invention can express any one of the above-mentioned polypeptides or homologs, several of the above-mentioned polypeptides or homologs, or all of the above-mentioned polypeptides or homologs. In some embodiments, a transgenic eukaryote also expresses other enzymes in the ecdysteroid biosynthetic pathway. Such transgenic organisms can serve as a source for enzymes, e.g., for use in the above-described inhibitor screens.
In general, a transgenic organism that expresses an exogenous polypeptide contains a nucleic acid construct that encodes a polypeptide not normally encoded by its genome. The nucleic acid construct is capable of being transcribed in the organism to make mRNA that is then translated to produce the exogenous polypeptide. A transgenic organism can contain one or multiple nucleic acid constructs, and each nucleic acid construct can encode one or multiple exogenous polypeptides.
A nucleic acid construct (e.g., vector) encoding an exogenous polypeptide can be introduced into a eukaryotic organism by a variety of techniques known to those of ordinary skill in this art, such as calcium phosphate or lithium acetate precipitation, electroporation, lipofection, particle bombardment, and electrospraying.
One of skill can screen transgenic organisms to determine whether they have the desired phenotype (e.g., whether the polypeptide encoded by the introduced nucleic acid construct is expressed, and whether an expressed exogenous polypeptide is functional). For example, one can use antibodies to detect expression of the polypeptide. One can also devise assays that measure the activity of the encoded polypeptide. In some applications, the screening method allows large numbers of samples to be processed rapidly, since transgenic organisms often may not have the desired phenotype.
Nucleic Acid Constructs
A nucleic acid encoding a novel polypeptide of the invention may be obtained by, for example, the polymerase chain reaction (PCR). PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, eds. Dieffenbach & Dveksler, Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. In addition, various known PCR strategies can be used to introduce site-specific nucleotide sequence modifications into template nucleic acid.
Genes encoding ecdysteroid biosynthetic enzymes can also be chemically synthesized. Manual synthesis of nucleic acid fragments may be accomplished using well-established procedures. For example, a synthetic gene can be enzymatically assembled in a DNA vector from chemically synthesized oligonucleotide duplex segments. Automated chemical synthesis can be performed using various commercially available machines.
To design a synthetic gene for enhanced expression in a target organism, the DNA sequence can be modified from a naturally occurring ecdysteroid biosynthetic polypeptide-encoding gene or designed de novo to: 1) contain codons preferred by highly expressed genes in the target organism; 2) have an A+T content in nucleotide base composition that approximates that of the target organism; 3) have an initiation sequence like those of the target organism; 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA; and/or 5) avoid sequences that produce secondary structure hairpins and RNA splice sites. Not all of the above-mentioned design features need be incorporated into a synthetic gene in order to obtain enhanced expression. A synthetic gene may be synthesized for other purposes in addition to that of achieving enhanced levels of expression.
In some embodiments, the codons for a synthetic gene reflect the distribution frequency of codon usage of highly expressed genes in the target organism. The distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression. For optimal expression, the synthetic gene is typically designed so that its distribution frequency of codon usage deviates no more than 30% (e.g., 0–10%, 10–20%, 20–30%) from that of highly expressed genes in the target organism. In general, genes within a taxonomic group exhibit similarities in codon choice, regardless of the function of these genes.
In embodiments where a synthetic gene is to be expressed in a dicot plant, a synthetic gene can be designed to incorporate to advantage codons used preferentially by highly expressed dicot proteins. In embodiments where enhanced expression is desired in a monocot, codons preferred by highly expressed monocot proteins are employed in designing the synthetic gene. An estimate of the overall use of the genetic code by a taxonomic group can be obtained by summing codon frequencies of all its sequenced genes. Monocot and dicot codon preferences are analyzed and reported, for example, in U.S. Pat. No. 6,013,523.
In some embodiments, the A+T content in DNA base composition of a synthetic gene reflects that normally found in genes for highly expressed proteins native to the target organsim (e.g., 55–60% in many plants). Typically, the synthetic has an A+T content near this value, and not so high as to cause destabilization of RNA and, thereby, reduce protein expression levels.
For some applications it may be useful to direct exogenous polypeptides to different cellular compartments, or to facilitate its secretion from the cell. To this end, the skilled artisan can make chimeric genes encoding an ecdysteroid biosynthetic enzyme with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell, 56:247–253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol., 42:21–53), or nuclear localization signals (Raikhel (1992) Plant Phys., 100:1627–1632). While the references cited give examples of each of these, the list is not exhaustive and additional targeting signals of use may be discovered in the future.
Nucleic acid constructs may contain cloning vector segments. Cloning vector segments are commercially available and are used routinely by those of ordinary skill. Nucleic acid constructs of the invention may also contain sequences encoding other polypeptides. Such polypeptides may, for example, facilitate the introduction or maintenance of the nucleic acid construct into a host organism. Such polypeptides may also be used to facilitate the introduction of the nucleic acid construct into a target organism. Other polypeptides may also affect the expression, activity, or biochemical or physiological effect of the encoded CBF polypeptide. Alternatively, other polypeptide coding sequences may be provided on separate nucleic acid constructs.
Nucleic acid constructs may also contain one or more regulatory elements operably linked to an ecdysteroid biosynthetic enzyme coding sequence. Such regulatory elements may include promoter sequences, enhancer sequences, response elements, protein recognition sites, or inducible elements that modulate expression of a nucleic acid sequence. As used herein, “operably linked” refers to positioning of a regulatory element in a construct relative to a nucleic acid coding sequence in such a way as to permit or facilitate expression of the encoded polypeptide. The choice of element(s) that may be included depends upon several factors, including, but not limited to, replication efficiency, selectability, inducibility, desired expression level, and cell or tissue specificity.
Suitable regulatory elements include promoters that initiate transcription only in certain cell types. In some embodiments, a cell type or tissue-specific promoter may also drive transcription of operably linked sequences in additional cell types or tissues. Other useful promoters may drive transcription in the same cell type or tissue through more than one developmental stage.
In some embodiments, a nucleic acid construct contains a promoter and a recognition site for a transcriptional activator, both of which are operably linked to the coding sequence for an ecdysteroid biosynthetic enzyme. In these embodiments, transgenic organisms that express the ecdysteroid biosynthetic enzyme contain a second nucleic acid construct that encodes a transcriptional activator. A transcriptional activator is a polypeptide that binds to a recognition site on DNA, resulting in an increase in the level of transcription from a promoter associated in cis with the recognition site.
