Novel Ecdysone Receptor-Based Inducible Gene Expression System

Abstract

the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to a novel inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.

Description

[0001]This application claims priority to co-pending U.S. provisional application Ser. No. 60/191,355, filed Mar. 22, 2000 and to co-pending U.S. provisional application Ser. No. 60/269,799, filed Feb. 20, 2001.

FIELD OF THE INVENTION

[0002]This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to a novel ecdysone receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.

BACKGROUND OF THE INVENTION

[0003]In the field of genetic engineering, precise control of gene expression is a valuable tool for studying, manipulating, and controlling development and other physiological processes. Gene expression is a complex biological process involving a number of specific protein-protein interactions. In order for gene expression to be triggered, such that it produces the RNA necessary as the first step in protein synthesis, a transcriptional activator must be brought into proximity of a promoter that controls gene transcription. Typically, the transcriptional activator itself is associated with a protein that has at least one DNA binding domain that binds to DNA binding sites present in the promoter regions of genes. Thus, for gene expression to occur, a protein comprising a DNA binding domain and a transactivation domain located at an appropriate distance from the DNA binding domain must be brought into the correct position in the promoter region of the gene.

[0004]The traditional transgenic approach utilizes a cell-type specific promoter to drive the expression of a designed transgene. A DNA construct containing the transgene is first incorporated into a host genome. When triggered by a transcriptional activator, expression of the transgene occurs in a given cell type.

[0005]Another means to regulate expression of foreign genes in cells is through inducible promoters. Examples of the use of such inducible promoters include the PR1-a promoter, prokaryotic repressor-operator systems, immunosuppressive-immunophilin systems, and higher eukaryotic transcription activation systems such as steroid hormone receptor systems and are described below.

[0006]The PR1-a promoter from tobacco is induced during the systemic acquired resistance response following pathogen attack. The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants. Gene regulation systems based on promoters induced by heat shock, interferon and heavy metals have been described (Wurn et al., 1986, Proc. Natl. Acad. Sci. USA 83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61; Filmus et al., 1992 Nucleic Acids Research 20:27550-27560). However, these systems have limitations due to their effect on expression of non-target genes. These systems are also leaky.

[0007]Prokaryotic repressor-operator systems utilize bacterial repressor proteins and the unique operator DNA sequences to which they bind. Both the tetracycline ("Tet") and lactose ("Lac") repressor-operator systems from the bacterium Escherichia coli have been used in plants and animals to control gene expression. In the Tet system, tetracycline binds to the TetR repressor protein, resulting in a conformational change which releases the repressor protein from the operator which as a result allows transcription to occur. In the Lac system, a lac operon is activated in response to the presence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside. Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e. tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression. For similar reasons, utility of such systems in animals is limited.

[0008]Immunosuppressive molecules such as FK506, rapamycin and cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc. Using this information, a general strategy has been devised to bring together any two proteins simply by placing FK506 on each of the two proteins or by placing FK506 on one and cyclosporine A on another one. A synthetic homodimer of FK506 (FK1012) or a compound resulted from fusion of FK506-cyclosporine (FKCsA) can then be used to induce dimerization of these molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw et al., 1996 Proc Natl Acad Sci USA 93:4604-7). Gal4 DNA binding domain fused to FKBP12 and VP16 activator domain fused to cyclophilin, and FKCsA compound were used to show heterodimerization and activation of a reporter gene under the control of a promoter containing Gal4 binding sites. Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limits its use for various mammalian gene switch applications.

[0009]Higher eukaryotic transcription activation systems such as steroid hormone receptor systems have also been employed. Steroid hormone receptors are members of the nuclear receptor superfamily and are found in vertebrate and invertebrate cells. Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms. In order to overcome such difficulties, an alternative system has been developed using insect ecdysone receptors (EcR).

[0010]Growth, molting, and development in insects are regulated by the ecdysone steroid hormone (molting hormone) and the juvenile hormones (Dhadialla, et al., 1998. Annu. Rev. Entomol. 43: 545-569). The molecular target for ecdysone in insects consists of at least ecdysone receptor (EcR) and ultraspiracle protein (USP). EcR is a member of the nuclear steroid receptor super family that is characterized by signature DNA and ligand binding domains, and an activation domain (Koelle et al. 1991, Cell, 67:59-77). EcR receptors are responsive to a number of steroidal compounds such as ponasterone A and muristerone A. Recently, non-steroidal compounds with ecdysteroid agonist activity have been described, including the commercially available insecticides tebufenozide and methoxyfenozide that are marketed world wide by Rohm and Haas Company (see International Patent Application No. PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs have exceptional safety profiles to other organisms.

[0011]International Patent Application No. PCT/US97/05330 (WO 97/38117) discloses methods for modulating the expression of an exogenous gene in which a DNA construct comprising the exogenous gene and an ecdysone response element is activated by a second DNA construct comprising an ecdysone receptor that, in the presence of a ligand therefor, and optionally in the presence of a receptor capable of acting as a silent partner, binds to the ecdysone response element to induce gene expression. The ecdysone receptor of choice was isolated from Drosophila melanogaster. Typically, such systems require the presence of the silent partner, preferably retinoid X receptor (RXR), in order to provide optimum activation. In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and regulates expression of target genes in a ligand dependent manner. International Patent Application No. PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor isolated from the silk moth Bombyx mori is functional in mammalian systems without the need for an exogenous dimer partner.

