DE102004061879A1 - New polynucleotide encoding insect thioredoxin reductase, useful as a target for identification of insecticides, also new protein, vector and recombinant cells - Google Patents

Abstract

Isolated polynucleotide (I) comprising a sequence that encodes a polypeptide (II) of sequence (2) of 494 amino acids (aa), reproduced. Independent claims are also included for the following: (1) isolated polynucleotide (I) comprising a sequence with at least 75% identity with region 21-1505 of sequence (1), reproduced; (2) isolated polynucleotide (Ib) comprising a sequence that hybridizes under stringent conditions to the 21-1505 region of sequence (1); (3) recombinant vector containing (I), (Ia) or (Ib); (4) recombinant host cell that contains the vector of (3); (5) method for preparing an insect thioredoxin reductase (III) by culturing cells of (4); (6) purified protein having a sequence at least 80% identical with sequence (2); and (7) method for detecting an agent (X) that reduces the enzymatic activity of (III). ACTIVITY : Insecticide. No details of tests for insecticidal activity are given. MECHANISM OF ACTION : Thioredoxin reductase inhibitor.

Description

Territory of invention

The The invention relates to insecticidal enzymes, and more particularly to an insecticidal one Thioredoxin reductase.

background the invention

thioredoxin reductases (TrxRs) are members of the flavin-containing disulfide reductases and their function is to maintain the redox environment the cell. Defense against oxidative stress in mammals includes the regeneration of the main ones Thiol reducing agents glutathione and thioredoxin by glutathione reductase or thioredoxin reductase (TrxR). Drosophila has no glutathione reductase on; therefore, TrxR is the only enzyme that contains dithiols from Drosophila reduced in response to oxidative stress. Thioredoxinreduktaseenzyme are homodimeric proteins and they catalyze the transfer of electrons from NADPH to disulfide a FAD co-factor. TrxRs show broad substrate specificity.

pesticide development was conventionally on chemical and physical properties of the pesticide itself focused, which is a relatively time-consuming and expensive process. As a consequence, efforts were made focused on the modification of existing ones, well validated compounds instead of developing new pesticides. There is a need in the field of new pesticidal compounds, which are safer, more selective and more efficient than currently available Pesticides are. The present invention addresses this need the provision of new pesticide targets from invertebrates, such as. the "tobacco budworm "Heliothis virescens and by providing methods for identifying of compounds which bind to such targets or the activity of such Modulate targets.

literature

• Bauer et al., (2003) Eur J Biochem. 270 (21): 4272-81; Hillebrand et al., (2003) J. Biol. Chem. 278 (9): 6809-15; Fujiwara et al., (2001) J Biochem 129 (5): 803-12; Sun et al., (2001) J. Biol. Chem. 276 (5): 3106-14.

SUMMARY THE INVENTION

The The present invention provides nucleic acid molecules encoding an insect thioredoxin reductase as well as thioredoxin reductase polypeptides derived from these be encoded. The invention further provides methods for the identification of agents containing a level of thioredoxin reductase mRNA, polypeptide or level of enzyme activity. Such agents are candidates for insecticidal Links.

It it is an object of the invention to provide isolated insecticidal nucleic acid molecules and proteins, which targets for Pesticides are to provide. The isolated insect nucleic acid molecules, such as provided herein, are useful for the production of insect proteins, which are coded by them. Insect proteins are useful in assays to identify compounds that have a biological activity of the proteins which assays identify compounds which useful as pesticides can be. It is an object of the present invention to invertebrate genes Animals available to ask for Encode polypeptides involved in genetic screening procedures can be used to characterize mechanistic pathways into which such genes can be involved as well as other interactive genetic mechanistic pathways. It is a further object of the invention to provide methods for screening compounds that interact with a central enzyme of a invertebrate animal interact. Connections with a central Enzyme of an invertebrate animal can interact as therapeutics or pesticides.

Short description the drawings

1 represents the nucleotide sequence of Heliothis thioredoxin reductase cDNA (SEQ ID NO: 01).

2 represents the amino acid sequence of Heliothis thioredoxin reductase (SEQ ID NO: 02).

3A - 3C represent the nucleotide sequence of H. virescens N6HIS-TrxR-1 in an A2.2 expression vector (SEQ ID NO: 03).

4 represents the amino acid sequence of H. virescens N6HIS-TrxR-1 (2-494) (SEQ ID NO: 04).

DEFINITIONS

As used herein, the term "isolated" means that a polynucleotide is described, a polypeptide, an antibody or a host cell that is in an environment different from that in which the polynucleotide, polypeptide, antibody or host cell occur naturally. As used herein, refers to "Substantially purified" means a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that has been removed from its natural environment and is at least 60% free, 75% free or 90% free of other components with which it is naturally derived is associated.

The Designation "polynucleotide" and "nucleic acid molecule", which are interchangeable here used refer to polymeric forms of nucleotides in any length, either ribonucleotides or deoxynucleotides. Consequently, this term excludes but are not limited to single, double or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the polynucleotide can Sugars and phosphate groups (as typically found in RNA or DNA are found), or modified or substituted Sugar or phosphate groups. Alternatively, the backbone of the Polynucleotides comprise a polymer of synthetic subunits, such as. Phosphoramidites, and therefore may be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24: 2318-2323. A polynucleotide can be modified nucleotides include, e.g. methylated nucleotides and nucleotide analogs, Uracyl or sugar and linking Groups such as e.g. Fluororibose and thioate and nucleotide branches. The sequence of the nucleotides may be by non-nucleotide components be interrupted. A polynucleotide may further be after polymerization be modified, such. by conjugation with a labeled one Component. Other types of modifications included in this Definition are caps, substitutions of one or more of the naturally occurring ones Nucleotides with an analog and the introduction of agents for Attachment of polynucleotides to proteins, metal ions, labeling components, other polynucleotides or a solid support.

For hybridization probes may be desirable be, nucleic acid analogs to use for stability and the binding affinity to improve. A number of modifications have been described the chemistry of the phosphodiester backbone, the sugar or the prevents heterocyclic bases.

Under the full changes in the backgrass chemistry are phosphorothioates; Phosphorodithioate, where both of the non-bridging Oxygens are substituted with sulfur; phosphoramidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O-5'-S phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoramidate one. peptide nucleic acids replace the entire phosphodiester backbone with a peptide linkage.

sugar modifications are also used to improve stability or affinity. The α-anomer of Deoxyribose can be used where the base is inverted in the Regard to the natural β-anomer. The 2'-OH of the ribose sugar can changed to be 2'-O-methyl or 2'-O-allyl sugar which gives resistance to degradation without affecting affinity. The modification of the heterocyclic bases needs the correct base pair formation maintained. Some significant substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine one. 5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine could as the affinity and the biological activity be shown improving when they deoxythymidine or deoxycytidine substituted.

The Designation "polypeptide" and "protein" as here interchangeable used refers to a polymeric form of amino acids of any Length, which coded and non-coded amino acids lock in, chemically or biochemically modified or derivatized amino acids and Polypeptides having modified backbones. The Designation closes Including fusion proteins, including but not limited to fusion proteins with a heterologous Amino acid sequence Fusions with heterologous or homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins and the like.

A "host cell" as used here refers to microorganisms or eukaryotic cells or cell lines cultivated as unicellular Entities, which can be used as receiver for recombinant Vectors or other transfer polynucleotides or used accordingly have been and are closing the offspring of the original cell transfected one. It is understood that the descendants of a single cell not necessarily complete identical in morphology or in genomic or total DNA complement with the original parent cell, due to natural, random or desired Mutation. A "recombinant host cell" is a host cell, in which a desired Nucleic acid molecule or a desired recombinant vector has been introduced.

By "transformation" is a permanent or temporary genetic change indu decorated in a cell followed by introduction of a new DNA (ie DNA exogenous to the cell). Genetic alteration can be realized either by introducing the new DNA into the genome of the host cell or by temporarily or stably maintaining the new DNA as an episomal element. When the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introducing the DNA into the genome of the cell.

Before the present invention is further described, it should be understand that this invention is not limited to particular embodiments, as they are described, as these, of course, vary can. It should also be understood that the terminology used here only for the purpose of describing specific embodiments and not limiting, since the scope of the invention is limited only by the appended claims will be.

Where a range of values is provided, it should be understood that any intervening value, to the tenth of the unit of the lower limit, as long as the context does not clearly dictate something else, between the upper and lower limits of the range, as well as each specified or intervening value in the specified range within the Invention is included. The upper and lower limits of this smaller areas can independently be enclosed by each other in the smaller areas and are also within the invention, subject to any specifically excluded borders in the said area. Where the mentioned Range includes one or both of the boundaries, ranges are one of exclude both of these included limits, also in the invention locked in.

Solang not otherwise defined, all technical and scientific Terms used here have the same meaning as commonly used understood by those skilled in the art to which the invention belongs becomes. Although any procedures and materials that are similar or equivalent to those described here, also in the Practice or to test the present invention can, The preferred methods and materials are now described. All publications mentioned here are included by reference herein to the methods and / or materials to describe in their context the publications be quoted.

It It should be noted that as used in the appended claims Singular forms "a", "and" and "the" include plural references, as long as the context does not clearly indicate the opposite. Hence the term "pesticidal Agent "a variety of such agents, and the reference to "thioredoxin reductase" includes the reference to one or more several such enzymes and equivalents thereof, known to those skilled in the art, etc.

The publications discussed here are alone in terms of their Revelation provided before the filing date of the present application. Nothing here's a concession be constructed that the present invention is not authorized is to precede such a publication force of the previous invention. Furthermore you can the data of the provided publication is different from the current ones Publication data that may require independent verification.

DETAILED DESCRIPTION OF THE INVENTION

A cDNA coding for a full-length open The reading frame of a TrxR was obtained from a Heliothis virescens cDNA library amplified and completely sequenced.

The The present invention provides the insecticidal nucleic acid and Protein compositions of thioredoxin reductase ready, as well A method of identifying agents which cause the level of the insecticidal thioredoxin reductase mRNA, protein or degree the enzyme activity modulate.

ISOLATED NUCLEIC ACID MOLECULES OF THE INVENTION

The The invention provides isolated insecticidal nucleic acid molecules comprising nucleotide sequences the insecticidal thioredoxin reductase (also referred to herein as TrxR), in particular nucleic acid sequences of Lepidoptera TrxR and more specifically nucleic acid sequences of Heliothis virescens TrxR, as well as methods of using these nucleic acid molecules.

The The present invention provides isolated nucleic acid molecules comprising nucleotide sequences include the insecticidal proteins, which are potential pesticide targets represent, encode. The isolated nucleic acid molecules have a multiplicity of Uses on, for example, as hybridization probes, for example for the identification of nucleic acid molecules which nucleotide sequence identity share; in expression vectors, to the polypeptides which by encodes the nucleic acid molecules to produce; and a host cell or animal for use in assays as described below.