The recognition site for the transcriptional activator polypeptide is positioned with respect to the promoter so that upon binding of the transcriptional activator to the recognition site, the level of transcription from the promoter is increased. The position of the recognition site relative to the promoter can be varied for different transcriptional activators, in order to achieve the desired increase in the level of transcription.
Many transcriptional activators have discrete DNA binding and transcription activation domains. The DNA binding domain(s) and transcription activation domain(s) of transcriptional activators can be synthetic or can be derived from different sources (e.g., two-component system or chimeric transcriptional activators). In some embodiments, a two-component system transcriptional activator has a DNA binding domain derived from the yeast gal4 gene and a transcription activation domain derived from the VP16 gene of herpes simplex virus. Populations of transgenic organisms or cells having a first nucleic acid construct that encodes a chimeric polypeptide and a second nucleic acid construct that encodes a transcriptional activator polypeptide can be produced by transformation, transfection, or genetic crossing. See, e.g., WO 97/31064.
For some applications, it may be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in a target organism for some applications. To this end, a skilled artisan can employ antisense RNA or cosuppression technologies. An “antisense” molecule generally contains nucleic acids or nucleic acid analogs that can specifically hybridize to a target nucleic acid. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense oligonucleotide is specifically hybridizable when (a) binding of the molecule to the target DNA or RNA interferes with the normal function of the target DNA or RNA, and (b) there is sufficient complementarity to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, i.e., under conditions in which in vitro assays are performed or under physiological conditions for in vivo assays or therapeutic uses.
The specific hybridization of an antisense molecule with its target nucleic acid can interfere with the normal function of the target nucleic acid. For a target DNA, an antisense molecule can disrupt replication and transcription. For a target RNA, an antisense molecule can disrupt, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA, and catalytic activity of the RNA. The overall effect of such interference with target nucleic acid function is, in the case of a nucleic acid encoding an ecdysteroid biosynthetic enzyme, modulation of the expression of a ecdysteroid biosynthetic enzyme. In the context of the present invention, “modulation” means a decrease in the expression of a gene and/or a decrease in cellular levels of the protein encoded by a gene.
Plants
Among the eukaryotic organisms provided by the invention are transgenic plants that express ecdysteroid biosynthetic enzymes substantially identical to the CYP302a1 protein (SEQ ID NO:1), the CYP306a1 protein (SEQ ID NO:2), CYP307a1 protein (SEQ ID NO:3), the CYP314a1 protein (SEQ ID NO:4), and the CYP315a1 protein (SEQ ID NO:5).
Techniques for introducing exogenous nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Pat. Nos. 5,204,253 and 6,013,863. If a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures by techniques known to those skilled in the art. Transgenic plants may be entered into a breeding program, e.g., to introduce a nucleic acid encoding a polypeptide into other lines, to transfer the nucleic acid to other species or for further selection of other desirable traits. Alternatively, transgenic plants may be propagated vegetatively for those species amenable to such techniques. Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid encoding a novel polypeptide.
A suitable group of plants with which to practice the invention include dicots, such as safflower, alfalfa, soybean, rapeseed (high erucic acid and canola), or sunflower. Also suitable are monocots such as corn, wheat, rye, barley, oat, rice, millet, amaranth or sorghum. Also suitable are vegetable crops or root crops such as potato, broccoli, peas, sweet corn, popcorn, tomato, beans (including kidney beans, lima beans, dry beans, green beans) and the like. Thus, the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna and Zea. 
Ecdysteroid biosynthetic enzymes can be expressed in plants in a cell- or tissue-specific manner according to the regulatory elements chosen to include in a particular nucleic acid construct introduced into the plant. Suitable cells, tissues and organs in which to express an ecdysteroid biosynthetic enzyme include, without limitation, embryo, cotyledons, endosperm, seed coat, vascular bundle, cambium, phloem, cortex, floral tissue, root tissue, leaf mesophyll cells, and leaf epidermal cells.
In some embodiments, a promoter that is specific to a plant vegetative tissue such as vascular bundle, cambium, phloem, cortex, leaf mesophyll, or leaf epidermis directs transcription of an ecdysone biosynthetic enzyme. In other embodiments, a promoter that is specific to a plant reproductive tissue such as fruit, ovule, seed, flower, endosperm directs transcription of an ecdysone biosynthetic enzyme. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano et al., (1989) Plant Cell, 1:855–866; Bustos et al., (1989) Plant Cell, 1:839–854; Green et al., (1988) EMBO J., 7:4035–4044; Meier et al., (1991) Plant Cell, 3, 309–316; and Zhang et al., (1996) Plant Physiology, 110:1069–1079.
Exemplary plant reproductive tissue promoters include those derived from the following genes: Brassica napus 2 s storage protein (see, Dasgupta (1993) Gene, 133:301–302); Arabidopsis 2 storage protein; soybean β-conglycinin; Brassica napus oleosin 20kD gene (see, GenBank No. M63985); soybean oleosin A (see, Genbank No. U09118); soybean oleosin B (see, GenBank No. U09119); Arabidopsis oleosin (see, GenBank No. Z17657); maize oleosin 18 kD (see, GenBank No. J05212; Lee (1994) Plant Mol. Biol., 26:1981–1987; the gene encoding low molecular weight sulfur rich protein from soybean, (see, Choi (1995) Mol. Gen, Genet., 246:266–268); and fruit-specific E8, a tomato gene expressed during fruit ripening, senescence and abscission of leaves and flowers (Blume (1997) Plant J., 12:731-746). See also, WO 98/08961; WO 98/28431; WO 98/36090; U.S. Pat. No. 5,907,082; and WO 00/24914.
Exemplary plant vegetative tissue promoters include those derived from the following genes: potato storage protein patatin gene (see, Kim (1994) Plant Mol. Biol., 26:603–615; Martin (1997) Plant J., 11:53–62); root Agrobacterium rhizogenes ORF13 (see, Hansen (1997) Mol. Gen. Genet., 254:337–343); genes active during taro corm development (see, Bezerra (1995) Plant Mol. Biol., 28:137–144); de Castro (1992) Plant Cell, 4:1549–1559); root meristem and immature central cylinder tobacco gene TobRB7 (see, Yamamoto (1991) Plant Cell, 3:371–382); ribulose biphosphate carboxylase genes RBCS1, RBCS2, and RBCS3A expressed in tomato leaves (see, Meier (1997) FEBS Lett., 415:91–95); ribulose biphosphate carboxylase genes expressed in leaf blade and leaf sheath mesophyll cells (see, Matsuoka (1994) Plant J., 6:311–319); leaf chlorophyll a/b binding protein (see, e.g., Shiina (1997) Plant Physiol., 115:477–483; Casal (1998) Plant Physiol., 116:1533–1538); Arabidopsis Atmyb5, expressed in developing leaf trichomes, stipules, in epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage (see, Li (1996) FEBS Lett., 379:117–121); and a maize leaf-specific gene described by Busk (1997) Plant J., 11:1285–1295.