[0012]U.S. Pat. No. 5,880,333 discloses a Drosophila melanogaster EcR and ultraspiracle (USP) heterodimer system used in plants in which the transactivation domain and the DNA binding domain are positioned on two different hybrid proteins. Unfortunately, this system is not effective for inducing reporter gene expression in animal cells (for comparison, see Example 1.2, below).

[0013]In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR as in International Patent Application No. PCT/US98/14215 or as modified EcR as in International Patent Application No. PCT/US97/05330) were incorporated into a single molecule and the other heterodimeric partners, either USP or RXR, were used in their native state.

[0014]Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and that these systems are not applicable for use in both plants and animals (see U.S. Pat. No. 5,880,333). For most applications that rely on modulating gene expression, these EcR-based systems are undesirable. Therefore, a need exists in the art for improved systems to precisely modulate the expression of exogenous genes in both plants and animals. Such improved systems would be useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic animals. Improved systems that are simple, compact, and dependent on ligands that are relatively inexpensive, readily available, and of low toxicity to the host would prove useful for regulating biological systems.

[0015]Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. However, the citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

[0016]The present invention relates to a novel ecdysone receptor-based inducible gene expression system, novel receptor polynucleotides and polypeptides for use in the novel inducible gene expression system, and methods of modulating the expression of a gene within a host cell using this inducible gene expression system. In particular, Applicants' invention relates to an improved gene expression modulation system comprising a polynucleotide encoding a receptor polypeptide comprising a truncation mutation.

[0017]Specifically, the present invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a first polypeptide comprising: i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and a ligand binding domain comprising a ligand binding domain from a nuclear receptor; and b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second polypeptide comprising: i) a transactivation domain; and ii) a ligand binding domain comprising a ligand binding domain from a nuclear receptor other than an ultraspiracle receptor; wherein the DNA binding domain and the transactivation domain are from a polypeptide other than an ecdysone receptor, a retinoid X receptor, or an ultraspiracle receptor; wherein the ligand binding domains from the first polypeptide and the second polypeptide are different and dimerize.

[0018]In a specific embodiment, the ligand binding domain of the first polypeptide comprises an ecdysone receptor (EcR) ligand binding domain

[0019]In another specific embodiment, the ligand binding domain of the second polypeptide comprises a retinoid X receptor (RXR) ligand binding domain.

[0020]In a preferred embodiment, the ligand binding domain of the first polypeptide comprises an ecdysone receptor ligand binding domain and the ligand binding domain of the second polypeptide comprises a retinoid X receptor ligand binding domain

[0021]The present invention also relates to a gene expression modulation system according to the invention further comprising c) a third gene expression cassette comprising: i) a response element to which the DNA-binding domain of the first polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second polypeptide; and the gene whose expression is to be modulated.

[0022]The present invention also relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide, wherein the truncation mutation affects ligand binding activity or ligand sensitivity.

[0023]In particular, the present invention relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of said EcR or RXR polypeptide. In a specific embodiment, the present invention relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of said EcR or RXR polypeptide. In another specific embodiment, the present invention relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of said EcR or RXR polypeptide.

[0024]The present invention also relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of said EcR or RXR polypeptide. In a specific embodiment, the present invention relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of said EcR or RXR polypeptide. In another specific embodiment, the present invention relates to an isolated polynucleotide encoding a truncated EcR or a truncated RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of said EcR or RXR polypeptide.

[0025]The present invention also relates to an isolated polynucleotide encoding a truncated RXR polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the truncated retinoid X receptor polypeptide and a dimerization partner. In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide.

[0026]The present invention also relates to an isolated polypeptide encoded by a polynucleotide according to Applicants' invention. In particular, the present invention relates to an isolated truncated EcR or truncated RXR polypeptide comprising a truncation mutation, wherein the EcR or RXR polypeptide is encoded by a polynucleotide according to the invention.

[0027]Thus, the present invention also relates to an isolated truncated EcR or truncated RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity of said EcR or RXR polypeptide.

[0028]Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention. Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the gene to be modulated comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand that independently combines with the ligand binding domains of the first polypeptide and the second polypeptide of the gene expression modulation system; wherein the gene to be expressed is a component of a chimeric gene comprising: i) a response element comprising a domain to which the DNA binding domain from the first polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second polypeptide; and the gene whose expression is to be modulated, whereby a complex is formed comprising the ligand, the first polypeptide, and the second polypeptide, and whereby the complex modulates expression of the gene in the host cell.

[0029]Applicants' invention also provides an isolated host cell comprising an inducible gene expression system according to the invention. The present invention also relates to an isolated host cell comprising a polynucleotide or polypeptide according to the invention. Accordingly, Applicants' invention also relates to a non-human organism comprising a host cell according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-CfaRDEF chimeric polypeptide and a second gene expression cassette encoding a VP16AD-MmRXRDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.1).

[0031]FIG. 2: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-CfEcRDEF chimeric polypeptide and a second gene expression cassette encoding a VP16AD-CfUSPDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.2).

[0032]FIG. 3: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-MmRXRDEF chimeric polypeptide and a second gene expression cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.3).