The Designation "isolated Nucleic acid sequence "as used herein includes the reverse complement, the RNA equivalent, single or double stranded DNA or RNA sequences and DNA / RNA hybrids of the sequences being described, unless otherwise stated.

1 and 2 For example, the nucleotide (SEQ ID NO: 01) and amino acid (SEQ ID NO: 02) sequences each represent a TrxR of Heliothis virescens.

In some embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a nucleotide sequence which at least about 50%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least about 97%, at least approximately 98%, at least approximately 99 or more nucleotide sequence identity with the sequence as shown in SEQ ID NO: 01. In some embodiments, an insecticidal comprises TrxR nucleic acid molecule, a nucleotide sequence, at least about 50%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least about 97%, at least approximately 98%, at least approximately 99% or more nucleotide sequence identity with the sequence as in Nucleotides 21-1505 of SEQ ID NO: 01. In other embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a nucleotide sequence the sequence as shown in SEQ ID NO: 01. In other embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a nucleotide sequence the sequence as shown in nucleotides 21-1505 of SEQ ID NO: 01.

In other embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a fragment of at least approximately 18, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least approximately 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, or at least about 1450 contiguous Nucleotides of nucleotides of the sequence as in nucleotides 21-1505 represented by SEQ ID NO: 01.

In other embodiments comprises a TrxR insecticidal nucleic acid molecule encoding a nucleotide sequence for a Polypeptide having an amino acid sequence includes, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90% or at least about 95% amino acid sequence identity with the amino acid sequence as shown in SEQ ID NO: 02. In some embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a nucleotide sequence which for a Polypeptide encoding the sequence as shown in SEQ ID NO: 02, includes. In many of these embodiments The encoded polypeptide has TrxR activity.

In other embodiments For example, a TrxR insecticidal nucleic acid molecule comprises a nucleotide sequence which for a Polypeptide encoding a fragment of at least about 6, at least approximately 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least approximately 200, at least about 225, at least about 250, at least about 275, at least approximately 300, at least about 350, at least about 400 or at least about 450 related amino acids the sequence as in SEQ ID NO: 02, to the entire length of the Amino acid sequence as shown in SEQ ID NO: 02. In many of these embodiments The encoded polypeptide has TrxR enzyme activity.

Fragments of the central nucleic acid molecules can be used for a number of purposes. The interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to create loss-of-function phenotypes or to formulate biopesticides (as further discussed below). The central nucleic acid fragments are also useful as nucleic acid hybridization probes and replication / amplification primers. Certain "antisense" fragments, ie those which are reverse complements of parts of the coding sequence of SEQ ID NO: 01, are useful for inhibiting the function of a central protein. The fragments are of a length sufficient to specifically hybridize to a nucleic acid molecule having the sequence as shown in SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 01). The fragments consist of or include at least 12, at least 24, at least 36, or at least 96 contiguous nucleotides of SEQ ID NO: 01 (or nucleotides 21-1505). When the fragments are flanked by other nucleic acid sequences, the total length of the combined nucleic acid sequence is less than 15 kb, less than 10 kb, less than 5 kb, or less than 2 kb.

The central nucleic acid sequences can only from SEQ ID NO: 01 or from fragments (e.g., the nucleotides 21-1505 of SEQ ID NO: 01) thereof. Alternatively, the central nucleic acid sequences and fragments thereof are linked to other components, such as. Labels, peptides, agents that transport across cell membranes allow away hybridization-triggered cleavage agents or intercalating Agents. The central nucleic acid sequences and fragments thereof also to other nucleic acid sequences linked be (i.e. they can Part of larger sequences be) and consist of synthetic / non-natural Sequences and / or are isolated and / or purified, i. no longer accompanied by at least some of the materials, with which they are in their natural environment are associated. In general, the isolated nucleic acids constitute at least approximately 0.5% or at least about 5% by weight of total nucleic acid, the is present in a given fraction and is usually recombinant, i. that she is a non-natural Sequence or a natural one Comprise sequence linked to nucleotide (s) which are different to those who are attached to it on a natural chromosome are.

derivatives of nucleic acid molecules of the central Nucleic acid molecules include sequences which is linked to the nucleic acid sequence of SEQ ID NO: 01, or to nucleic acid molecules containing the open reading frame of SEQ ID NO: 01 (e.g., nucleotides 21-1505 of SEQ ID NO: 01), under stringent conditions such that the nucleic acid hybridization derivative with the central nucleic acid is related by a certain degree of sequence identity. One nucleic acid molecule is "hybridizable" to another nucleic acid molecule, e.g. a cDNA, a genomic DNA, or RNA, if a single-stranded form of the nucleic acid molecule to the anneal other nucleic acid molecule can. The stringency of hybridization refers to the conditions, below which nucleic acids are hybridizable. The degree of stringency can be determined by temperature, Ionic strength, pH and the presence of denaturing agents, e.g. formamide, while hybridization and washing. Like here are used under the name "stringent hybridization conditions" those that are normally used by a person skilled in the art, by at least 90% sequence identity between the complementary pieces to achieve DNA or DNA and RNA. "Moderately stringent hybridization conditions" are used to find derivatives that have at least 70% sequence identity. After all become "low stringency Hybridization conditions "used to isolate derivatives of nucleic acid molecules, at least about 50% identity with the central nucleic acid sequence share.

The ultimate hybridization stringency reflects both the current ones Hybridization conditions as well as the washing conditions on follow the hybridization, and it is well known in the art, How the conditions must be varied to the desired To get results. Conditions that are commonly used are available in ready Procedure texts (for example, Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments is a nucleic acid molecule of the invention able to hybridize to a nucleic acid molecule, which contains a nucleotide sequence as shown in SEQ ID NO: 01 under stringent conditions, which include: prehybridization of filters containing nucleic acid for 8 hours to about Night at 65 ° C in a solution comprising 6X single strong citrate (SSC, single strength citrate) (1X SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0), 5X Denhardts Solution, 0.05% sodium pyrophosphate and 100 μg / ml herring sperm DNA; hybridization for 18-20 Hours at 65 ° C in a solution containing 6X SSC, 1X Denhardt's Solution, 100 μg / ml Yeast tRNA and 0.05% sodium pyrophosphate; and washing the filters at 65 ° C for 1 h in a solution containing 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate, sodium dodecyl sulfate).

Nucleic acid derivative sequences having at least about 70% identity with SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 01) are capable of hybridizing to a nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 01) to hybridize under moderately stringent conditions comprising pretreatment of the filters containing nucleic acid for 6 hours at 40 ° C in a solution containing 35% formamide , 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA and 500 μg / ml denatured salmon sperm DNA; Hybridization for 18-20 h at 40 ° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0 , 2% BSA, 100 μg / ml salmon sperm DNA and 10% (w / v) dextran sulfate; followed washing twice for 1 hour at 55 ° C in a solution containing 2X SSC and 0.1% SDS.

Other Exemplary Derivatnukleinsäuresequenzen are able to SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 01) under low stringency conditions which including: incubation for 8 hours to overnight at 37 ° C in a solution comprising 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate and 20 μg / ml sheared salmon sperm DNA; Hybridization in the same buffer for 18 up to 20 hours; Wash the filters in 1 x SSC at approximately 37 ° C for 1 hour.

As used here is the "percentage (%) Nucleic acid sequence identity "with respect to a central sequence or a special part of a central one Sequence defined as the percentage of nucleotides in question coming nucleic acid derivative sequence that is identical with the nucleotides of the central sequence (or a special Part of it) is, after adjustment of the two sequences and the introduction from gaps, if necessary, to achieve the maximum percent sequence identity as described by the program WU-BLAST-2.0a19 (Altschul et al., J. Biol. Biol. (1997) 215: 403-410; http://blast.wustl.edu/blast/README.html; generally referred to herein as "BLAST") with all the variable parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and will be through the program itself depending on the composition of the particular Sequence and the composition of the special database, against which the sequence of interest is sought established. A percentage (%) Value for the nucleic acid sequence identity is determined by the Number of matching identical nucleotides divided by the sequence length for which the percentage identity is determined.

In an exemplary embodiment coded the derivative of the nucleic acid for a Polypeptide having an amino acid sequence represented in SEQ ID NO: 02 or for a fragment or derivative thereof, as further described below. A derivative of a central Nucleic acid molecule or Fragments thereof may have 100% sequence identity with SEQ ID NO: 01 or the Nucleotides 21-1505 of SEQ ID NO: 01 include, but may be a derivative be in the sense that there are one or more modifications to the Base or sugar group or the phosphate backbone. Examples modifications are well known in the art (Bailey, Ullmann's Encyclopedia of Industrial Chemistry (1998), 6th Ed. Wiley and Sons). Such derivatives can be used be to modified stability or any other desired To give property.

As used here a "derivative" of a nucleic acid or amino acid sequence orthologous sequences of SEQ ID NO: 01 and SEQ ID NO: 02, which are of other species were obtained. In some embodiments, the ortholog is off a heliothin species, for example from Heliocoverpa armigera and Heliothis zea, which together with Heliothis virescens three the main worldwide grain pests are. Orthologous genes from these three species are extremely similar. (The International Meeting on Genomics of Lepidoptera, Lyon, France August 16-17, 2001; "International Lepidopteran Genome Project Proposal ", Rev. September 10, 2001; available at world wide web site ab.a.u-tokyo.ac.jp/lep-genome/.)

In In another example, it may be desirable to have a pesticidal To develop agent that specifically a non-heliothin insect species has the goal. In such a case, it can be most efficient develop biochemical screening assays (i.e., assays, to molecules Identify which protein target continues as below described to inhibit) which the orthologous protein of this Use insect. While orthologues in two species have essentially the same functions can have can differences in their protein structure properties, e.g. Interaction with affecting other proteins, linkage binding and stability. Therefore, the results of biochemical assays are the most significant for the specific protein that is used in the assay. As used here close Orthologist Nukleinsäuren- and polypeptide sequences.