Cell type or tissue-specific promoters derived from viruses can also be suitable regulatory elements. Exemplary viral promoters include: the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA, 92:1679–1683; the phloem-specific tungro bacilliform virus (RTBV) promoter; the cassaya vein mosaic virus (CVMV) promoter, expressed most strongly in vascular elements, leaf mesophyll cells, and root tips (Verdaguer (1996) Plant. Mol. Biol., 31:1129–1139).
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Mutations in dib, spo, shd, sad and phm Affect Drosophila Morphogenesis
Wild type and mutant Drosophila strains, available from the Bloomington Drosophila Stock Center (Indiana University), were cultured on standard cornmeal/yeast extract/dextrose medium. At particular stages of development, embryos were stained with spectrin antibody. Mutations in dib, spo, shd, sad, and phm were observed to prevent full differentiation of the embryonic cuticle. dib, spo, shd, sad, and phm embryos appeared to develop normally to approximately stage 14. At this point, many morphogenetic movements including dorsal closure, head involution, midgut morphogenesis and hindgut looping become abnormal. In particular, no denticle belts, dorsal hairs or differentiated mouth parts were evident in dib, spo, shd, sad, and phm homozygotes. Only a thin cuticular remnant containing both dorsal and anterior holes was secreted by most of the mutant embryos.
Embryos were also stained with Acridine orange, DAPI and reaper to examine whether there was an increase in apoptosis during these late stages. Neither unusual DAPI staining, nor increased numbers of AO-positive or reaper-expressing cells were observed, suggesting that abnormal numbers of cells were not dying during this time.
To determine if the abnormal morphogenesis phenotypes were caused by removal of maternal components, germline clones were made. Homozygous mutant germline clones were generated in the background of a dominant female-sterile mutant ovo D. For example, virgin females w, hs-Flp/w: FRT79, dib F8/TM3 were crossed to males FRT79, ovo D w+/TM3. The first instar larvae of this exemplary cross were heat shocked at 37° C. for 90 minutes and the non TM3 females were crossed to males dib P3/TM3, ftz-lacZ. Eggs were stained with anti-β-gal antibody in order to distinguish the homozygous dib germline clone embryos from the heterozygous embryos.
Embryos derived from mutant germlines exhibited exactly the same range and timing of defects as did simple zygotic mutants, suggesting that dib, spo, shd, sad, and phm are not required in the germline during oogenesis or during early embryogenesis before stage 14.
Example 2
Identification of a Novel P450 Gene
The dib locus is localized to the 64A3 interval on the left arm of the third Drosophila chromosome. A phage walk spanning about 30 kb was established in the region. Several cDNAs were isolated and positioned within the walk by Southern hybridization analysis and sequencing. P-element-mediated transformation was used to make transgenic lines carrying various overlapping genomic DNA fragments. P elements carrying DNA from several phages were able to rescue the l(3)64Ak complementation group, previously shown to be allelic to dib. A single transcription unit defined by a 1.7 kb cDNA was localized to the DNA contained on each of these phages.
The 1.7 kb cDNA was sequenced, and conceptual translation of the Dib product revealed that it is a new member of the cytochrome P450 superfamily. Genomic DNA obtained from lines carrying three different dib alleles was also sequenced. Each dib allele resulted from the introduction of a stop codon. See FIG. 1.
Example 3
dib, spo, shd, sad and phm Display Reduced ECD-dependent Transcription
Expression of the 20E-inducible genes IMP-E1 and L1 in wild type and in dib, spo, shd, sad and phm mutant embryos was observed by whole-mount RNA in situ hybridization and antibody staining. IMP-E1 and L1 encode novel secreted proteins that respond either directly (IMP-E1) (i.e., inducible in the presence of cycloheximide), or indirectly (IMP-L1), to 20E in culture. Both genes are expressed in complex patterns in the embryo. Since the timing of the epidermal expression coincides with the rise in embryonic ecdysteroid titers, these patterns were examined in wild type and in dib, spo, shd, sad and phm mutant backgrounds.
Embryos were fixed for 20 minutes in a 1:2 mixture of PBS:heptane that contained 4% formaldehyde. Devitellinizing was accomplished by washing the embryos several times in methanol. Imaginal disks and ovaries were dissected into phosphate-buffered saline (PBS), fixed in 4% formaldehyde and stored in ethanol at about 20° C. until use. Sense and anti sense digoxigenin-labeled RNA probes (Boehringer Mannheim) were generated by transcription of pBluescript dib, IMP-E1 or L1 subclones using T3, T7 or the Sp6 promoter. Embryos collected from heterozygotes containing a balancer chromosome marker with either Ubx-lacZ (for third chromosome mutants), or wg-lacZ (for second chromosome mutants), were double stained with anti-β-gal antibody and RNA probe in order to distinguish the homozygous mutants from those that carried the lacZ balancer chromosome. RNA hybridization and detection was carried out using standard methods. The rabbit anti-Spectrin antibody (a gift from T. Hays) was used at 1/100 dilution and the mouse β-gal monoclonal antibody (Promega) was used at 1/1000 dilution. Staining for both antibodies was visualized using an HRP-coupled secondary antibody (Vector Laboratories) and diaminobenzidine as substrate. Embryos, disks and ovaries were mounted for photography in 10% PBS and 90% glycerol.
Epidermal expression of both IMP-E1 and IMP-L1 was observed to be greatly reduced or absent in dib, spo, shd, sad and phm mutant backgrounds.
Example 4
dib, spo, shd, sad and phm Mutants Display Reduced ECD and 20E Titers
Levels of ECD and 20E in 7- to 16-hour old embryos were measured by an enzyme immunoassay (i.e., EIA). Embryos were collected from parents heterozygous for each of the mutant strains (in this example, dib F8) and from a TM3 balancer marked with a arm-GFP reporter. Embryos were sorted by fluorescence into GFP-negative pools containing the dib homozygous mutant embryos and a GFP-positive pool corresponding to dib/TM2, arm-GFP heterozygotes and TM2, arm-GFP homozygotes. Embryos were dechorionated and frozen at about 20° C. until used.
Embryos were homogenized in 0.5 ml of methanol and incubated at 4° C. for 2 hours. The supernatant was saved and the embryos were again incubated in 0.5 ml methanol overnight. Both supernatants were pooled and dried under N2. Ecdysteroids were analyzed with an enzyme immunoassay modified from that of Porcheron et al. (1989) Insect Biochem., 19:117–122, using a 20-hydroxyecdysone-peroxidase conjugate as a tracer and either DBL2-polyclonal anti-ECD antiserum or EC19 monoclonal anti-20E antibodies (see Aribi et al. (1997) Biochim. Biophys. Acta, 1335:246–252; Pascual et al. (1995) Z. Naturforsch., [C] 50:862–7). Briefly, dried samples were solubilized in a phosphate buffer (0.1 M, pH 7.4), placed on microplate wells (previously coated with secondary anti-IgG antibodies), then a known amount of tracer was added along with the primary anti-ecdysteroid antibody. After a 3-hour incubation at room temperature, bound peroxidase activity was revealed using tetramethylbenzidine as substrate and microplates were analyzed using a SpectraMax 340 microplate reader (Molecular Devices). Results were compared with data obtained from reference concentrations of ecdysone or 20-hydroxyecdysone and were normalized to the weight of the samples.
Homozygous mutants contained very low concentrations of both E and 20E, compared to control embryos. The results for the dib and spo mutants are shown below in Tables 1 and 2. The residual low levels of these products seen in the homozygous mutants could correspond to either remaining maternal products, to other metabolites weakly recognized by the antibodies, or to low-level synthesis of each product by another less-efficient pathway.