[0033]FIG. 4: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-MmRXRDEF chimeric polypeptide and a second gene expression cassette encoding a VP16AD-DmEcRCDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.4).

[0034]FIG. 5: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-CfUSPDEF chimeric polypeptide and a second gene expression cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.5).

[0035]FIG. 6: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a Gal4DBD-CfEcRDEF-VP16AD chimeric polypeptide; prepared as described in Example 1 (switch 1.6).

[0036]FIG. 7: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.7).

[0037]FIG. 8: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a VP16AD-DmEcRCDEF chimeric polypeptide and a second gene expression cassette encoding a MmRXR polypeptide; prepared as described in Example 1 (switch 1.8).

[0038]FIG. 9: An ecdysone receptor-based gene expression system comprising a first gene expression cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide and a second gene expression cassette encoding a MmRXR polypeptide; prepared as described in Example 1 (switch 1.9).

[0039]FIG. 10: An ecdysone receptor-based gene expression system comprising a gene expression cassette encoding a Gal4DBD-CfEcRCDEF chimeric polypeptide; prepared as described in Example 1 (switch 1.10).

[0040]FIG. 11: Expression data of GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE truncation mutants transfected into NIH3T3 cells along with VP16MmRXRDE, pFRLUc and pTKRL plasmid DNAs.

[0041]FIG. 12: Expression data of GAL4CfEcRA/BCDEF, GAL4CfEcRCDEF, GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE truncation mutants transfected into 3T3 cells along with VP16MmRXRE, pFRLUc and pTKRL plasmid DNAs.

[0042]FIG. 13: Expression data of VP16MmRXRA/BCDEF, VP16MmRXRCDEF, VP16MmRXRDEF, VP16MmRXREF, VP16MmRXRBam-EF, VP16MmRXRAF2del constructs transfected into NIH3T3 cells along with GAL4CfEcRCDEF, pFRLUc and pTKRL plasmid DNAs.

[0043]FIG. 14: Expression data of VP16MmRXRA/BCDEF, VP16MmRXRCDEF, VP16MmRXRDEF, VP16MmRXREF, VP16MmRXRBam-EF, VP16MmRXRAF2del constructs transfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLUc and pTKRL plasmid DNAs.

[0044]FIG. 15: Expression data of various truncated CfEcR and MmRXR receptor pairs transfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLUc and pTKRL plasmid DNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0045]Applicants have now developed an improved ecdysone receptor-based inducible gene expression system comprising a truncation mutant of an ecdysone receptor or a retinoid X receptor (RXR) polypeptide that affects ligand binding activity or ligand sensitivity. This mutational effect may increase or reduce ligand binding activity or ligand sensitivity and may be steroid or non-steroid specific. Thus, Applicants' invention provides an improved ecdysone receptor-based inducible gene expression system useful for modulating expression of a gene of interest in a host cell. In a particularly desirable embodiment, Applicants' invention provides an inducible gene expression system that has a reduced level of background gene expression and responds to submicromolar concentrations of non-steroidal ligand. Thus, Applicants' novel inducible gene expression system and its use in methods of modulating gene expression in a host cell overcome the limitations of currently available inducible expression systems and provide the skilled artisan with an effective means to control gene expression.

[0046]The present invention provides a novel inducible gene expression system that can be used to modulate gene expression in both prokaryotic and eukaryotic host cells. Applicants' invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms.

Definitions

[0047]In this disclosure, a number of terms and abbreviations are used. The following definitions are provided and should be helpful in understanding the scope and practice of the present invention.

[0048]In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value or range.

[0049]The term "substantially free" means that a composition comprising "A" (where "A" is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of "B" (where "B" comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A". Preferably, "A" comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.

[0050]The term "isolated" for the purposes of the present invention designates a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present).

[0051]For example, a polynucleotide present in the natural state in a plant or an animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids in which it is naturally present. The term "purified" does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.

[0052]A polynucleotide is in the "purified" state after purification of the starting material or of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

[0053]A "nucleic acid" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes but is not limited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA. DNA may be linear, circular, or supercoiled.

[0054]A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

[0055]The term "fragment" will be understood to mean a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.

[0056]As used herein, an "isolated nucleic acid fragment" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0057]A "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene or "heterologous" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

[0058]"Heterologous" DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.

[0059]The term "genome" includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.

[0060]A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., 1989 infra). Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0061]Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_m of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, e.g., 40% formamide, with 5× or 6×SCC. High stringency hybridization conditions correspond to the highest T_m, e.g., 50% formamide, 5× or 6×SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.

[0062]The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.

[0063]In a specific embodiment, the term "standard hybridization conditions" refers to a T_m of 55° C., and utilizes conditions as set forth above. In a preferred embodiment, the T_m is 60° C.; in a more preferred embodiment, the T_m is 65° C.

[0064]Post-hybridization washes also determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleic acids comprise complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.

[0065]The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).

[0066]In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferable a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

[0067]The term "probe" refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.

[0068]As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 18 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g., with ³²P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, or to detect the presence of a nucleic acid. An oligonucleotide can also be used to form a triple helix with a DNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

[0069]A "primer" is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.

[0070]"Polymerase chain reaction" is abbreviated PCR and means an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.

[0071]"Reverse transcription-polymerase chain reaction" is abbreviated RT-PCR and means an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above. RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions; to determine the relative amount of that target molecule within the starting pool of nucleic acids.