Methods of identifying the orthologs in other species are known in the art. Usually, orthologues in different species retain the same function due to the presence of one or more protein motifs and / or three-dimensional structures. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as heliothis, may correspond to more genes (paralogues) in another. As used herein, the term includes "orthologous" paralogues. When sequence data is available for a particular species, orthologues are generally identified by sequence homology analysis, such as by BLAST analysis, using protein bait sequences. Sequences are confirmed to be a potential ortholog if the most appropriate sequence of the forward BLAST result reflects the original sequence in the backward BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95: 5849-5856, Huynen MA et al. , Genome Research (2000) 10: 1204-1210). Multiple sequence alignment programs such as CLUSTAL-W (Thompson JD et al., 1994, Nucleic Acids Res 22: 4673-4680) can be used to highlight conserved regions and / or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing many homologous sequences from different species (for example, retrieved by BLAST analysis), orthologous sequences from two species generally appear closest to each other on the pedigree with respect to all other sequences from these two species.

structural Convolutions or other analyzes of protein folding (e.g., using software from ProCeryon, Biosciences, Salzburg, Austria) can also identify potential orthologs. Nucleic acid hybridization method can also be used to find orthologous genes, for example if sequence data is not available are. Degenerate PCR and screening of cDNA or genomic DNA libraries are general methods for finding related gene sequences and are well known in the art (see, for example Sambrook et al. Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989; Dieffenbach C and Dveksler G (Eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, 1989). For example, methods for generating a cDNA library from an insect species of interest and scanning the library with partially homologous gene probes in Sambrook et al. described. A highly conserved part of the Heliothis TrxR coding sequence can be used as a probe. TrxR orthologous nucleic acids can bind to the nucleic acid of SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 1) hybridize to high, moderate or low stringency conditions.

To Amplification or isolation of a segment of a putative orthologue The segment can be cloned and sequenced by standard techniques be used as a probe to obtain a complete cDNA or isolate genomic clone. Alternatively, it is possible to have an EST project to initiate a database of sequence information for the species of interest. In other approaches, antibodies are used bind specifically known TrxR polypeptides for the isolation of Orthologists used (Harlow E and Lane D, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988, New York; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, New York).

Western film Blot analyzes can determine that a TrxR orthologue (i.e., an orthologous protein) in a crude extract of a tissue of a particular species. If the reactivity is examined, the sequence which the candidate Ortholog coded, by screening expression libraries, which represent the special species, be isolated. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gt11 as described in Sambrook et al. As soon as that in question upcoming orthologist (s) identified by any of these means is / are the candidate ortholog sequence as Bait (the "query") for the backward BLAST against the sequence of Heliothis or another species used in which TrxR nucleic acid and / or Polypeptide sequences were identified.

Insulation, Production and Expression of the Central Nucleic Acid Molecules The Central Nucleic Acid Molecules, Fragments or derivatives thereof can be obtained from a suitable cDNA library that is derived from any one of prepared suitable insect species was (including, but not limited to Drosophila and Heliothis). In many embodiments For example, a Lepidopteran species is used, e.g. a heliothin species. Where the central nucleic acid molecule from a Heliothin species can be any of a variety of field and laboratory strands of the various Heliothis species, including, however not limited to a Heliothis virescens, Heliothis maritima, Heliothis ononis, Heliothis peltigera, Heliothis phloxiphaga, Helicoverpa punctigera, Heliothis subflexa, Helicoverpa armigera and Helicoverpa zea.

A Expression library can be prepared using known methods be constructed. For example, mRNA can be isolated to cDNA ligated into a suitable expression vector, for expression in a host cell into which they are introduced should produce. Various screening assays can be used to select the gene or gene product (e.g., oligonucleotides of at least about 20 to 80 bases designed to identify the desired gene or labeled antibodies, which bind specifically to the gene product). The gene and / or gene product can then be removed from the host cell using known techniques be recovered.

A polymerase chain reaction (PCR) can also be used to isolate a central nucleic acid molecule where oligonucleotide primers representing fragmentary sequences of interest amplify RNA or DNA sequences from a source, such as a genomic or cDNA library (as described in U.S. Pat Sam brook et al., supra). In addition, degenerate primers can be used to amplify homologs from any species of interest. Once a PCR product of appropriate size and sequence is obtained, it can be cloned and sequenced by standard techniques and used as a probe to isolate a complete cDNA or genomic clone.

fragmentary Sequences of the central nucleic acid molecules and derivatives of it can under Using known methods are synthesized. For example can Oligonucleotides using an automated DNA synthesizer available commercial operators (eg Biosearch, Novato, CA; Perkin-Elmer Applied Biosystems, Foster City, Calif.) become. Antisense RNA sequences can be transmitted intracellularly Transcription of an exogenous sequence are produced, for example from vectors that you want Antisense nucleic acid sequences contain. Newly generated sequences can be generated using standard techniques be identified and isolated.

One isolated central nucleic acid molecule can be found in any suitable cloning vector, for example in bacteriophage, e.g. Lambda derivatives, or plasmids such as e.g. pBR322, pUC plasmid derivatives and the Bluescript vector (Stratagene, San Diego, CA). Recombinant molecules can be transformed into host cells via transformation, Transfection, infection, electroporation, etc. are introduced or in transgenic animals, e.g. a fly. The transformed Cells can be cultivated to great Generate quantities of the desired nucleic acid. Suitable methods for isolating and producing the desired nucleic acid sequences are well known in the art (Sambrook et al., supra; DNA Cloning: A Practical Approach, Vol. 1, 2, 3, 4 (1995) Glover, ed., MRL Press, Ltd., Oxford, U.K.).

The Nucleotide sequence that is a desired protein or fragment or derivative thereof may be expressed in any expression vector suitable for the Transcription and translation of the introduced protein-coding Sequence are introduced. Alternatively, the necessary transcriptional and translational signals through the natural central gene and / or whose flanking regions are supplied. A variety of Host vector systems can be used to encode protein-coding Sequence to express (for example, mammalian cell systems infected with virus (for example vaccine virus, adenovirus, etc.); Insect cell systems infected with virus (for example baculovirus); microorganisms such as. Yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. The expression a central protein can be controlled by a suitable promoter / enhancer element become. About that In addition, a host cell strain can be selected that expresses the expression the inserted one Sequence is modulated, or modified, and the gene product specifically desired Processed way. Exemplary host cells include E. coli, Lepidoptera Sf-9 or S-21 cells and Drosophila S2 cells.

Around the expression of a desired Gene product can be detected, the expression vector, a promoter which is operably linked to a desired nucleic acid molecule, one or more replication origins and one or more selectable markers (for example, thymidine kinase activity, resistance against antibiotics etc.). Alternatively, recombinant expression vectors by testing on the expression of a central gene product based on the physical or functional properties of a central Protein in in vitro assay systems (eg immunoassays) be identified.

A central protein, fragment or derivative may optionally be expressed as a fusion or chimeric protein product (ie, it is linked via a peptide bond to a heterologous protein sequence or another, ie, non-TrxR protein). In one embodiment, the central protein is expressed as a fusion protein with a "tag", which facilitates purification, such as glutathione-S-transferase or (His). ₆ A chimeric product may be prepared by ligation of the appropriate nucleic acid sequences encoding desired amino acid sequences together in the appropriate coding frame using standard techniques and expression of the chimeric product. A chimeric product can also be produced by protein synthesis techniques, for example, by the use of a peptide synthesizer.

As soon as a recombinant vector expressing a central nucleic acid molecule is identified, the encoded central polypeptide can be isolated and using standard methods (e.g. Ion exchange, affinity and gel exclusion chromatography; centrifugation; differential solubility; Electrophoresis). The amino acid sequence of the protein can be from the nucleic acid sequence derived from the recombinant nucleic acid molecule which is contained in the recombinant vector and can therefore by standard chemical methods (Hunkapiller et al., Nature (1984) 310: 105-111) be synthesized. Alternatively, native desired proteins from natural Sources are purified by standard methods (for example Immunoaffinity).

Recombinant VECTORS AND HOST CELLS

Also provided are constructs ("recombinant vectors") containing the central ¹ nucleic acids incorporated into a vector, as well as host cells comprising these constructs. The central constructs are used for a number of different applications, including duplication, protein production, etc. Viral and non-viral vectors can be prepared and used, including plasmids. The choice of plasmid will depend on the type of cell in which the duplication is desired and the purpose of the amplification. Certain vectors are important for amplification and for generating large quantities of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal. The choice of the appropriate vector is well within the expertise. Many such vectors are commercially available.

Around making the constructs becomes the partial or full length polynucleotide inserted into a vector, typically with the aid of DNA ligase binding to a restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence may be homologous recombination can be introduced in vivo. typically, this is done by attaching homologous regions to the vector at the Flanks of the desired Achieved nucleotide sequences. Regions of homology are through ligation of oligonucleotides added or by polymerase chain reaction using primers, for example, both the region of homology and a Section of the desired Nucleotide sequence.

Also are provided expression cassettes or systems that - in addition other applications - application in the synthesis of central proteins. To the expression becomes the gene product encoded by a polynucleotide of the invention in any suitable expression system, including, for example bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells are described in U.S. Patent No. 5,654,173. In the expression vector becomes a The term "central" stands in the following for the English. Term "subject" TrxR-encoding polynucleotide, for example, as in SEQ ID NO: 01 (or nucleotides 21-1505 of SEQ ID NO: 01), operatively linked to a regulatory Sequence linked, which is suitable, the desired expression properties to obtain. these can Promoters (attached either at the 5 'end of the sense strand or at the 3' end of the antisense strand), Include enhancers, terminators, operators, repressors and inducers. The Promoters can be regulated or constitutive. In some situations it can desirable be conditioned, active promoters such. tissue-specific or to use development stage-specific promoters. These be to the desired Nucleotide sequence linked using the techniques described above for linking Vectors. Any techniques known in the art are, can be used. In other words, the expression vector becomes a transcriptional and translational initiation region, which Inducible or constitutive may be where the coding region operatively linked under the transcriptional control of the transcriptional initiation region and provide a transcriptional and translational termination region. These control regions can Naturally for the be central TrxR gene, or may be off be derived from exogenous sources.

expression vectors usually have suitable Restriction sites localized near the promoter sequence the insertion of nucleic acid sequences, to encode the heterologous proteins. A selectable Marker operable in the expression host may be present and although for the detection of host cells containing the recombinant vector include. A variety of markers are known and can be found in the vector, such markers include those that confer antibiotic resistance, for example resistance to Ampicillin, tetracycline, chloramphenicol, kanamycin, neomycin; such as Markers which allow histochemical detection, etc. expression vectors can et al For the production of central proteins, central fusion proteins, such as described above, and in screening assays, as described above, be used.

expression cassettes can which comprise a transcription initiation region, the gene or fragment thereof and a transcriptional termination region. Of particular interest is the use of sequences involving the Allow expression of functional epitopes or domains, usually at least about 8 amino acids long, more usually at least about 15 amino acids long, until about 25 amino acids and until the complete open reading frame of the gene. After the introduction of the DNA, the Cells containing the construct, using a selectable Markers selected become; the cells are expanded and then used for expression.