TABLE 1







Ecdysteroid levels measured by EIA on single pools of 7- to 12-hour


homo- or heterozygous dib embryos










Phenotypes
dib/dib
dib/gfp
Oregon (WT)





Pool size (mg)
26
71.1
8.1–112


ECD-equivalents
1.52 (±0.18; n = 8)
11.04 (±0.59; n = 8)
15.35 (±1.19; n = 6)


(pg/mg), using L2


polyclonal serum


20E-equivalents
0.71 (±0.11; n = 3)
 2.92 (±0.05; n = 3)
 3.68 (±0.25; n = 3)


(pg/mg), using EC19


mAb





Results expressed as pg equivalents. For dib/dib and dib/GFP embryos the same sample was measured several times (3–8 replicates) and the results are expressed as a mean ± the standard deviation. Statistical analysis by Student's t-test indicates that the two experimental pool means differ from that of the control population (P < 0.05).













TABLE 2







Ecdysteroid levels measured by EIA on single pools of 7- to 16-hour


homo- or heterozygous spo embryos










Phenotypes
spo/spo
spo/gfp
Oregon (WT)





Pool size (mg)
18.8
57
45


ECD-equivalents
 5.3 (±0.5)
17.8 (±0.5)
26.1 (±0.2)


(pg/mg), using L2


polyclonal serum


20E-equivalents
0 (*)
11.9 (±0.2)
28.2 (±2.6)


(pg/mg), using EC19


mAb





(*) Values were below the detection limit of the method. For each sample, 6 replicates have been measured, allowing calculation of the standard error of the corresponding pool mean (indicated by ±).






Example 5
Activity of CYP302a1, CYP306a1, CYP307a1, CYP314a1, & CYP315a1
Schneider line-2 cells (S2 cells) were grown in M3 medium (Shields and Sang M3 insect medium, Sigma) with 10% insect medium supplement (IMS, Sigma) and 1% penicillin/streptomycin (Gibco BRL). A DNA-DDAB (dimethyldioctadecyl-ammonium bromide) mixture was prepared as described by Han (1996) Nucleic Acid Res., 24:4362–4363. DDAB suspension (250 μg/ml) was mixed with M3 in a 1:2 ratio and left at room temperature for 5 min. A series of microfuge tubes was prepared, each containing 670 ng of cDNA (dib, spo, shd, sad, and phm; GFP cDNA was used as a control) in pUAST plasmid. DDAB/M3 mixture (140 μl per tube) was added to each microfuge tube and incubated at room temperature for 15 min. S2 cell suspension was prepared at 2.7×106 cells/ml and split into a titer plate. Immediately after plating, DDAB/M3/DNA mixtures were transferred to the wells. The S2 cells were incubated at 25° C. for 72 hours.
At 72 hours, the medium was aspirated and replaced with 1 ml/well of M3 medium with an appropriate substrate. Cells transfected with dib, sad, phm, and shd were incubated with tritiated 2,22-dideoxyecdysone, 2,22,25-trideoxyecdysone, 25-hydroxycholeserol, or ECD (200,000–300,000 cpm/well). Cells transfected with shd and spo were incubated with 10 μg of unlabelled cholesterol, desmosterol, sitosterol, or fucosterol. The incubation was stopped at 2 hours and 8 hours. The S2 cells were scraped from the wells and transferred to 2 ml cryogenic vials. Two additional washes were done with 400 μl PBS and 400 μl methanol, and the contents transferred to the vials, which were then frozen at −80° C.
Methanol extracts were analyzed by HPLC, and reaction products were identified based on their mobility characteristics relative to standards. CYP302a1, encoded by dib, can catalyze the conversion of 2,22-dideoxyecdysone to 2-deoxyecdysone. See FIG. 2. CYP315a1, encoded by sad, can convert 2,22-dideoxyecdysone to 22-deoxyecdysone. See FIG. 3. CYP315a1 can also convert 2-deoxyecdysone to ecdysone. The spo, shd and phm genes appear to encode enzymes that catalyze earlier steps in the ecdysteroid biosynthetic pathway.
Example 6
Screen for Inhibitors of CYP302a1
A Format 2 assay is used to identify inhibitors of CYP302a1. A substrate-specific antibody is immobilized in the wells of a 96-well titer plate, along with a tracer. The antibody binds 2,22-dideoxyecdysone and has a very low cross-reactivity for 2-deoxyecdysone. The tracer is synthesized from 2,22-dideoxyecdysone. For an EIA, the labeling enzyme (e.g., peroxidase) is linked to 2,22-dideoxyecdysone via the C3 or C6 functionalities. Drosophila S2 cells transfected with the dib gene are incubated with 2,22-dideoxyecdysone and various inhibitors in a separate 96-well titer plate. After the reaction is complete, the reaction mixture is heated to inactivate the CYP302a1 enzyme and the reactions are discretely transferred to the wells of antibody-coated titer plate. Following this incubation, the wells are aspirated and washed. In RIA, scintillant is added to the wells and antibody-bound radiolabeled tracer is measured. In EIA, following the washing step, substrate for the labeling enzyme is added to the antibody-bound tracer and following incubation, the product is quantified (e.g., by UV absorption for peroxidase conjugate tracers).
Example 7
Screen for Inhibitors of CYP315a1
A Format 1 assay is used to identify inhibitors of CYP315a1. A product-specific antibody, Horn H22, is immobilized in the wells of a 96-well titer plate. It is able to bind ecdysone and has a very low cross-reactivity for 2-deoxyecdysone. Drosophila S2 cells transfected with the sad gene are added to the antibody-coated wells. Various inhibitors are added to the wells for pre-incubation with the S2 cells, along with ecdysone tracer. For an EIA, a labeling enzyme (e.g., peroxidase) is linked via the side-chain at carbon 22 or carbon 26. Unlabeled 2-deoxyecdysone is then added to the wells. The wells are aspirated and washed. In RIA, scintillant is added to the wells and antibody-bound 3H-ECD is measured. In EIA, following the washing step, a substrate for the labeling enzyme is simply added to the antibody-bound tracer and following incubation, the product is quantified (e.g., by UV absorption for peroxidase conjugate tracers).
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention.