[0072]A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0073]"Open reading frame" is abbreviated ORF and means a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0074]The term "head-to-head" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-head orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5' end of the other polynucleotide. The term "head-to-head" may be abbreviated (5')-to-(5') and may also be indicated by the symbols ( →) or (3'5'5'→3').

[0075]The term "tail-to-tail" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a tail-to-tail orientation when the 3' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide. The term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be indicated by the symbols (→ ) or (5'→3'3'5').

[0076]The "head-to-tail" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-tail orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide. The term "head-to-tail" may be abbreviated (5')-to-(3') and may also be indicated by the symbols (→ →) or (5'→3'5'→3').

[0077]The term "downstream" refers to a nucleotide sequence that is located 3' to reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0078]The term "upstream" refers to a nucleotide sequence that is located 5' to reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0079]The terms "restriction endonuclease" and "restriction enzyme" refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.

[0080]"Homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

[0081]Several methods known in the art may be used to propagate a polynucleotide according to the invention. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As described herein, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[0082]A "vector" is any means for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives, or the Bluescript vector. For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.

[0083]Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include but are not limited to retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

[0084]The term "plasmid" refers to an extra chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0085]A "cloning vector" is a "replicon", which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type and expression in another ("shuttle vector").

[0086]Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0087]A polynucleotide according to the invention can also be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., 1987. PNAS 84:7413; Mackey, et al., 1988. Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031; and Ulmer et al., 1993. Science 259:1745-1748). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989. Science 337:387-388). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

[0088]Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).

[0089]It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., 1992. Hum. Gene Ther. 3:147-154; and Wu and Wu, 1987. J. Biol. Chem. 262:4429-4432).

[0090]The term "transfection" means the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

[0091]"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0092]The term "genetic region" will refer to a region of a nucleic acid molecule or a nucleotide sequence that comprises a gene encoding a polypeptide.

[0093]In addition, the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.

[0094]The term "selectable marker" means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.

[0095]The term "reporter gene" means a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable marker genes may also be considered reporter genes.

[0096]"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters". Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters". Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0097]A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0098]A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0099]"Transcriptional and translational control sequences" are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0100]The term "response element" means one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of the first chimeric gene. This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides. The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem. The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element will be incorporated. The DNA binding domain of the first hybrid protein binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element. Examples of DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), Genes Dev. 5, 120-131); AGGTCAN.sub.(n)AGGTCA, where N(_n) can be one or more spacer nucleotides (see D'Avino P P., et. al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et. al., (1994). Mol. Cell Biol. 14, 4465-4474).

[0101]The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0102]The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.

[0103]The terms "cassette", "expression cassette" and "gene expression cassette" refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. "Transformation cassette" refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[0104]For purposes of this invention, the term "gene switch" refers to the combination of a response element associated with a promoter, and an EcR based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.

[0105]The terms "modulate" and "modulates" mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.

[0106]The plasmids or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell. The term "expression vector" means a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host. The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like. Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to: viral promoters, plant promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoter, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOXI promoter (useful for expression in Pichia); b-lactamase, lac, ara, tet, trp, lP_L, lP_R, T7, tac, and trc promoters (useful for expression in Escherichia coli); and light regulated-, seed specific-, pollen specific-, ovary specific-, pathogenesis or disease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase, stress inducible, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses, the cytomegalovirus early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter, and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo AII control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell α-actin, and the like. In a preferred embodiment of the invention, the promoter is selected from the group consisting of a cauliflower mosaic virus 35S promoter, a cassava vein mosaic virus promoter, and a cauliflower mosaic virus 35S minimal promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, and an albumin promoter. In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.

[0107]Enhancers that may be used in embodiments of the invention include but are not limited to: tobacco mosaic virus enhancer, cauliflower mosaic virus 35S enhancer, tobacco etch virus enhancer, ribulose 1,5-bisphosphate carboxylase enhancer, rice tungro bacilliform virus enhancer, and other plant and viral gene enhancers, and the like.

[0108]Termination control regions, i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included. In a preferred embodiment of the invention, the termination control region may be comprise or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, nopaline synthase (nos), cauliflower mosaic virus (CaMV), octopine synthase (ocs), Agrocateum, viral, and plant terminator sequences, or the like.

[0109]The terms "3' non-coding sequences" or "3' untranslated region (UTR)" refer to DNA sequences located downstream (3') of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0110]"Regulatory region" means a nucleic acid sequence which regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region). In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.

[0111]A regulatory region from a "heterologous source" is a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.

[0112]"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" MA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.

[0113]A "polypeptide" is a polymeric compound comprised of covalently linked amino acid residues. Amino acids have the following general structure:

##STR00001##

Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. A polypeptide of the invention preferably comprises at least about 14 amino acids.

[0114]A "protein" is a polypeptide that performs a structural or functional role in a living cell.

[0115]An "isolated polypeptide" or "isolated protein" is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

[0116]"Fragment" of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of 10, 15, 20, 30 to 40, 50, 100, 200 or 300 amino acids.

[0117]A "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art. A variant polypeptide preferably comprises at least about 14 amino acids.

[0118]A "heterologous protein" refers to a protein not naturally produced in the cell.