The expression systems described above can be used with prokaryotes or with eukaryotes in accordance with conventional routes, depending on the purpose of expression. For large scale production of proteins or for use in screening assays as described herein, a unicellular organism such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors or cells from higher organisms, such as vertebrates, for example, COS 7 cells, HEK 293, CHO, Xenopus oocytes, Lepidoptera Sf-9 or S-21 cells, Drosophila S2 cells, as host cells for expression. In some situations, it is desirable to express the gene in eukaryotic cells, whereby the expression protein will benefit from the natural folding and post-translational modifications. Small peptides can also be synthesized in the laboratory. Polypeptides that are subgroups of the complete protein sequence can be used to identify and study portions of the protein that are important to the function.

Specific expression systems of interest include bacterial, yeast, insect and mammalian cell-derived systems. Representative systems of each of these categories are provided below:
Bacteria. Expression systems in bacteria include those described in: Chang et al., Nature (1978) 275: 615; Goeddel et al., Nature (1979) 281: 544; Goeddel et al., Nucleic Acids Res. (1980) 8: 4057; EP 0 036 776 ; U.S. Patent No. 4,551,433; DeBerer et al., Proc. Natl. Acad. Sci. (USA) (1983) 80: 21-25; and Siebenlist et al., Cell (1980) 20: 269.
Yeasts. Expression systems in yeast include those described in: Hinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75: 1929; Ito et al., J. Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol. (1986) 6: 142; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202: 302; Das et al., J. Bacteriol. (1984) 158: 1165; De Louvencourt et al., J. Bacteriol. (1983) 154: 737; Van den Berg et al., Bio / Technology (1990) 8: 135; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Cregg et al., Mol. Cell. Biol. (1985) 5: 3376; U.S. Patent Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300: 706; Davidow et al., Curr. Genet. (1985) 10: 380; Gaillardin et al., Curr. Genet. (1985) 10:49; Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112: 284-289; Tilburn et al., Gene (1983) 26: 205-221; Yelton et al., Proc. Natl. Acad. Sci. (USA) (1984) 81: 1470-1474; Kelly and Hynes, EMBO J. (1985) 4: 475479; EP 0 244,234 and WO 91/00357.
Insect cells. Expression of heterologous genes in insects is achieved as described in: U.S. Patent No. 4,745,051; Friesen et al., "The Regulation of Baculovirus Gene Expression," in: The Molecular Biology of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839 ; EP 0 155,476 ; and Vlak et al., J.Gen. Virol. (1988) 69: 765-776; Miller et al., Ann. Rev. Microbiol. (1988) 42: 177; Carbonell et al., Gene (1988) 73: 409; Maeda et al., Nature (1985) 315: 592-594; Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82: 8844; Miyajima et al., Gene (1987) 58: 273; and Martin et al., DNA (1988) 7: 99. Various baculoviral strands and variants and corresponding permissive insect host cells of hosts are described in: Luckow et al., Bio / Technology (1988) 6: 47-55, Miller et al., Generic Engineering (1986) 8: 277-279 and Maeda et al., Nature (1985) 315: 592-594. Various insect cells, including Lepidoptera Sf-9 cells and S-21 cells as well as Drosophila S2 cells, have been extensively described in the art. See, for example, "Insect Cell Culture Engineering", Goosen, Daugulis and Faulkner, eds. (1993) Marcel Dekker.
Mammalian cells. Mammalian expression is achieved as described in Dijkema et al., EMBO J. (1985) 4: 761, Gorman et al., Proc. Natl. Acad. Sci. (U.S.A.) (1982) 79: 6777, Boshart et al., Cell (1985) 41: 521 and U.S. Patent No. 4,399,216. Other features of mammalian expression are realized as described in Ham and Wallace, Meth. Enz. (1979) 58: 44, Bames and Sato, Anal. Biochem. (1980) 102: 255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195 and US RE 30,985.

Plant cells. Plant cell culture has been clearly described in various publications including For example, Plant Cell Culture: A Practical Approach, (1995). R.A. Dixon and R.A. Gonzales, ed., IRL Press; and U.S. Patent No. 6,069,009.

To the production of the expression vector becomes the expression vector introduced into a suitable host cell for the production of the central polypeptide, i.e. a host cell is transformed with the expression vector. The introduction of the recombinant vector into a host cell becomes in any favorable Way, including but not limited to calcium phosphate precipitation, electroporation, Microinjection, the use of lipids (for example Lipofectin), Infection and the like.

If any of the above-mentioned host cells or other suitable host cells or organisms used to produce the polynucleotides or nucleic acids of the Invention to replicate and / or express, is the resulting and replicated nucleic acid, RNA, the expressed protein or polypeptide as a product of Host cell or organism within the scope of the invention. The product is made by any suitable means in the state the technique is known, recovered.

The invention further provides recombinant host cells as described above containing a central recombinant vector comprising a central TrxR nucleic acid molecule, for example as part of a recombinant vector, either extrachromosomally or integrated into the genome of the host cell. Recombinant host cells are generally isolated but may also be part of a multicellular organism, such as a transgenic animal. Thus, the invention further provides transgenic non-human animals, especially insects, which comprise a central TrxR nucleic acid molecule.

The central nucleic acid molecules can be used become transgenic, non-human animals or to produce plants, or site-specific gene modifications in cell lines. Transgenic animals and plants can be homologous by recombination where the endogenous site is altered. Alternatively it will a nucleic acid construct fortuitously integrated into the genome. Vectors for stable integration include plasmids, Retroviruses and other animal viruses, YACs and the like. transgenic Insects are significant for Screening assays as described below. The trans genesis of Insects have been described, for example, in "Insect Transgenesis: Methods and Applications" Handler and James, eds. (2000) CRC Press.

ISOLATED POLYPEPTIDES OF THE INVENTION

The The invention further provides isolated polypeptides which an amino acid sequence of SEQ ID NO: 02 or fragments, variants or derivatives (for example Orthologues) include or consist thereof. compositions comprising any of these proteins can be essential from a central one Protein, fragments or derivatives exist or may be additional Components include (for example, pharmaceutically acceptable carriers or excipients, Culture media, carriers used in pesticide formulations, etc.).

One Derivative of a central protein typically shares a special one Degree of sequence identity or sequence similarity with SEQ ID NO: 02 or a fragment thereof. As used here the percent (%) Amino acid sequence identity "with regard to a central sequence, or a special section of a central one Sequence, defined as the percentage of amino acids in the candidate derivative amino acid sequence, identical to the amino acids in the central sequence (or a special section of it) is, after adjusting the sequences and inserting Gaps, if necessary to achieve the maximum percent sequence identity, as demonstrated by BLAST (Altschul et al., supra) using the same parameters as discussed above for derivatives of nucleic acid sequences is produced. A% value for the Amino acid sequence identity is determined by the number of matching identical amino acid sequences divided by the sequence length, for which the percentage identity is specified.

"Percent (%) amino acid sequence similarity" is determined by execution the same calculation as for the determination of the% amino acid sequence identity, wherein but conservative amino acid substitutions additionally to the identical amino acids included in the calculation. A conservative amino acid sequence is one in which an amino acid sequence instead of another amino acid sequence is substituted, the similar Exhibits properties, so that the folding or the activity of the protein not changed significantly become. Aromatic amino acids that substitute each other, are phenylalanine, tryptophan and tyrosine; exchangeable hydrophobic amino acids are leucine, isoleucine, methionine and valine; exchangeable polar amino acids are glutamine and asparagine; are exchangeable basic amino acids Arginine, lysine and histidine; exchangeable acidic amino acids are aspartic acid and glutamic acid; and are exchangeable small amino acids Alanine, serine, threonine, cysteine and glycine.

In some embodiments shares a central protein variant or derivative at least about 70%, at least about 75%, at least 80% of the sequence identity or similarity, at least 85%, at least 90%, at least approximately 95%, at least about 97%, at least approximately 98% or at least about 99% sequence identity or similarity with a coherent one Section of at least 25 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, at least 350 amino acids at least 400 amino acids or at least 450 amino acids and in some cases with the entire length of SEQ ID NO: 02. In some embodiments, a polypeptide comprises invention an amino acid sequence such as shown in SEQ ID NO: 02.

In some embodiments, a TrxR polypeptide of the invention comprises a fragment of at least about 6, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 400, or at least about 450 contiguous amino acids of the sequence shown in SEQ ID NO: 02, to the full length of the sequence shown in SEQ ID NO: 02. In many versions The TrxR polypeptide has TrxR enzyme activity.

The Fragment or derivative of a central protein is preferably "functionally active," meaning that the central protein derivative or fragment one or more functional activities associated with a central full-length, wild-type protein, comprising the amino acid sequence of SEQ ID NO: 02. As an example, a fragment or derivative antigenicity so that it can be used in immunoassays, for immunization, for inhibition the activity a central protein, etc. can be used as further described below with respect to the generation of antibodies to central proteins becomes. In many embodiments is a functionally active fragment or derivative of a central Protein one that associates one or more biological activities with a central protein, e.g. catalytic activity. For the here close to existing purposes functionally active fragments include those fragments which have a or show more structural features of a central protein, such as. Transmembrane or calcium ion channel domains. Protein domains can be identified are using the PFAM program (see for example Bateman A., et al., Nucleic Acids Res, 1999, 27: 260-2; and the world wide web at pham.wustl.edu).

The functional activity The central proteins, derivatives and fragments can be differentiated by different Methods that are known in the art are examined (Current Protocols in Protein Science (1998) Coligan et al., Eds. John Wiley & Sons, Inc., Somerset, New Jersey). Enzymatic assays, such as the assays, described in examples or variants thereof, are for use suitable. In the assay described in the Examples is the NADPH dependent Reduction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) spectrophotometrically measured by TrxR after the formation of the product thionitrobenzoic (TNB). Thus, in many embodiments, the assays involve training of TNB, which by measuring the absorbance at 410 nm below Using a spectrophotometer is followed.

A non-limiting example of a suitable assay is an assay which gives an increase in absorbance at 410 nm, which results from the TrxR-mediated reduction of DTNB in an NADPH-dependent reaction yielding two moles of TNB and one mole of NADP. TNB absorbs a maximum of 410 m and has an extinction coefficient of 13600 M ^-1 cm ^-1 . Reduction of DTNB by TrxR-1 is measured by determining the enzyme catalyzed increase in absorbance at 410 nm. As a non-limiting example of such an assay, the following components are added in the following order: a) containing 5 μl of a solution 10 × a compound in 5% dimethyl sulfoxide (DMSO) (v / v); b) 20 μl of a solution containing 0.1 M potassium phosphate (Kp _i ), pH 7.4, 0.5 mmol ethylenediamine tetraacetic acid (EDTA), 0.625 mmol dihydronicotinamide adenine dinucleotide phosphate (NADPH), 0.05 mmol dithiothreitol (DTT), 0.005 mmol Flavin adenine dinucleotide (FAD) and 0.022 mg / ml TrxR; and c) 25 μl of a solution containing 0.1 mmol Kp _i , pH 7.4, 0.5 mmol EDTA, 2 mmol 5,5'-dithiobis (2-nitrobenzoic acid (DTNB), 0.1 mg / ml bovine serum albumin ( BSA), 0.2% (v / v) dimethyl sulfoxide (DMSO), 0.02% (w / v) Tween 20. The resulting reaction mixture is kept at room temperature (22 ° -24 ° C) for 30 minutes. The reduction of DTNB by TrxR is detected by measuring the absorbance at 410 nm.