TABLE 3





Novel P450 Enzyme Amino Acid and cDNA sequences
















CYP302a1 protein









(SEQ ID NO: 1)









MLTKLLKISCTSRQCTFAKPYQAIPGPRGPFGMGNLYNYLPGIGSYSWLRLHQAGQDKYEKYGAIVRETIVPGQDIVWLYDPK



 


DIALLLNERDCPQRRSHLALAQYRKSRPDVYKTTGLLPTNGPEWWRIRAQVQKELSAPKSVRNFVRQVDGVTKEFIRFLQESR


 


NGGAIDMLPKLTRLNLELTSLLTFGARLQSFTAQEQDPSSRSTRLMDAAETTNSCILPTDQGLQLWRFLETPSFRKLSQAQSY


 


MEGVAMELVEENVRNCSVGSSLISAYVKNPELDRSDVVGTAADLLLAGIDTTSYASAFLLYHIARNPEVQQKLHEEAKRVLPS


 


AKDELSMDALRTDITYTRAVLKESLRLNPIAVGVGRILNQDAIFSGYFVPKGTTVVTQNMVRCRLEQHFQDPLRFQPDRWLQH


 


RSALNPYLVLPFGHGMRACIARRLAEQNMHILLLRLLREYELIWSGSDDEMGVKTLLINKPDAPVLIDLRLRRE


 


CYP302a1 cDNA








(SEQ ID NO:6)









CGTTGCTGTCGGAATTTGCCGCGAAAAGACCAGAGTAACGACGAAAAATGTTGACCAAACTGTTAAAGATTAGCTGCACCTCG



 


AGGCACTGCACCTTTGCCAAGCCGTATCAGGCGATACCAGGACCACGAGGACCCTTTGGAATGGGTAATCTATACAATTACCT


 


GCCCGGAATCGGATCCTATTCCTGGCTAAGATTGCACCAAGCCGGCCAGGATAAGTATGAGAAATATGGCGCAATTGTGCGGG


 


AAACTATAGTTCCTGGGCAGGACATTGTCTGGTTGTACGATCCCAAGGACATAGCTTTGCTGCTCAACCAGCCGGATTGTCCG


 


CAGCGAAGAAGTCACCTGCCACTGGCTCAATATCGCAAGAGCCGACCGGATGTCTATAAAACCACCGGCTTGCTGCCCACCAA


 


TGGTCCGGAATGGTGGCGTATACGTGCCCAGGTGCAAAAGGAGCTGAGTGCACCAAAGAGTGTGCGGAACTTCGTTCGCCAAG


 


TGGATGGAGTGACCAAGGAGTTCATTAGATTTCTACAAGAATCTCGCAATGGTGGTGCCATTGATATGCTGCCCAAGCTCACC


 


AGATTGAATTTGGAATTAACCTCTTTGCTTACCTTTGGAGCCCGTCTGCAGTCTTTTACTGCCCAGGAACAAGATCCTAGTTC


 


CCGATCCACTCGCTTGATGGATGCGGCCGAGACCACCAATAGCTGCATCCTGCCCACAGATCAGGGCCTCCAGCTGTGGCGAT


 


TTCTGGAGACACCTAGCTTTCGCAAACTAAGCCAGGCCCAATCATATATGGAGGGTGTGGCCATGGAGTTAGTGGAGGAGAAT


 


GTTAGGAATGGTTCAGTGGGATCTTCACTGATCTCGGCTTATGTAAAAAATCCCGAGCTTGATCGCACTGACGTGGTGGGCAC


 


CGCTGCAGATTTACTCTTGGCTGGCATCGATACCACTTCGTATGCCTCGGCATTTCTGCTCTATCACATAGCTCGAAATCCGG


 


AGGTGCAGCAAAAACTGCACGAGGAGGCCAAGAGAGTGCTTCCGAGTGCCAAGGACGAGCTATCCATGGATGCCCTACGAACT


 


GATATCACCTATACGAGGGCTGTCCTCAAGGAATCACTACGCTTGAATCCCATTGCCGTGGGCGTGGGCAGGATTCTTAATCA


 


GGATGCGATCTTCAGTGGCTACTTCGTGCCAAAGGGGACCACCGTGGTTACCCAGAACATGGTACGCTGCCGGCTGGAGCAGC


 


ACTTCCAGGATCCCCTGCGCTTCCAACCAGATCGATGGCTCCAGCACCGTAGTGCCCTCAATCCCTATCTGGTCCTTCCCTTC


 


GGTCACGGAATGCGGGCCTGCATTGCCCGCCGTTTGGCCGAGCAGAATATGCACATTTTGCTTCTCAGGCTGCTGCGTGAATA


 


CGAATTGATTTGGAGCGGATCCGATGATGAGATGGGTGTGAAGACCCTGTTGATAAATAAACCCGATGCTCCAGTGCTGATCG


 


ATCTGCGATTGCGTAGAGAATAAGGTTATTAGGTATAAGTAAGTCGCCAGAGCCTTAAGACTGAGATACTAGACTCGTGTCAC


 


GTTTAAATAATTCGTTTATTAATTATTTTAAATTAGCTCATAATTAATTATAATTAGTGTAAATTATATATAACTAGACTTGC


 


ATTTTATGTGATTGAAAGGCTGTGGGTGTTTTGGTCTAAGTACTAAAGAGAACGACTAAAAGGCAAAAAAAAAAAAAAAA


 


CYP306a1 protein








(SEQ ID NO:2)









MSADIVDIGHTGWMPSVQSLSILLVPGALVLVILYLCERQCNDLMGAPPPGPWGLPFLGYLPFLDARAPHKSLQKLAKRYGGI



 


FELKMGRVPTVVLSDAALVRDFFRRDVMTGRAPLYLTHGIMGGFGIICAQEDIWRHARRETIDWLKALGMTRRPGELRARLER


 


RIARGVDECVRLFDTEAKKSCASEVNPLPALHHSLGNIINDLVFGITYKRDDPDWLYLQRLQEEGVKLIGVSGVVNFLPWLRH


 


LPANVRNIRFLLEGKAKTHAIYDRIVEACGQRLKEKQKVFKELQEQKRLQRQLEKEQLRQSKEADPSQEQSEADEDDEESDEE


 


DTYEPECILEHFLAVRDTDSQLYCDDQLRHLLADLFGAGVDTSLATLRWFLLYLAREQRCQRRLHELLLPLGPSPTLEELEPL


 


AYLRACISETMRIRSVVPLGIPHGCKENFVVGDYFIKGGSMIVCSEWAIHMDPVAFPEPEEFRPERFLTADGAYQAPPQFIPF


 


SSGYRMCPGEEMARMILTLFTGRILRRFHLELPSGTEVDMAGESGITLTPTPHMLRFTKLPAVEMRHAPDGAVVQD


 