[0119]A "mature protein" refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0120]The term "signal peptide" refers to an amino terminal polypeptide preceding the secreted mature protein. The signal peptide is cleaved from and is therefore not present in the mature protein, Signal peptides have the function of directing and translocating secreted proteins across cell membranes. Signal peptide is also referred to as signal protein.

[0121]A "signal sequence" is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide. The term "translocation signal sequence" is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.

[0122]The term "homology" refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.

[0123]As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667.). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.

[0124]Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., 1987, Cell 50:667). As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies and homologous proteins from different species (Reeck et al., supra). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.

[0125]In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., 1989, supra.

[0126]As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0127]Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 70% identical to the DNA sequence of the nucleic acid fragments reported herein. Preferred substantially nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein. Even more preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.

[0128]Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program.

[0129]The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

[0130]A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.

[0131]The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Meg align program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0132]The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include but is not limited to the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters which originally load with the software when first initialized.

[0133]"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

Gene Expression Modulation System of the Invention

[0134]Applicants have now shown that separating the transactivation and DNA binding domains by placing them on two different proteins results in greatly reduced background activity in the absence of a ligand and significantly increased activity over background in the presence of a ligand. Applicants' improved gene expression system comprises two chimeric gene expression; the first encoding a DNA binding domain fused to a nuclear receptor polypeptide and the second encoding a transactivation domain fused to a nuclear receptor polypeptide. The interaction of the first protein with the second protein effectively tethers the DNA binding domain to the transactivation domain. Since the DNA binding and transactivation domains reside on two different molecules, the background activity in the absence of ligand is greatly reduced.

[0135]In general, the inducible gene expression modulation system of the invention comprises a) a first chimeric gene that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first hybrid polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) a ligand binding domain comprising the ligand binding domain from a nuclear receptor; and b) a second chimeric gene that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising: i) a transactivation domain; and a ligand binding domain comprising the ligand binding domain from a nuclear receptor other than ultraspiracle (USP); wherein the transactivation domain are from other than EcR, RXR, or USP; and wherein the ligand binding domains from the first hybrid polypeptide and the second hybrid polypeptide are different and dimerize.

[0136]This two-hybrid system exploits the ability of a pair of interacting proteins to bring the transcription activation domain into a more favorable position relative to the DNA binding domain such that when the DNA binding domain binds to the DNA binding site on the gene, the transactivation domain more effectively activates the promoter (see, for example, U.S. Pat. No. 5,283,173). This two-hybrid system is a significantly improved inducible gene expression modulation system compared to the two systems disclosed in International Patent Applications PCT/US97/05330 and PCT/US98/14215.

[0137]The ecdysone receptor-based gene expression modulation system of the invention may be either heterodimeric and homodimeric. A functional EcR complex generally refers to a heterodimeric protein complex consisting of two members of the steroid receptor family, an ecdysone receptor protein obtained from various insects, and an ultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid X receptor protein (see Yao, et al. (1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71, 63-72). However, the complex may also be a homodimer as detailed below. The functional ecdysteroid receptor complex may also include additional protein(s) such as immunophilins. Additional members of the steroid receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR. Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators). These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription. They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions. Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al, Curr. Opin. Cell Biol. 9:222-232, 1997). Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand. These corepressors may interact with the unliganded ecdysone receptor to silence the activity at the response element. Current evidence suggests that binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity. Examples of corepressors include N-CoR and SMRT (for review, see Horwitz et al. Mol Endocrinol. 10: 1167-1177, 1996). These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion. Homodimer complexes of the ecdysone receptor protein, USP, or RXR may also be functional under some circumstances.

[0138]The ecdysone receptor complex typically includes proteins which are members of the nuclear receptor superfamily wherein all members are characterized by the presence of an amino-terminal transactivation domain, a DNA binding domain ("DBD"), and a ligand binding domain ("LBD") separated from the DBD by a hinge region. As used herein, the term "DNA binding domain" comprises a minimal peptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see Evans, Science 240:889-895 (1988)). The "A/B" domain corresponds to the transactivation domain, "C" corresponds to the DNA binding domain, "D" corresponds to the hinge region, and "E" corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to "F".

[0139]The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins. This EcR receptor, like a subset of the steroid receptor family, also possesses less well defined regions responsible for heterodimerization properties. Because the domains of EcR, USP, and RXR are modular in nature, the LBD, DBD, and transactivation domains may be interchanged.

[0140]Gene switch systems are known that incorporate components from the ecdysone receptor complex. However, in these known systems, whenever EcR is used it is associated with native or modified DNA binding domains and transactivation domains on the same molecule. USP or RXR are typically used as silent partners. We have now shown that when DNA binding domains and transactivation domains are on the same molecule the background activity in the absence of ligand is high and that such activity is dramatically reduced when DNA binding domains and transactivation domains are on different molecules, that is, on each of two partners of a heterodimeric or homodimeric complex. This two-hybrid system also provides improved sensitivity to non-steroidal ligands for example, diacylhydrazines, when compared to steroidal ligands for example, ponasterone A ("PonA") or muristerone A ("MurA"). That is, when compared to steroids, the non-steroidal ligands provide higher activity at a lower concentration. In addition, since transactivation based on EcR gene switches is often cell-line dependent, it is easier to tailor switching system to obtain maximum transactivation capability for each application. Furthermore, this two-hybrid system avoids some side effects due to overexpression of RXR that often occur when unmodified RXR is used as a switching partner. In this two-hybrid system, native DNA binding and transactivation domains of EcR or RXR are eliminated. As a result, these chimeric molecules have less chance of interacting with other steroid hormone receptors present in the cell resulting in reduced side effects.