The Central proteins and polypeptides may be naturally occurring Sources are obtained or produced synthetically. For example can Wild-type proteins can be obtained from biological sources which expressing the proteins, for example from Heliothis. The central Proteins can also be obtained from synthetic agents, for example by Expressing a recombinant gene that is the protein of interest in a suitable host as described above. Any suitable protein purification procedures can be used where appropriate Protein purification methods in the guidelines for protein purification (Deuthser ed.) (Academic Press, 1990). For example can be a lysate from the original source prepared and purified by HPLC using exclusion chromatography, Gel electrophoresis, affinity chromatography and the same.

A derivative of a central protein can be produced by various methods known in the art. The manipulations that result in their production may appear at the level of the gene or protein. For example, a cloned central gene sequence can be cut at appropriate sites with restriction endonuclease (s) (Wells et al., Philos, Trans Soc R. London SerA (1986) 317: 415), followed by further enzymatic modification, if desired isolated and ligated in vitro, and expressed to produce the desired derivative. Alternatively, a central gene can be mutated in vitro or in vivo to create and / or destroy translation, initiation and / or termination sequences, or to create variations in coding regions and / or to form or add previously existing endonuclease restriction sites destroy to allow further in vitro modifications union. A variety of mutagenesis techniques are well known in the art such as chemical mutagenesis, in vitro site-directed mutagenesis (Carter et al, Nucl Acids Res (1986) 13:... 4331), use of TAB ^® linkers (available from Pharmacia and Upjohn, Kalamazoo, MI), etc.

At the protein level, manipulations include post-translational modification, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, coupling to antibody molecules or other cellular ligands, etc. Any of the many chemical modifications may be carried out by known techniques (for example, specific chemical cutting by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH _4, acetylation, Formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.) , Derivative proteins can also be synthesized chemically using a peptide synthesizer to introduce, for example, nonclassic amino acid or chemical amino acid analogs as substitutions or additions into the central protein sequence.

Chimera or Fusion proteins can which are a central protein or a fragment of which (preferably comprising one or more structural or functional domains the central protein) bound to its amino or carboxyl terminus via a Peptide bond to an amino acid sequence of a different protein. chimera Proteins can produced by any known method including: recombinant Expression of a nucleic acid encoding for the Protein (comprising an amino acid sequence encoding a central protein linked in the frame with a coding Sequence of another protein); Ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences into each other in a suitable coding frame, and expressing of the chimera product; and protein synthesis techniques, for example, by use a peptide synthesis automaton.

Generegulatory ELEMENTS OF CENTRAL NUCLEIC ACID MOLECULES

The The invention further comprises gene regulatory DNA elements, such as e.g. Enhancers or promoters that direct the transcription of the central nucleic acid molecules. Such regulatory elements can used to identify tissues, cells, genes and factors that are specific control the production of a central protein. Analyze the components specific for a specific protein function are, can for understanding to lead, how to manipulate these regulatory processes, specifically for pesticides and therapeutic applications, as well as understanding how dysfunction in these Procedures to diagnose.

gene fusions with central regulatory elements can be realized. For compact genes, having relatively few and small intervening sequences, such as. this one for Heliothis described, it is typically the case that the regulatory Elements, which are spatial and control temporal expression patterns, in the DNA directly upstream be found to the coding region and in the extreme vicinity of the neighboring Genes extend. Regulatory regions can be used to gene fusions to construct where the regulatory DNAs are operatively linked to a coding Region of a reporter protein are fused, its expression easy to detect, and these constructs are called transgenes introduced into the animal of choice.

A full regulatory DNA region may be used or the regulatory region can be divided into smaller segments to identify subelements specific for the Control expression in a given cell type or stage of development. Reporter proteins necessary for the construction of this Gene fusions can be used shut down E. coli beta-galactosidase and Green Fluorescent Protein (GFP) one. these can are easily detected in situ and thus are important for histological studies and can used to sort cells which are a central protein express (O'Kane and Gehring PNAS (1987) 84 (24): 9123-9127; Chalfie et al., Science (1994) 263: 802-805; and Cumberledge and Krasnov (1994) Methods in Cell Biology 44: 143-159). Recombinase proteins, e.g. FLP or CRE can in the control of gene expression by teine, e.g. FLP or CRE can in the control of gene expression by site-specific recombination (Golic and Lindquist (1989) Cell 59 (3): 499-509; White et al., Science (1996) 271: 805-807). Toxic proteins, like e.g. Reaper and hid-cell-death proteins are important to cells to maim, which normally express a central protein to the physiological function of cells (Kingston, In Current Protocols in Molecular Biology (1998) Ausubel et al., John Wiley & Sons, Inc. sections 12.0.3-12.10) or any other protein where desired, the function of this special protein specifically in cells that are a key protein synthesize, investigate.

Alternatively, a binary reporter system may be used, similar to that described below, where a central regulatory element is operatively fused to the coding region of an exogenous transcriptional activator protein, such as the GAL4 or tTA activators described below, to create a central regulatory element ("driver gene"). For the other half of the binary system, the exogenous activator directs a separate "target gene" operatively fused to a coding region of a reporter protein to a related regulatory element for the exogenous activator protein, such as a UAS _G and tTA response element, respectively. An advantage of a binary system is that a single driver gene construct can be used to activate the transcription of preconstructed target genes encoding various reporter proteins, each with its own uses as discussed above.

headquarters Regulatory element reporter gene fusions are also significant in testing genetic interactions where the task is to identify those genes that have a special role in the Have control of the expression of the central genes, or Growth and differentiation of tissues, which is a key protein express, promote. Central gene regulatory DNA elements are also important in protein-DNA binding assays to gene regulatory proteins to identify those that control the expression of central genes. The gene regulatory proteins can be detected under Use of a variety of methods involving specific protein-DNA interactions which are well known to those skilled in the art (Kingston, supra), including In vivo footprinting assays based on the protection of DNA sequences based on chemical and enzymatic modifications, within living or permeabilized cells; and in vitro footprinting Assays based on the protection of DNA sequences from chemical or enzymatic Modifications using protein extracts, nitrocellulose filter binding assays and gel electrophoresis mobility shift assays are based on using radioactively labeled regulatory DNA elements mixed with protein extracts. Candidate genes for regulatory proteins can be purified using a combination of conventional and DNA Affinity Purification Techniques. molecular Cloning strategies can also be used to identify the proteins that are specific bind central gene regulatory DNA elements. For example, can a Drosophila cDNA library in an expression vector for cDNAs which encode central gene regulatory element DNA binding activity. In a similar way and way, the yeast "one-hybrid" system can be used (Li and Herskowitz, Science (1993) 262: 1870-1874; Luo et al., Biotechniques (1996) 20 (4): 564-568; Vidal et al., Proc. Natl. Acad. Sci. USA (1996) 93 (19): 10315-10320).

ANTIBODY SPECIFIC FOR CENTRAL PROTEINS

The The present invention provides antibodies that isolated antibody could be, specifically bind to a central protein. The central proteins, Fragments and derivatives thereof can used as an immunogen to monoclonal or polyclonal antibody and antibody yields or Generate derivatives (e.g., chimeric, single-stranded, Fab fragments). As used here the term "antibody" antibody of any kind Isotypes, fragments of antibodies which maintaining specific binding to antigen, including, however not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized Antibody, stranded antibody and fusion proteins containing an antigen-binding portion of a antibody and a non-antibody protein include. Also provided are "artificial" antibodies, for example Antibodies and Antibody fragments made and selected in vitro. In some embodiments become such antibodies on the surface of a bacteriophage or other viral particle. In many embodiments are such artificial antibodies as Fusion proteins with a viral or bacteriophage structural protein present, including, but not limited to the M13 gene III protein. Procedure for Producing such artificial antibody are well known in the art. See for example US patent Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988; 5,498,538; 5,403,484; 5,571,698 and 5,625,033.

The antibodies may be detectably labeled, for example with a radioisotope, an enzyme which produces a detectable product, a green fluorescent protein or the like. The antibodies may further be conjugated to other groups, for example members of specific binding pairs, for example biotin (member of the biotin-avidin-specific binding pair) and the like. The antibodies may also be bound to a solid support including but not limited to polystyrene plates or beads and the like. For example, fragments of a central protein, for example those identified as hydrophilic, are used as immunogens for antibody production using methods known in the art, such as hybridomas; the production of monoclonal antibodies in germ-free animals (PCT / US90 / 02545); the use of human hybridomas (Cole et al., Proc Natl Acad Sci USA (1983) 80: 2026-2030; Cole et al., Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss. Pages 77-96), and the production of humanized antibodies (Jones et al., Nature (1986) 321: 522-525, U.S. Patent 5,530,101). In a particular embodiment, central polypeptide fragments provide specific antigens and / or immunogens, especially when coupled to carrier proteins. For example, peptides are covalently linked to the keyhole limpet antigen (KLH) and the conjugate is emulsified in Freund's complete adjuvant. Laboratory animals, for example mice, rats or rabbits are immunized according to conventional protocol and blood is collected. The presence of specific antibodies is checked by solid phase immunosorbent assays using immobilized corresponding polypeptides. Specific activity or function of the antibodies produced may be determined by conventional in vitro, cell-based or in vivo assays, for example, in vitro binding assays, etc. The binding affinity can be tested by determining the equilibrium constants of antigen-antibody associations (usually at least about 10 ⁷ M ^-1 , at least about 10 ⁸ M ^-1 or at least about 10 ⁹ M ^-1 ).

SCREENING PROCEDURES

The The present invention further provides methods for identifying of agents that exhibit activity of a central TrxR polypeptide reduce the levels of TrxR mRNA and / or polypeptide levels reduce in a cell, especially an insect cell. The Invention further provides methods for identifying molecules which interact with a central TrxR.