CYP306a1 cDNA








(SEQ ID NO:7)









ATGTCGGCGGACATCGTCGATATTGGCCACACCGGTTGGATGCCCTCGGTGCAGAGCCTGAGTATTCTGCTGGTTCCGGGTGC



 


GCTCGTCCTGGTGATTCTCTACCTGTGCGAGCGCCAGTGCAATGACCTCATGGGTGCCCCACCGCCGGGTCCCTGGGGCCTGC


 


CCTTTCTGGGTTACCTGCCCTTCCTGGACGCCCGTGCGCCGCACAAGTCACTCCAGAAGCTGGCCAAGCGGTATGGTGGAATT


 


TTCGAGCTGAAAATGGGCAGGGTGCCGACCGTAGTCCTCTCGGATGCCGCCTTGGTGCGGGATTTCTTTCGGCGCGATGTGAT


 


GACTGGCCGTGCGCCGCTCTACCTCACCCACGGCATCATGGGTGGATTTGGCATCATCTGCGCCCAGGAGGACATTTGGCGAC


 


ATGCACGGCGCGAGACTATCGATTGGCTAAAGGCCTTGGGCATGACCCGTCGGCCGGGGGAACTGCGCGCGCGGCTCGAGCGG


 


CGCATAGCCCGCGGAGTCGACGAGTGCGTACGGCTTTTCGATACTGAGGCAAAGAAGAGCTGTGCGTCGGAAGTGAATCCGCT


 


GCCGGCGCTCCATCACTCGCTGGGCAACATAATCAACGACCTGGTCTTCGGGATCACCTACAAGCGCGACGACCCCGACTGGC


 


TGTACCTGCAGCGGCTGCAGGAGGAGGGCGTCAAGCTGATTGGCGTCTCCGGGGTGGTCAACTTTTTGCCCTGGCTGCGTCAC


 


CTGCCCGCCAACGTGCGCAACATCCGCTTCCTGCTGGAGGGCAAGGCCAAGACGCACGCCATCTACGACCGCATTGTGGAGGC


 


CTGTGGCCAGCGGCTGAAGGAGAAGCAGAAGGTGTTCAAGGAGCTCCAGGAGCAGAAGCGGCTGCAAAGGCAGCTAGAGAAGG


 


AGCAGCTCAGGCAGTCAAAGGAAGCGGATCCAAGCCAGGAGCAGAGTGAGGCAGACGAGGATGACGAGGAGAGCGATGAGGAG


 


GACACGTACGAGCCGGAGTGCATCCTGGAGCACTTCCTAGCCGTTCGAGACACGGATTCGCAGCTCTACTGCGACGACCAGCT


 


GCGCCATCTGCTGGCCGATCTCTTTGGAGCCGGGGTGGACACCTCGCTGGCCACCCTGCGCTGGTTCCTGCTCTACTTGGCCC


 


GCGAACAACGCTGCCAGCGGCGCCTGCATGAGCTCCTCCTGCCGCTGGGTCCGTCTCCCACTTTGGAGGAACTGGAGCCGCTG


 


GCCTACCTAAGGGCTTGCATTTCCGAGACGATGCGCATACGCAGCGTTGTCCCACTGGGCATTCCGCACGGATGCAAAGAGAA


 


CTTCGTCGTGGGCGATTATTTTATCAAGGGTGGTTCGATGATCGTTTGCTCGGAGTGGGCTATCCACATGGACCCAGTGGCCT


 


TCCCGGAACCGGAGGAGTTCCGTCCGGAGCGCTTCTTGACCGCCGATGGAGCCTACCAGGCGCCGCCACAGTTCATCCCATTC


 


TCGTCCGGCTATCGAATGTGTCCCGGCGAAGAGATGGCTCGCATGATACTCACGCTCTTTACGGGTCGCATCCTCAGGCGCTT


 


CCACTTGGAACTGCCCTCGGGCACTGAGGTGGACATGGCGGGTGAGAGCGGCATCACCCTGACCCCCACTCCGCACATGCTGC


 


GATTCACCAAGCTGCCGGCGGTGGAGATGCGCCATGCACCCGACGGAGCTGTGGTGCAGGATTAG


 


CYP307a1 protein








(SEQ ID NO:3)









MLAALIYTILAILLSVLATSYICIIYGVKRRVLQPVKTKNSTEINHNAYQKYTQAPGPRPWPIIGNLHLLDRYRDSPFAGFTA



 


LAQQYGDIYSLTFGHTRCLVVNNLELIREVLNQNGKVMSGRPDFIRYHKLFGGERSNSLALCDWSQLQQKRRNLARRHCSPRE


 


SSCFYMKMSQIGCEEMEHWNRELGNQLVPGEPINIKHLILKACANMFSQYMCSLRFDYDDVDFQQIVQYFDEIFWEINQGHPL


 


DFLPWLYPFYQRHLNKIINWSSTIRGFIMERIIRHRELSVDLDEPDRDFTDALLKSLLEDKDVSRNTIIFMLEDFIGGHSAVG


 


NLVMLVLAYIAKNVDIGRRIQEEIDAITEEKNRSINLLDMNAMPYTMATIFEVLRYSSSPIVPHVATEDTVISGYGVTKGTIV


 


FINNYVLNTSEKFWVNPKEFNPLRFLEPSKEQSPKNSKGSDSGIESDNEKLQLKRNIPHFLPFSIGKRTCIGQNLVRGFGFLV


 


VVNVMQRYNISSHNPSTIKISPESLALPADCFPLVLTPREKIGPL


 


CYP307a1 cDNA








(SEQ ID NO:8)









AGTTGTGTTTTGTGCTTCCTACTTTCAAGAGCTCAGCAAAAATGCTGGCTGCTTTGATTTACACTATTTTGGCGATTTTACTG



 


AGTGTTCTGGCCACGTCCTACATATGCATTATATATGGAGTCAAGCGCCGCGTTCTGCAGCCCGTTAAAACAAAGAATTCAAC


 


CGAAATCAATCACAATGCTTATCAAAAATATACCCAGGCTCCAGGACCACGACCATGGCCCATCATTGGTAATCTTCATCTGC


 


TGGATCGATACAGGGATAGTCCCTTTGCGGGATTCACGGCGTTGGCACAGCAATACGGAGACATATACTCCCTGACCTTCGGA


 


CACACCCGCTGTCTGGTGGTGAACAACTTGGAGCTGATCCGCGAGGTGCTCAATCAAAATGGCAAGGTGATGAGCGGGCGGCC


 


AGACTTCATACGATATCATAAACTATTTGGTGGCGAGCGAAGCAATTCGTTGGCTCTGTGCGATTGGTCACAGCTGCAGCAGA


 


AGAGAAGGAATCTGGCCAGGCGTCACTGCTCGCCCAGGGAATCTTCCTGCTTCTACATGAAAATGTCCCAGATTGGTTGCGAG


 