[0141]Specifically, Applicants' invention relates to a gene expression modulation system comprising: a) a first gene expression cassette that is capable of being expressed in a host cell, wherein the first gene expression cassette comprises a polynucleotide that encodes a first polypeptide comprising i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and ii) a ligand binding domain comprising a ligand binding domain from a nuclear receptor; and b) a second gene expression cassette that is capable of being expressed in the host cell, wherein the second gene expression cassette comprises a polynucleotide sequence that encodes a second polypeptide comprising i) a transactivation domain; and a ligand binding domain comprising a ligand binding domain from a nuclear receptor other than ultraspiracle (USP); wherein the DNA binding domain and the transactivation domain are from other than EcR, RXR, or USP; wherein the ligand binding domains from the first polypeptide and the second polypeptide are different and dimerize.

[0142]The present invention also relates to a gene expression modulation system according to the present invention further comprising c) a third gene expression cassette comprising: i) the response element to which the DNA-binding domain of the first polypeptide binds; a promoter that is activated by the transactivation domain of the second polypeptide; and iii) the gene whose expression is to be modulated.

[0143]In a specific embodiment, the gene whose expression is to be modulated is a homologous gene with respect to the host cell. In another specific embodiment, the gene whose expression is to be modulated is a heterologous gene with respect to the host cell.

[0144]In a specific embodiment, the ligand binding domain of the first polypeptide comprises an ecdysone receptor ligand binding domain.

[0145]In another specific embodiment, the ligand binding domain of the first polypeptide comprises a retinoid X receptor ligand binding domain.

[0146]In a specific embodiment, the ligand binding domain of the second polypeptide comprises an ecdysone receptor ligand binding domain.

[0147]In another specific embodiment, the ligand binding domain of the second polypeptide comprises a retinoid X receptor ligand binding domain.

[0148]In a preferred embodiment, the ligand binding domain of the first polypeptide comprises an ecdysone receptor ligand binding domain, and the ligand binding domain of the second polypeptide comprises a retinoid X receptor ligand binding domain.

[0149]In another preferred embodiment, the ligand binding domain of the first polypeptide is from a retinoid X receptor polypeptide, and the ligand binding domain of the second polypeptide is from an ecdysone receptor polypeptide.

[0150]Preferably, the ligand binding domain is an EcR or RXR related steroid/thyroid hormone nuclear receptor family member ligand binding domain, or analogs, combinations, or modifications thereof. More preferably, the LBD is from EcR or RXR. Even more preferably, the LBD is from a truncated EcR or RXR. A truncation mutation may be made by any method used in the art, including but not limited to restriction endonuclease digestion/deletion, PCR-mediated/oligonucleotide-directed deletion, chemical mutagenesis, UV strand breakage, and the like.

[0151]Preferably, the EcR is an insect EcR selected from the group consisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR, a Homopteran EcR and a Hemipteran EcR. More preferably, the EcR for use is a spruce budworm Choristoneura fumiferana EcR ("CfEcR"), a Tenebrio molitor EcR ("TmEcR"), a Manduca sexta EcR ("MsEcR"), a Heliothies virescens EcR ("HvEcR"), a silk moth Bombyx mori EcR ("BmEcR"), a fruit fly Drosophila melanogaster EcR ("DmEcR"), a mosquito Aedes aegypti EcR ("AaEcR"), a blowfly Lucilia capitata EcR ("LcEcR"), a Mediterranean fruit fly Ceratitis capitata EcR ("CcEcR"), a locust Locusta migratoria EcR ("LmEcR"), an aphid Myzus persicae EcR ("MpEcR"), a fiddler crab Uca pugilator EcR ("UpEcR"), or an ixodid tick Amblyomma americanum EcR ("AmaEcR"). Even more preferably, the LBD is from spruce budworm (Choristoneura fumzferana) EcR ("CfEcR") or fruit fly Drosophila melanogaster EcR ("DmEcR").

[0152]Preferably, the LBD is from a truncated insect EcR. The insect EcR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the insect EcR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the insect EcR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the insect EcR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0153]In a preferred embodiment, the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0154]In another preferred embodiment, the ecdysone receptor ligand binding domain comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0155]Preferably, the RXR polypeptide is a mouse Mus musculus RXR ("MmRXR") or a human Homo sapiens RXR ("HsRXR"). The RXR polypeptide may be an RXR.sub.α, RXR.sub.β, or RXR.sub.γ isoform.

[0156]Preferably, the LBD is from a truncated RXR. The RXR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the RXR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the RXR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the RXR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0157]In a preferred embodiment, the retinoid X receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0158]In another preferred embodiment, the retinoid X receptor ligand binding domain comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0159]For purposes of this invention EcR and RXR also include synthetic and chimeric EcR and RXR and their homologs.