Method of identification of molecules, which interact with a central protein

A Variety of methods can be used to identify molecules or to screen, e.g. Proteins or other molecules that interact with a central protein or derivatives of the fragments from that. The assays can purified protein or cell lines or model organisms such as e.g. Heliothis, Drosophila and C. elegans which are genetically were manipulated to express a central protein. Suitable screening methodologies are well known in the art for testing proteins or other molecules, which interact with a central gene and protein (see For example, International PCT Publication No. WO 96/34099). The newly identified interacting molecules can be new targets for pharmaceutical or pesticidal agents. Irgenwelche of the variety the exogenous molecules, both natural occur and / or are synthetic (for example, libraries of small molecules or peptides, or phage display libraries) can be screened for the binding activity. In a typical binding experiment, a central protein or Fragment with candidate molecules under Conditions that are favorable for the Binding are mixed, and sufficient time is allowed Thus, any binding can occur and assays are performed to test the bound complexes.

assays To find interacting proteins can be performed by any known methods known in the art For example, immunoprecipitation with an antibody which the protein binds in a complex, followed by size fractionation analysis the immunoprecipitated Proteins (for example, denaturing or non-denaturing Polyacrylamide gel electrophoresis), Western blot analysis, non-denaturing Gel electrophoresis, two-hybrid systems (Fields and Song, Nature (1989) 340: 245-246; U.S. Pat. No. 5,283,173; for review see Brent and Finley, Annu. Rev. Genet. (1977) 31: 663-704), Etc.

immunoassays

immunoassays can used to identify proteins that have a central protein interact or bind to it. Various assays are available for Testing the ability a protein to bind to a central wild type protein or to compete with the bond or to bind to one against the central protein-targeted antibodies. Suitable assays include radioimmunoassays, ELI-SA (Enzyme Linked Immuno Sorbent Assay), immunoradiometric assays, gel diffusion precipitin reactions, Immunodiffusion assays, in situ immunoassays (e.g. Use of colloidal gold, enzyme or radioisotoplates), Western blots, precipitation reactions, Agglutination assays (for example, gel agglutination assays, hemagglutination assays), Complement fixation assays fixation assays, immunofluorescence assays, Protein A assays, immunoelectrophoresis assays, etc.

One or more of the molecules in the immunoassay may be attached to a label, which label may directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescent chemiluminescent substances, enzymes, specific binding molecules, particles such as magnetic particles and the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary members would normally be labeled with a molecule that matches the detection ensured with known procedures.

identification of possible Pesticide or drug targets

The The present invention further provides methods for identifying of agents having an enzymatic activity of a reduce central TrxR, which is the level of TrxR mRNA and / or Reduce polypeptide in a cell, especially an insect cell.

As soon as identify new target genes or genes interacting with a target can, can they as potential pesticide or drug targets or as biopesticides be rated. Furthermore you can transgenic plants expressing central proteins with regard to their activity against insect pests (Estruch et al., Nat. Biotechnol (1997) 15 (2): 137-141).

The central proteins are validated pesticide targets, since the destruction of the central genes in Drosophila are deadly ends when they are homozygous. The mutation to lethality of this Gene implies that drugs that act as agonists or antagonists act on the gene product, may be effective pesticidal agents.

As As used herein, the term "pesticide" generally refers to chemical, biological agents and other compounds that negatively affect insect viability influence, for example, kill, paralyze, sterilize or otherwise pest species in the field of agricultural crop protection or human and animal health. Exemplary pest species shut down Parasites and disease vectors, e.g. Mosquitoes, fleas, ticks, parasitic Nematodes, Sand fleas, mites etc., a. pest species shut down even those that are aesthetic or hygienic reasons eradicated (for example, ants, cockroaches, clothing moths, Mildew, etc.), for reasons home and garden applications and the protection of structures (including forest damaging pests such as. Termites, and surface-growing Marine organisms).

pesticidal Connections can traditionally include small organic molecular pesticides (typified by classes of compounds such as e.g. Organophosphates, pyrethroids, Carbamates, and organochlorine compounds, benzoylureas, etc.). Close other pesticides proteinaceous toxins, e.g. Bacillus thuringiensis Crytoxins (Gill et al., Annu Rev Entomol (1992) 37: 615-636) and Photorabdus luminescent toxins (Bowden et al., Science (1998) 280: 2129-2132); and nucleic acids, such as. central dsRNA or antisense nucleic acids that affect the activity of a central nucleic acid molecule influence take one.

The Terms "candidate agent", "agent", "substance" and "compound" are used here to each other used interchangeably. Candidate agents include various chemical classes, typically synthetic, semisynthetic or of course occurring inorganic or organic molecules. Candidate agents can be small be organic compounds that have a molecular weight of more than 50 and less than about 2500 daltons. Candidate agents may include functional groups, the necessary for the structural interaction with proteins are, in particular Hydrogen bonds and can at least one amine, carbonyl, hydroxyl or carboxyl group and include can contain at least two of the functional chemical groups. The Candidate agent may be cyclic hydrocarbons or heterocyclic Structures include and / or aromatic or polyaromatic structures substituted with one or more of the above functional Groups. Candidate agents are also found among the biomolecules inclusively Peptides, saccharides, fatty acids, Steroids, purines, pyrimidines, derivatives, structural analogs or combinations from that.

Candidate agents be of a big one Obtained variety of sources, including libraries of synthetic or natural Links. For example, various means are available for random or directed syntheses of a large Variety of organic compounds and biomolecules including the Expression of randomized oligonucleotides and oligopeptides. alternative are libraries of natural Compounds in the form of bacterial, fungal, plant and animal Extracts available or are easily produced. In addition, natural or synthetic produced libraries or compounds slightly modified by conventional chemical, physical and biochemical agents and can used to generate combinatorial libraries. Known pharmacological agents can directed or random subjected to chemical modifications, e.g. acylation, Alkylation, esterification, amidation, etc., to structural analogs to produce.

Candidate agents which reduce the enzyme activity of a central polypeptide and / or reduce a level of TrxR mRNA and / or polypeptide by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more are candidate pesticides.

Candidate agents the the enzyme activity reduce a central TrxR polypeptide and / or a level In addition, TrxR reduce mRNA and / or polypeptide beyond in terms of toxicity tested against vertebrate species, e.g. Mammalian species, etc .; and regarding Bioavailability.

A A variety of other reagents may be included in the screening assay be. Close this Reagents such as salts, neutral proteins, e.g. Albumin, detergents etc., one that can be used to optimize protein-protein binding to enable and / or nonspecific or background interactions reduce. Reagents that improve the efficiency of the assay, such as. Protease inhibitors, nuclease inhibitors, antimicrobial Agents, etc., can be used. The components are in any order admitted that needed the activity provides. Incubations will be at any suitable temperature carried out, typically between 4 ° C and 40 ° C. Incubation periods are selected for optimal Activity, but can be optimized to enable fast high-throughput screening. Typically, a time between 0.1 and 1 hour will be sufficient.

Assays of Links against upgraded TrxR

The This invention provides methods for screening for agents that the enzymatic activity modulate a central TrxR. Such agents are considered pesticidal Agents significant.

The The present invention provides methods for identifying agents providing an enzymatic activity of a TrxR polypeptide of the invention modulate. The term "modulate" includes a Magnification or a reduction in the measurement of TrxR activity in comparison to a suitable control.

The Method generally comprises: a) contacting a test agent with a sample containing a TrxR polypeptide; and b) Check for an activity TrxR activity of the TrxR polypeptide in the presence of the test agent. An increase or a reduction in TrxR activity compared to TrxR activity in the suitable control (e.g., a sample comprising a TrxR polypeptide in the absence of an agent to be tested) indicates that the Agent an enzymatic activity of the TrxR modulated.

An "agent, this is a TrxR activity of a TrxR polypeptide ", as used herein, any molecule, e.g. a synthetic or natural organic or inorganic compound, a protein or a pharmaceutical which the ability have the TrxR activity a TrxR polypeptide as described herein. In general a variety of mixtures is enforced in parallel, with different Concentrations to give a differentiated response to different concentrations to obtain. Typically, one of these concentrations serves as a negative control, i. it is at a zero concentration or below the detection limit.

Assays of Compounds against insects

potential insecticidal compounds can insects are administered in a variety of ways, including orally (including adding to a synthetic diet, applied to plants or prey consumed by the test organisms) topically (including spraying, direct Applying the compound to the animal, enabling the animal's contact with a treated surface) or by injection. Insecticides are typically very hydrophobic Molecules and have to commonly in organic solvents disbanded which are evaporated in the case of methanol or acetone leaves, or which may be present in low concentrations to facilitate the intake (ethanol, dimethyl sulfoxide).

Of the The first step in an insect assay is usually the determination the minimum lethal dose (MLD, the minimum lethal dose) of an insect chronic exposure to the compound. The connections become common diluted in DMSO and applied to the food surface, which carries 0-48 hour old embryos and larvae. In addition to the determination of MLD This step allows the determination of the fraction evolving Eggs, the behavior of the larvae, for example, how they move, eat compared to untreated larvae, the fraction that survived until pupation, and the fraction that hatches (To survive of the adult insect from the pupation stage). Based on these results can more detailed assays with shorter exposure time be designed, and the larvae can be dissected, after to look for obvious morphological defects. Once the MLD is determined specific acute and chronic assays are designed.

In a typical acute assay, the compounds are applied to the food surface for embryos, larvae or adults, and the animals become after 2 hours and after incubation observed overnight. For use on embryos, defects in development and the percentage that survives until adulthood are determined. Defects in behavior, mobility and pupation are observed for larvae. For adult use, defects in levels and / or enzyme activity are observed and effects on behavior and / or fertility are recorded.

For one Chronic exposure assay will be adults for 48 hours placed on vials containing the compound, then on one transferred to clean container and in terms of fertility, Defects in levels and / or activity of a central enzyme and watched for death.

Assays of Compounds using cell cultures

Links, the the activity of a central protein (e.g., block or amplify) and / or can also modulate a level of TrxR mRNA or polypeptide be tested using cell cultures. exemplary Cells are cultured insect cells, e.g. Drosophila S2 cells. In some embodiments a recombinant vector containing a sequence that contains all or one Part of a central TrxR, in cells in an in vitro culture and the resulting recombinant host cells used to screen test agents. For example, different Added compounds to cells that express a central protein, and can on their ability be screened, the activity to modulate the central genes based on the measurement of biological activity a central protein. For example, connections can be screened be on their ability the activity of TrxR genes based on measurements of TrxR enzyme activity, TrxR mRNA levels or TrxR polypeptide levels.

assays to change in a biological activity of a central protein performed on cultured cells which are the endogenous normal or mutant central protein express. Such studies can also be carried out on cells that are transfected with vectors that are capable are to express the central protein or functional domains of a the central proteins in normal or mutated form. Furthermore can Cells to amplify the signal measured in such assays are co-transfected be with nucleic acid molecules or a central recombinant vector encoding a central protein.

alternative can Cells that express a central protein are lysed; the Central protein can be purified and used in vitro of methods known in the art are tested (Kanemaki M., et al., J Biol Chem, (1999) 274: 22437-22444).