GAAATGGAGCACTGGAATCGGGAGCTGGGAAACCAACTCGTTCCTCGAGAGCCGATCAACATCAAGCATCTGATTCTGAAGGC


 


CTGTGCAAATATGTTTAGTCAGTACATGTGTTCGTTGAGGTTCGACTACGATGATGTGGACTTCCAACAGATTGTTCAATACT


 


TCGATGAGATATTCTGGGAAATCAATCAGGGACATCCGCTGGATTTTCTACCCTGGCTATATCCCTTCTACCAGCGACACCTG


 


AACAAGATCATCAACTGGTCCTCGACTATCAGGGGATTCATAATGGAAAGGATTATCCGGCATCGGGAGCTGAGCGTCGATTT


 


GGATGAACCAGATCGGGACTTCACAGATGCTCTACTTAAAAGCCTGCTTGAAGATAAAGATGTCTCCCCGGCAACGATTATCT


 


TCATGCTCGAGGATTTCATTGGTGGACATTCAGCGGTTGGAAATCTAGTAATGCTAGTGCTGGCCTATATAGCCAAAAATGTG


 


GATATTGGAACGAGAATACAAGAGGAAATTGACCCAATTACTGAAGAGAAAAATAGGTCAATTAATTTGCTGGACATGAATGC


 


TATGCCCTACACGATCGCGACGATTTTCGAGGTGCTGCCATATTCATCCTCCCCAATTCTTCCACATGTGGCCACCGAGCACA


 


CAGTGATCTCTGGCTATGGGGTACCAAGGGCACCATTGTGTTCATCACAATTATGTGCTAAACACCAGCGAGAGAAATTCTCG


 


GTAAATCCCAAGGAATTTAATCCATTAAGATTTTTGGAACCGTCAAAGGAACAAAGCCCAAAAAATTCCAAAGGTTCTGATTC


 


TGGCATCGAAAGTGATAATGAAAAACTTCAACTAAAGAGCAATATTCCGCACTTTCTGCCCTTTAGCATCGCGAAGCGGACTT


 


GCATCGGCCACAATTTGGTGAGAGGATTTGGTTTTCTGGTCGTGGTCAACGTAATGCAGAGATATAATATCAGCAGTCATAAT


 


CCTTCGACGATTAAGATCAGTCCGGAGAGTTTGGCACTCCCTGCCGATTGTTTTCCATTGGTCTTGACACCCAGGGAAAAGAT


 


CGGACCACTATAATAAATTATAAATTAAAAACCAATACCATTAAGCACTAGCAATTAAAATATTACACATAAATAGCCCAAAC


 


AGCTTCAGAATAAGATTACTGCGTTATCTATTATCAGACATAACGAATAAGCTCAATCAAATAGTAAAACTATTTTACTGTTG


 


AGTATATTTTCAATTACTTAACGCAAATACAAAATTTTTGTATTTCATGTCTATATTTTGTACGATACCAAACACGCAAAGTC


 


TTATAGATGCTCACCACACTATAAAAATTTCACATACATTCGATTCTTGAGGATCTTAGGATTAGATTAATTCGTTAAGAACT


 


TCGTTACAGTAAATGACTCAAATATGTATAATGTGCACGCCGATGTAAAATTCAAGTGTAATTCTTAAGCGAAATATTTACAA


 


TTTTTATTTTCTTTTAGAATCAATAAATGTGGGCCCACATGGGCCTTATATAAATGACATAGAAACCTAAAGCTGAATAAAAT


 


CCAAAA


CYP314a1 protein








(SEQ ID NO:4)









MAVILLLALALVLGCYCALHRHKLDIYLLRPLLKNTLLEDFYHAELIQPEAPKRRRRGIWDIPGPKRIPFLGTKWIFLLFFRR



 


YKMTKLHEVYADLNRQYGDIVLEVMPSNVPIVHLYNRDDLEKVLKYPSKYPFRPPTEIIVMYRQSRPDRYASVGIVNEQGPMW


 


QRLRSSLTSSITSPRVLQNPLPALNAVCDDFIELLRARRDPDTLVVPNFEELANLMGLEAVCTLMLGRRMGFLAIDTKQPQKI


 


SQLAAAVKQLFISQRDSYYGLGLWKYFPTKTYRDFARAEDLIYDVISEIIDHELEELKKSAACEDDEAAGLRSIFLNILELKD


 


LDIRDKKSAIIDFIAAGIETLANTLLFVLSSVTGDPGAMPRILSEFCEYRDTNILQDALTNATYTKACIQESYRLRPTAFCLA


 


RILEEDMELSGYSLNAGTVVLCQNNIACHKDSNFQCAKQFTPERWIDPATENFTVNVDNASIVVPFGVGRRSCPGKRFVEMEV


 


VLLLAKMVLAFDVSFVKPLETEFEFLLAPKTPLSLRLSDRVF


 


CYP314a1 cDNA








(SEQ ID NO:9)









ATGGCCGTGATACTGTTGCTGGCCCTCGCACTCGTACTTGTCTGCTACTGCGCTCTCCATCCGCACAAATTGGCGGATATCTA



 


CCTCCGGCCGCTCCTGAAGAACACGCTCCTTGAGGACTTCTACCATGCCGAGCTGATCCAGCCCGAGGCGCCAAAGAGGCGCA


 


GGCGCCGCATCTCGGACATACCCGGGCCAAAGAGGATTCCCTTCCTGCCCACTAAGTGGATATTCCTCCTCTTCTTTCGACCG


 


TACAAGATGACCAAGCTGCACCAGGTATATGCGGATTTGAACAGACAATATGGGCACATAGTGCTGGAGCTGATCCCCTCCAA


 


TGTGCCAATAGTGCACCTGTACAATCGCGATGATCTGGAGAAGGTGCTGAAGTACCCCAGCAAATACCCATTCCGACCTCCCA


 


CCGAGATCATCGTGATGTACCGTCAGTCCCGACCGGATCGCTATGCAAGTGTTGGAATTGTGAATGAGCAAGGACCAATGTGG


 


CAGCGCCTACCATCTTCCCTGACCTCCAGCATTACTTCTCCCCGGGTCCTGCAGAATTTCCTGCCAGCCTTGAATGCGGTTTG


 


TCATCATTTTACCGAACTACTCCGAGCCAGGCGGGATCCGCATACACTGGTGGTTCCCAATTTCGAAGAGCTGGCCAATCTGA


 


TGGGTCTGGAAGCTGTGTGCACTTTAATGCTGGGCAGAAGGATGGGTTTCCTGGCTATCGATACCAAGCAGCCGCAAAACATA


 


AGCCAACTGGCAGCTGCTGTTAAACAGCTTTTCATATCCCAAAGGGACTCGTACTACGGTCTGGGTCTGTGGAAATACTTTCC


 