[0160]The DNA binding domain can be any DNA binding domain with a known response element, including synthetic and chimeric DNA binding domains, or analogs, combinations, or modifications thereof. Preferably, the DBD is a GAL4 DED, a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 41 (polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD [(SEQ ID NO: 43 (polynucleotide) or SEQ ID NO: 44 (polypeptide)].

[0161]The transactivation domain (abbreviated "AD" or "TA") may be any steroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, or an analog, combination, or modification thereof. Preferably, the AD is a synthetic or chimeric AD, or is obtained from a VP16, GAL4, or NF-κB. Most preferably, the AD is a VP16 AD [SEQ ID NO: 45 (polynucleotide) or SEQ ID NO: 46 (polypeptide)].

[0162]The response element ("RE") may be any response element with a known DNA binding domain, or an analog, combination, or modification thereof. Preferably, the RE is an RE from GAL4 ("GAL4RE"), LexA, a steroid/thyroid hormone nuclear receptor RE, or a synthetic RE that recognizes a synthetic DNA binding domain. More preferably, the RE is a GAL4RE comprising a polynucleotide sequence of SEQ ID NO: 47 or a LexA 8× operon comprising a polynucleotide sequence of SEQ ID NO: 48. Preferably, the first hybrid protein is substantially free of a transactivation domain and the second hybrid protein is substantially free of a DNA binding domain. For purposes of this invention, "substantially free" means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.

[0163]The ligands for use in the present invention as described below, when combined with the ligand binding domain of an EcR, USP, RXR, or another polypeptide which in turn are bound to the response element linked to a gene, provide the means for external temporal regulation of expression of the gene. The binding mechanism or the order in which the various components of this invention bind to each other, that is, ligand to receptor, first polypeptide to response element, second polypeptide to promoter, etc., is not critical. Binding of the ligand to the ligand binding domains of an EcR, USP, RXR, or another protein, enables expression or suppression of the gene. This mechanism does not exclude the potential for ligand binding to EcR, USP, or RXR, and the resulting formation of active homodimer complexes (e.g. EcR+EcR or USP+USP). Preferably, one or more of the receptor domains can be varied producing a chimeric gene switch. Typically, one or more of the three domains, DBD, LBD, and transactivation domain, may be chosen from a source different than the source of the other domains so that the chimeric genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski, et al. (1988) Nature, 335:563-564) or LexA protein from E. coil (see Brent and Ptashne (1985), Cell, 43:729-736), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim, et al. (1997), Proc. Natl. Acad. Sci., USA, 94:3616-3620) to accommodate chimeric receptors. Another advantage of chimeric systems is that they allow choice of a promoter used to drive the gene expression according to a desired end result. Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled. When genes, operatively linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the system of this invention. Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cells) or specific to certain developmental stages of the organism.

Gene Expression Cassettes of the Invention

[0164]The novel ecdysone receptor-based inducible gene expression system of the invention comprises a novel gene expression cassette that is capable of being expressed in a host cell, wherein the gene expression cassette comprises a polynucleotide encoding a hybrid polypeptide. Thus, Applicants' invention also provides novel gene expression cassettes for use in the gene expression system of the invention.

[0165]Specifically, the present invention provides a gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide. The hybrid polypeptide comprises either 1) a DNA-binding domain that recognizes a response element and a ligand binding domain of a nuclear receptor or 2) a transactivation domain and a ligand binding domain of a nuclear receptor, wherein the transactivation domain is from a nuclear receptor other than an EcR, an RXR, or a USP.

[0166]In a specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and an ecdysone receptor ligand binding domain, wherein the DNA binding domain is from a nuclear receptor other than an ecdysone receptor.

[0167]In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain that recognizes a response element and a retinoid X receptor ligand binding domain, wherein the DNA binding domain is from a nuclear receptor other than a retinoid X receptor.

[0168]The DNA binding domain can be any DNA binding domain with a known response element, including synthetic and chimeric DNA binding domains, or analogs, combinations, or modifications thereof. Preferably, the DBD is a GAL4 DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroid hormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 41 (polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD [(SEQ ID NO: 43 (polynucleotide) or SEQ ID NO: 44 (polypeptide)].

[0169]In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and an ecdysone receptor ligand binding domain, wherein the transactivation domain is from a nuclear receptor other than an ecdysone receptor.

[0170]In another specific embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain and a retinoid X receptor ligand binding domain, wherein the transactivation domain is from a nuclear receptor other than a retinoid X receptor.

[0171]The transactivation domain (abbreviated "AD" or "TA") may be any steroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, or an analog, combination, or modification thereof. Preferably, the AD is a synthetic or chimeric AD, or is obtained from a VP16, GAL4, or NF-κB. Most preferably, the AD is a VP16 AD [SEQ ID NO: 45 (polynucleotide) or SEQ ID NO: 46 (polypeptide)].

[0172]Preferably, the ligand binding domain is an EcR or RXR related steroid/thyroid hormone nuclear receptor family member ligand binding domain, or analogs, combinations, or modifications thereof. More preferably, the LBD is from EcR or RXR. Even more preferably, the LBD is from a truncated EcR or RXR.