A size Variety of cell-based assays can be used to agents which modulate the level of TrxR mRNA Identification of agents containing the level of TrxR polypeptide modulate and identify agents that determine the degree of TrxR enzyme activity modulate in a eukaryotic cell, for example, under Use of an insect cell (for example Drosophila S2 cells) transformed with a construct containing a TrxR-encoding cDNA comprises, so that the cDNA is expressed or, alternatively, a Construct comprising a TrxR promoter operatively attached to a Reporter gene is bound.

Accordingly The present invention provides a method for identifying an agent, in particular a biologically active agent, which modulates the level of TrxR expression in a cell, wherein the method comprising: combining a candidate agent, to be tested with a cell comprising a nucleic acid, which encodes a TrxR polypeptide; and determining the effect of said agent on the TrxR expression (for example, determination of the Effects of the agent on a level of TrxR mRNA, a level of TrxR polypeptide or a degree of TrxR enzyme activity in the Cell).

The "modulation" of TrxR expression levels includes the rise of the mirror and the decrease in the level of TrxR mRNA and / or TrxR polypeptide encoded by the TrxR polynucleotide and / or the level of TrxR activity compared to a control sample, which does not have the agent to test for. An increase or a reduction of about 1.25x, usually at least about 1.5 times, usually at least about 2-fold, usually at least about 5 times, usually at least about 10 times or more in the level (i.e., amount) of TrxR mRNA and / or polypeptide and / or TrxR enzyme activity as a result of the contact the cell with a candidate agent to be tested compared to a control sample to which no agent is added is an indication that that the agent TrxR modulates the mRNA level, the TrxR polypeptide level or the degree of TrxR activity in the Cell. Of particular interest in many embodiments are candidate agents, reduce a level of TrxR mRNA and / or a level reduce TrxR polypeptide and / or a level of TrxR enzyme activity in one Reduce insect cell.

TrxR mRNA and / or polypeptide, their levels or activity measured can, through an endogenous TrxR polynucleotide or the TrxR polynucleotide can be encoded one that is included in a recombinant vector and in which Cell inserted is, i. the TrxR mRNA and / or the polypeptide can be encoded its by an exogenous TrxR polynucleotide. For example, can a recombinant vector isolated TrxR transcriptional regulatory Sequence, e.g. a promoter sequence that is operative with linked to a reporter gene is (for example, β-galactosidase, CAT, Luciferase or another gene whose product is easily tested can). In these embodiments includes the method for identifying an agent that has a Modulated levels of TrxR expression in a cell, the following: Combine a candidate agent to be tested with a cell comprising a nucleic acid which is a transcriptional regulatory element of a TrxR gene operatively linked to a reporter gene connected is; and determining the effect of said agent on reporter gene expression.

One Recombinant vector may be an isolated TrxR transcriptional regulatory sequence, e.g. a promoter sequence, operatively linked coding for sequences for a TrxR polypeptide; or the transcriptional control sequences may be operative connected be with coding sequences for a TrxR fusion protein comprising a TrxR polypeptide fused to a polypeptide which enables detection. In these embodiments The method comprises combining a candidate agent which to be tested with a cell comprising a nucleic acid, which is a transcriptional regulatory element of a TrxR gene comprising operatively linked to the TrxR polypeptide coding sequence connected is; and determining the effect of said agent on TrxR expression, where the determination is carried out can be measured by measuring an amount of TrxR mRNA, TrxR polypeptide, TrxR fusion polypeptide or TrxR enzyme activity generated by the cell.

cell-based Assays generally include the steps of contacting the cell with an agent to be tested, the training a test sample and after appropriate time the evaluation of the effect the agent on TrxR mRNA levels, TrxR polypeptide levels and / or Activity level. A control sample includes the same cell without the added one Candidate agent. TrxR expression levels are present in both the test sample as well as in the control sample. A comparison between the TrxR expression levels in the test sample and in the control sample is carried out. TrxR expression can be tested using conventional assays. For example, when a cell line is transformed with a construct, the resulted in the expression of TrxR, detected TrxR mRNA levels and as well as TrxR polypeptide levels and / or TrxR enzyme levels can be detected and be measured. An appropriate period for contacting of the agent can be determined empirically and is in general a time sufficient to prevent the agent from entering the To enable cell and to allow the agent to have a measurable effect on TrxR has mRNA and / or polypeptide levels and / or on the enzyme activity. In general is a suitable time between 10 minutes and 24 hours, more typically approximately 1-8 hours.

method for measuring TrxR mRNA levels are known in the art, some thereof have been described above, and any of these methods can be used in The methods of the present invention can be used to Agent to identify which TrxR mRNA levels in a cell modulated, including but not limited to PCR, e.g. a PCR that is detectable labeled oligonucleotide primer, and any of the plurality the hybridization assays. In similar Way you can TrxR polypeptide levels are measured using any one Standard methods, some of which have been described here, however not limited to an immunoassay, e.g. an ELISA, the a detectably labeled antibody specific for a TrxR polypeptide starts. TrxR enzyme activity can be as described above or as described in the examples be measured.

Links, which selectively modulate a level of a central TrxR-encoding nucleic acid molecule, or selectively modulating a level of a central protein or which selectively modulate a degree of TrxR enzyme activity identified as potential pesticide and drug candidates, the specificity for the have central protein. Whether a candidate compound selective modulates a level of a central TrxR-encoding nucleic acid molecule or selectively modulates a level of a central protein or can selectively modulate the level of TrxR enzyme activity by measuring the level of an mRNA or a protein, for example from GAPDH or other suitable control proteins or mRNAs, wherein a candidate agent is "selective" if it is not Substantially the production or the activity of any protein or inhibits a mRNA different from a TrxR protein or a TrxR-encoding mRNA.

The identification of small molecules and compounds as potential pesticides or Pharmaceutical compounds from large chemical libraries require high throughput screening (HTS) procedures (Bolger, Drug Discovery Today (1999) 4: 251-253). Some of the assays used herein may offer themselves for such screening procedures. For example, cells or cell lines expressing the wild-type or mutant central protein or a fragment thereof and a reporter gene can be exposed to compounds of interest, and depending on the reporter gene, interactions can be measured using a variety of methods, eg, color detection, fluorescence detection (eg, GFP), autoradiography, scintillation analysis, etc.

links identified using the methods described above are significant to pests to curb, and are useful, for example, as pesticides. Such compounds can harmful contain, for example, by reducing the growth of the pests and / or their fertility and / or their survivability.

CENTRAL NUCLEIC ACIDS ALS biopesticides

headquarters nucleic acids and fragments thereof, e.g. Antisense sequences or double-stranded RNA (dsRNA) used to inhibit central nucleic acid molecules in their function and can consequently be used as biopesticides. Method of use of dsRNA interference are reported in the published PCT application WO 99/32619. Biopesticides may be the nucleic acid molecules themselves, an expression construct capable of expressing the nucleic acid, or organisms transfected with the expression construct. The biopesticides can be applied directly to plant parts or to the soil, which surrounds the plants (for example, to reach plant parts, the grow below ground level), or directly on the pests.

One Approach well known in the art is short interfering RNA (siRNA) mediated gene silencing, whereby the expression products of a TrxR gene through specific double-stranded TrxR-derived siRNA nucleotide sequences be addressed, which is complementary to at least one 19-25 Nucleotide long segment (for example, a 20-21 nucleotide long Sequence) of the TrxR gene transcript including the 5 'untranslated (UT) Region, the ORF or the 3 'UT region, are. In some embodiments For example, short interfering RNAs are about 19-25 nucleotides long. Please refer For example, PCT applications WO 0/44895, WO 99/32619, WO 01/75164, WO 01/92513, WO 01/29058, WO 01/89304, WO 02/16620 and WO 02/29858 for descriptions the siRNA technology.

biopesticides comprising a central nucleic acid can are prepared in any suitable vector for introduction into a plant or an animal. To produce plants that the central nucleic acids express, close suitable vectors Agrobacterium tumefaciens Ti plasmid-based Vectors (Horsch et al., Science (1984) 233: 496-89; Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80: 4803), and recombinant cauliflower Mosaic Virus (Hohn et al., 1982, In Molecular Biology of Plant Tumors, Academic Press, New York, pp. 549-560; U.S. Patent No. 4,407,956 to Howell). Retrovirus-based vectors are important for introduction of genes in vertebrates (Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90: 8033-37).

transgenic Insects can can be generated using a transgene comprising a central one Gen operatively fused to a suitable inducible promoter. For example, a tTA-controlled promoter can be used to the expression of a central protein at an appropriate time in a life cycle of an insect. In this way can test efficiency as an insecticide for example in the Larval phase of the life cycle (i.e., when the for the Emte causes). Vectors for introducing genes into insects include P element (Rubin and Spradling, Science (1982) 218: 348-53; U.S. Patent No. 4,670,388), "Hermes" (O'Brochta et al., Genetics (1996) 142: 907-914), "minos" (U.S. Patent No. 5,348,874), "marine" (Robertson, Insect Physiol. (1995) 41: 99-105) and "sleeping beauty" (Ivics et al., Cell (1997) 91 (4): 501-510), "piggyBac" (Thibault et al., Insect Mol Biol (1999) 8 (1): 119-23) and "hobo" (Atkinson et al., Proc. Natl. Acad. Sci USA (1993) 90: 9693-9697). recombinant Virus systems for the expression of toxic proteins in infected Insect cells are well known and include the Semliki Forest Virus a (DiCiommo and Brenner, J. Biol. Chem. (1998) 273: 18060-66), the recombinant Sindbis virus (Higgs et al., Insect Mol. Biol. (1995) 4: 97-103; Seabaugh et al., Virology (1998) 243: 99-112), the recombinant pantropic retrovirus (Matsubara et al., Proc. Natl. Acad. Sci. USA (1996) 93: 6181-85; Jordan et al., Insect Mol. Biol. (1998) 7: 215-22) and the recombinant baculovirus (Cory and Bishop, Mol. Biotechnol. (1997) 7 (3): 303-13; U.S. Patent No. 5,470,735; US Patent Nos. 5,352,451; US patent No. 5,770,192; U.S. Patent No. 5,759,809; U.S. Patent No. 5,665,349; and U.S. Patent No. 5,554,592).

The following examples are shown in order to provide those skilled in the art with a complete disclosure and description of how to practice and use the present invention, and are not intended to limit the scope of what the inventors contemplate as their invention, nor is it intended the experiments below represent all or the only experiments that have been performed. Efforts have been made to ensure consistency in terms of numbers used (eg, amounts, temperature, etc.), but some experimental errors and deviations should be expected. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius and pressure is at or near atmospheric pressure. Standard abbreviations may be used, for example bp for base pair (s); kb for kilobase (n); pl for picoliter (s); s for second (s); min for minute (s); hr per hour (s); aa for amino acid (s); kg for kilobases (n); bp for base pair (s); nt for nucleotide (s); and the same.