CACCAAAACCTACAGAGACTTTGCCCGCGCCGAGGACTTGATCTATGATGTGATCTCCGAGATCATCGATCATGAGCTGGAGG


 


AACTCAAAACGTCCGCTGCCTGCGAGGATGACGAGGCTGCTGGATTACGAAGTATCTTTCTGAATATTCTCGAGCTCAAGGAT


 


CTGGATATCAGCGACAAAAACTCAGCGATCATAGACTTTATTGCCGCTGGCATAGAAACGTTAGCCAACACTTTCTTCTTTGT


 


ACTGAGTTCTGTTACTGCACATCCCGGTGCTATGCCACCAATCCTAAGTGAATTCTGCGAGTATCGGGACACGAATATCCTGC


 


AGGATGCACTAACGAATGCCACATACACAAAGGCCTGTATACAGGAGTCCTACAGACTGAGGCCCACAGCCTTTTGCCTGCCC


 


AGAATCCTGGAGGAGGACATGGAGCTCTCGGGCTACTCGCTTAATGCACGGACTGTGGTGCTCTGTCAGAATATGATAGCCTG


 


CCACAAGGACAGCAACTTCCAAGGGGCCAAGCAGTTTACCCCAGAGCGTTGGATTGATCCTGCCACGCAGAATTTCACGGTGA


 


ACGTCGGATAATGCCACTATTGTGGTGCCCTTCGCAGTGGGTCGAGCCATCGTGTCCAGGAGCGTTTTGTGGAAATGGAGGTG


 


GTGCTGCTGCTAGCTAAGATGGTCCTAGCCTTTCATGTGAGCTTTGTGAAGCCACTGGAAACGGAGTTCGACTTCCTGCTGGC


 


ACCCAAAACTCCACTCAGTCTAAGACTCAGCCATCGGGTTTTCTGA


 


CYP315a1 protein








(SEQ ID NO:5)









MTEKRERPGPLRWLRHLLDQLLVRILSLSLFRSRCDPPPLQRFPATELPPAVAAKYPIPRVKGLPVVGTLVDLIAACGGATHL



 


HKYIDARHKQYGPIFRERLGGTQDAVFVSSANLMRGVFQHEGQYPQHPLPDAWTLYNQQHACQRGLFFMEGAEWLHNRRILNR


 


LLLNGNLNWMDVHIESCTRRMVDQWKRRTAEAAAIPLAESGEIRSYELPLLEQQLYRWSIEVLCCIMFGTSVLTCPKIQSSLD


 


YFTQIVHKVFEHSSRLMTFPPRLAQILRLPIWRDFEANVDEVLREGAAIIDHCIRVQEDQRRPHDEALYHRLQAADVPGDMIK


 


RIFVDLVTAAGDTTAFSSQWALFALSKEPRLQQRLAKERATNDSRLMHGLIKESLRLYPVAPFIGRYLPQDAQLGGHFIEKDT


 


MVLLSLYTAGRDPSHFEQPERVLPERWCIGETEQVHKSHGSLPFAIGQRSCIGRRVALKQLHSLLGRCAAQFEMSCLNEMPVD


 


SVLRMVTVPDRTLRLALRPRTE 


 


CYP315a1 cDNA








(SEQ ID NO:10)









GCGCGGAGTCTTCCAGCACGAGGGTCAGTATCCGCAGCATCCGCTGCCGGATGCCTGGACGCTGTATAACCAGCAACATGCCT



 


GCCAACGGGGACTGTTCTTCATGGAGGGCGCCGAGTGGCTGCACAACCGACGCATACTTAATCGACTGCTGCTCAACGGAAAT


 


TTGAATTGGATGGACGTGCATATTGAGAGCTGTACCACACGAATGGTGGATCAGTGGAAAAGACGCACTGCGGAGGCGGCGGC


 


GATTCCGCTAGCGGAGAGTGGTGAAATACGAAGCTACGAACTGCCCCTGTTGGAACAACAGCTCTACCGTTGGTCCATAGAAG


 


TTCTGTGCTGCATCATGTTTGGCACCAGCGTGCTCACCTGCCCCAAGATCCAGTCCTCGCTGGACTACTTCACGCAGATTGTG


 


CACAAGGTGTTTGAGCATAGCTCGCGACTGATGACATTCCCGCCTCGCTTGGCCCAGATTTTGCGCCTGCCCATCTGGCGGGA


 


TTTCGAGGCCAATGTGGATGAGGTGCTGCGTGAGGGAGCTGCCATAATCGATCACTGCATCAGAGTGCAGGAGGACCAAAGGA


 


GACCGCACGATGAGGCGCTTTACCATCGCCTCCAGGCGGCGGATGTGCCAGGCGATATGATCAAGCGGATATTTGTAGACTTG


 


GTCATTGCAGCAGGTGACACGACCGCATTCAGCAGTCAGTGGGCTTTGTTTGCCCTTTCAAAGGAGCCGAGGCTCCAGCAACG


 


ACTGGCCAAGGAGCGAGCTACCAATGATTCTCGCCTGATGCACGGCCTGATCAAGGAGTCCCTGCGTCTGTACCCCGTAGCTC


 


CCTTCATTGGCCGATATCTGCCGCAGGACGCGCAACTTGGCGGTCACTTTATCGAAAAGGATACCATGGTGCTGCTCTCCTTG


 


TACACGGCAGGTCGCGATCCATCACACTTTGAGCAGCCGGAACGTGTGCTCCCGGAGCGCTGGTGCATTGGAGAGACGGAGCA


 


GGTGCATAAGTCACACGGCAGTCTGCCTTTTGCCATCGGCCAGCGGTCTTGCATTGGTCGCCGTGTGGCACTCAAGCAGCTCC


 


ACTCCCTGCTGGGCCGATGTGCTGCTCAGTTTGAGATGAGCTGCCTTAACGAGATGCCCGTTGACAGCGTACTCCGCATGGTC


 


ACCGTGCCCGATCGGACTTTGCGTTTAGCCCTTCGGCCGCGAACCGAGTGA

## Claims

1. A method for identifying inhibitors of ecdysteroid synthesis, said method comprising:
contacting a purified ecdysteroid biosynthetic enzyme with a candidate inhibitor molecule; and
determining whether or not said molecule inhibits the activity of said enzyme,
wherein said enzyme has an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:4, wherein said enzyme exhibits ecdysteroid biosynthetic activity in the absence of said candidate inhibitor molecule.

2. The method of claim 1, wherein said determining step uses a product-specific antibody or a substrate-specific antibody.

3. The method of claim 2, wherein said product-specific antibody or said substrate-specific antibody is attached to a solid substrate.

4. The method of claim 3, wherein said solid substrate is a microtiter plate.