Example 1: Cloning a cDNA encoding Heliothis TrxR

A cDNA encoding a full-length open reading frame of a TrxR was amplified from a Heliothis virescens cDNA library and sequenced in its entirety. The cDNA sequence is in 1 shown; the amino acid sequence coding for TrxR is in 2 shown.

eight full-length Heliothis virescens thioredoxin reductase-1 (TrxR-1) cDNAs were from pooled larvae amplified cDNAs from the head and thorax and subcloned. All clones were prepared using standard procedures sequenced. The most abundant full-length TrxR-1 cDNA (4328.HvirTrxR-1.wt.pCRll.clone # 4) that was obtained had one 1482bp ORF and encoded a polypeptide of 494aa. The predicted H. virescens TrxR-1 translation product is 66% identical to the predicted Drosophila melanogaster protein (gi | 10953879) and represents the orthologous sequence. 4328.HvirTrxR1.wt.pCRII.clone # 4 was in the sequence used for protein expression.

• Primersatz #1: (BamHI)
• Fwd: 5'-[Phosp]gatccGCACCTATTGATGGAAACTTTGAC (SEQ ID NO:05)
• Rev: 5'-cGCTGCAGCAGGAGGCG (SEQ ID NO:06)

• Primersatz #2 (EcoRI)
• Fwd: 5'-cGCACCTATTGATGGAAACTTTGAC (SEQ ID NO:07)
• Rev: 5'-[Phosp]aattcGCTGCAGCAGGAGGCG (SEQ ID NO:08)

Recombinant H. virescens full-length TrxR-1 (aa 2-494) was expressed as a protein in a frame with an N-terminal 6HIS tag in E. coli [NTH-TEV-TrxR-1 (A2-S494)]. The TrxR-1 ORF (aa 2-494) was amplified with primer sets as below and fused in a frame with N6HIS separated by a linker region encoding a TEV site and having a BamHI cloning site.

• Primer set # 1: (BamHI)
• Fwd: 5 '- [Phosp] gatccGCACCTATTGATGGAAACTTTGAC (SEQ ID NO: 05)
• Rev: 5'-cGCTGCAGCAGGAGGCG (SEQ ID NO: 06)

• Primer set # 2 (EcoRI)
• Fwd: 5'-cGCACCTATTGATGGAAACTTTGAC (SEQ ID NO: 07)
• Rev: 5 '- [Phosp] aattcGCTGCAGCAGGAGGCG (SEQ ID NO: 08)

To generate BamHi / EcoRI5 'overhangs, equimolar amounts of the separated TrxR PCR products were mixed, melted and annealed using the following thermal conditions: 1 cycle at 95 ° C for 5 minutes; and 1 cycle at 95 ° C to 15 ° C at -0.1 ° C / second. This annealed PCR product (1.5 kb) was transfected into a BamHI / EcoRI-restricted E. coli expression vector A2.2 (see SEQ ID NO: 03; 3 ) and transformed into Top10 E. coli cells. Positive clones were identified by PCR using the above conditions and verified by sequencing. Ligation into vector A2.2 fused the TrxR-1 ORF into a frame with an N-terminal 6HIS tag 5 'to the BamHI site and an in-frame stop codon immediately 3' to the EcoRI site. The EcoRI site fused two more residues (Glu, Phe) to the C-terminus of TrxR-1 ORF (see SEQ ID NO: 03; 3 ). For recombinant protein expression, the N6HIS-TrxR-1 expression construct was transformed into the BL21 (DE3) -RP ^™ E. coli tRNA codon-optimized expression strand (Novagen).

Example 2: Purification from TrxR

Four liters of E. coli cells expressing N6His-TrxR-1 were added at a 1: 5 ratio (cell weight / ml volume) in a buffer containing 50 mM Na phosphate pH 8.0, 300 mM NaCl, 5 μmol resuspended in FAD and 20 mM imidazole and protease inhibitor cocktail. The cells were lysed with two passages through the microfluidizer at 15,000 psi and were clarified by centrifugation at 30,000 xg for 45 minutes. The supernatant was first vacuum filtered through a 0.45 micron membrane, followed by incremental binding to 15 ml of nickel-NTA (Invitrogen) resin for one hour at + 4 ° C. The bound resin was washed with two column volumes of lysis buffer (50 mM Na phosphate pH 8.0, 300 mM NaCl, 5 mM FAD, 20 mM imidazole), then with another 10 column volumes of lysis buffer containing 25 mM imidazole. The bound proteins were eluted from the resin with 500 mM imidazole in lysis buffer and the eluate immediately buffer exchanged into 5 mM Na phosphate pH 8.0, 300 mM NaCl, and 5 μM FAD. The Bradford assay was used to determine the final protein construction of TrxR-1 at 11.8 mg / ml in approximately 45 ml. TrxR-1 was on a size exclusion chromatography column and had a retention time of a globular homodimer determined using a standard curve of known proteins. Purified TrxR-1 and tryptic digested fragments of TrxR-1 were analyzed by mass spectroscopy LC / MS and MS / MS. The results confirm the identity of purified TrxR-1.

Example 3: Assays for TrxR-1 Enzyme Activity and High Throughput screening

The TrxR-1 enzyme was used as a histidine-tagged protein in E. coli expressed. NADPH-dependent Reduction of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) by TrxR-1 is spectrophotometrically after the formation of the product thionitrobenzoic (TNB) measured.

The Assay protocol is designed for a 384 well microtiter plate format. The log consists of adding the TrxR-1 enzyme and NADPH together with the addition of DTNB, which triggers the reaction. The reaction mixtures are then incubated at room temperature (22-24 ° C), and though for 30 minutes, and the increase in absorbance at 410 nm measured. Inhibited reaction mixtures become lower Absorbency result.

TrxR-1 reduces DTNB in an NADPH-dependent reaction and results in 2 moles of TNB and one mole of NADP. TNB absorbs at a maximum at 410 nm and has an extinction coefficient of 13,600 M ^-1 cm ^-1 . The reduction of DTNB by TrxR-1 is measured by determining the enzyme catalyzed increase in absorbance at 410 nm. The proposed negative control reaction will not contain TrxR-1.

• a) 5 μl einer Lösung enthaltend 10 × eine Verbindung in 5 % Dimethylsulfoxid (DMSO) (v/v);
• b) 20 μl einer Lösung enthaltend 0,1 M Kaliumphosphat (Kp_i), pH 7,4, 0,5 mMol Ethylendiamintetraessigsäure (EDTA), 0,625 mMol Dihydronicotinamidadenindinukleo tidphosphat (NADPH), 0,05 mMol Dithiothreitol (DTT), 0,005 mMol Flavinadenindinukleotid (FAD) und 0,022 mg/ml TrxR; und
• c) 25 μl einer Lösung enthaltend 0,1 mMol Kp_i; pH 7,4, 0,5 mMol EDTA, 2 mMol 5,5'-Dithiobis(2-nitrobenzoesäure (DTNB), 0,1 mg/ml Bovinserumalbumin (BSA), 0,2 % (v/v) Dimethylsulfoxid (DMSO), 0,02 % (w/v) Tween 20.

In a typical assay, the following components are added, in the following order:

• a) 5 μl of a solution containing 10 × a compound in 5% dimethyl sulfoxide (DMSO) (v / v);
• b) 20 μl of a solution containing 0.1 M potassium phosphate (Kp _i ), pH 7.4, 0.5 mmol ethylenediaminetetraacetic acid (EDTA), 0.625 mmol dihydronicotinamide adenine dinucleotide phosphate (NADPH), 0.05 mmol dithiothreitol (DTT), 0.005 mmol Flavin adenine dinucleotide (FAD) and 0.022 mg / ml TrxR; and
• c) 25 μl of a solution containing 0.1 mmol Kp _i ; pH 7.4, 0.5 mmol EDTA, 2 mmol 5,5'-dithiobis (2-nitrobenzoic acid (DTNB), 0.1 mg / ml bovine serum albumin (BSA), 0.2% (v / v) dimethyl sulfoxide (DMSO ), 0.02% (w / v) Tween 20.

The resulting reaction mixture is kept at room temperature (22 ° -24 ° C) for 30 minutes. The reduction of DTNB by TrxR is measured by measuring the absorbance detected at 410 nm.

Example 4: Validation

mutations with loss of function in the Drosophila gene encoding TrxR-1 enzyme, were isolated and resulted in the persistence of the larvae in the stage L1. The expression data of the levels of mRNA indicate that the gene is expressed in all three larval stages.

While the present invention with reference to specific embodiments was described, should be for It will be understood by those skilled in the art that various changes can be made and equivalents can be substituted, without departing from the true spirit and scope of the invention. Furthermore can many modifications done become a special situation, a material, a composition of matter, a method, a method step or method steps the task of adapting the spirit and scope of the invention. All such modifications are to be understood as being within the scope of the claims attached here are intended.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (11)

An isolated polynucleotide comprising a nucleotide sequence, which encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 02 includes. An isolated polynucleotide comprising a nucleotide sequence, at least about 75% nucleotide sequence identity with the nucleotide sequence shown in nucleotides 21-1505 of SEQ ID NO: 01. An isolated polynucleotide comprising a nucleotide sequence, which hybridizes under stringent hybridization conditions to a nucleic acid molecule, which has the sequence in nucleotides 21-1505 of SEQ ID NO: 01. A recombinant vector comprising a polynucleotide according to any the claims 1 to 3. A recombinant host cell comprising a recombinant Vector according to claim 4th A method for producing an insect thioredoxin reductase comprising culturing the host cell of claim 5 under conditions for expressing said protein and recovering said Protein. A purified protein comprising an amino acid sequence, at least about 80% sequence identity having the sequence shown in SEQ ID NO: 02. A method for detecting an agent which the enzyme activity insect thioredoxin reductase (TrxR), said method contacting said TrxR or a fragment thereof, have the enzyme activity, comprising a test agent; and determining the effect if one occurs from said test agent on the enzyme activity of said TrxR polypeptide or fragment; where the amino acid sequence of said TrxR an amino acid sequence includes, at least approximately 80% identical to the sequence shown in SEQ ID NO: 02. The method of claim 8, further comprising selecting a test agent that reduces TrxR activity; the determination an effect, if any, of the test agent on survivability of the insect, with a test agent showing insect viability reduced as a pesticidal agent is identified. The method of claim 8, wherein said contacting comprises administering said test agent to cultured host cells, genetically engineered to produce said TrxR. A method for controlling a pest, comprising contacting a pest with a compound identified by a method according to claim 8.