[0173]Preferably, the EcR is an insect EcR selected from the group consisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR, a Homopteran EcR and a Hemipteran EcR. More preferably, the EcR for use is a spruce budworm Choristoneura fumiferana EcR ("CfEcR"), a Tenebrio molitor EcR ("TmEcR"), a Manduca sexta EcR ("MsEcR"), a Heliothies virescens EcR ("HvEcR"), a silk moth Bombyx mori EcR ("BmEcR"), a fruit fly Drosophila melanogaster EcR ("DmEcR"), a mosquito Aedes aegypti EcR ("AaEcR"), a blowfly Lucilia capitata EcR ("LcEcR"), a Mediterranean fruit fly Ceratitis capitata EcR ("CcEcR"), a locust Locusta migratoria EcR ("LmEcR"), an aphid Myzus persicae EcR ("MpEcR"), a fiddler crab Uca pugilator EcR ("UpEcR"), or an ixodid tick Amblyomma americanum EcR ("AmaEcR"). Even more preferably, the LBD is from spruce budworm (Choristoneura fumiferana) EcR ("CfEcR") or fruit fly Drosophila melanogaster EcR ("DmEcR").

[0174]Preferably, the LBD is from a truncated insect EcR. The insect EcR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the insect EcR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the insect EcR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the insect EcR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0175]In a preferred embodiment, the ecdysone receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0176]In another preferred embodiment, the ecdysone receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0177]Preferably, the RXR polypeptide is a mouse Mus musculus RXR ("MmRXR") or a human Homo sapiens RXR ("HsRXR"). The RXR polypeptide may be an RXR.sub.α, RXR.sub.β, or RXR.sub.γ isoform.

[0178]Preferably, the LBD is from a truncated RXR. The RXR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the RXR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the RXR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the RXR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0179]In a preferred embodiment, the retinoid X receptor ligand binding domain is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0180]In another preferred embodiment, the retinoid X receptor ligand binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0181]In a preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO: 43) and an ecdysone receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0182]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising a polypeptide sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and an ecdysone receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0183]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO: 43) and a retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0184]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a DNA-binding domain comprising a polypeptide sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and a retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0185]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 and an ecdysone receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0186]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising a polypeptide sequence of SEQ ID NO: 46 and an ecdysone receptor ligand binding domain comprising a polypeptide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0187]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 and a retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0188]In another preferred embodiment, the gene expression cassette encodes a hybrid polypeptide comprising a transactivation domain comprising a polypeptide sequence of SEQ ID NO: 46 and a retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0189]For purposes of this invention EcR and RXR also include synthetic and chimeric EcR and RXR and their homologs.

Polynucleotides of the Invention

[0190]The novel ecdysone receptor-based inducible gene expression system of the invention comprises a gene expression cassette comprising a polynucleotide that encodes a truncated EcR or RXR polypeptide comprising a truncation mutation and is useful in methods of modulating the expression of a gene within a host cell.

[0191]Thus, the present invention also relates to a polynucleotide that encodes an EcR or RXR polypeptide comprising a truncation mutation. Specifically, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity.

[0192]Preferably, the truncation mutation results in a polynucleotide that encodes a truncated EcR polypeptide or a truncated RXR polypeptide comprising a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the EcR or RXR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the EcR or RXR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the EcR or RXR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0193]In a specific embodiment, the EcR polynucleotide according to the invention comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In a specific embodiment, the polynucleotide according to the invention encodes a ecdysone receptor polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF, which comprises the last 33 carboxy-terminal amino acids of C domain), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), and SEQ ID NO: 20 (DmEcR-DE).

[0194]In another specific embodiment, the RXR polynucleotide according to the invention comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. In another specific embodiment, the polynucleotide according to the invention encodes a truncated RXR polypeptide comprising an amino acid sequence consisting of SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF), SEQ ID NO: 39 (HsRXR-truncated EF), and SEQ ID NO: 40 (HsRXR-E).

[0195]In particular, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation, wherein the mutation reduces ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide. In a specific embodiment, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated polynucleotide encoding an EcR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR polypeptide, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 8 (DmEcR-DEF), or SEQ ID NO: 9 (DmEcR-EF). In another specific embodiment, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated polynucleotide encoding an EcR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 4 (CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF).

[0196]The present invention also relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation, wherein the mutation enhances ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide. In a specific embodiment, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In another specific embodiment, the present invention relates to an isolated polynucleotide encoding an EcR or RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated polynucleotide encoding an EcR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF) or SEQ ID NO: 8 (DmEcR-DEF).

[0197]The present invention also relates to an isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the mutated retinoid X receptor polypeptide and a dimerization partner. Preferably, the isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23 (MraRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 28 (HsRXR-EF), or SEQ ID NO: 29 (HsRXR-truncated EF). In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide. Preferably, the dimerization partner is a truncated EcR polypeptide. More preferably, the dimerization partner is an EcR polypeptide in which domains A/B/C have been deleted. Even more preferably, the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).

Polypeptides of the Invention

[0198]The novel ecdysone receptor-based inducible gene expression system of the invention comprises a polynucleotide that encodes a truncated EcR or RXR polypeptide and is useful in methods of modulating the expression of a gene within a host cell. Thus, the present invention also relates to an isolated truncated EcR or RXR polypeptide encoded by a polynucleotide or a gene expression cassette according to the invention. Specifically, the present invention relates to an isolated truncated EcR or RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity encoded by a polynucleotide according to the invention.

[0199]The present invention also relates to an isolated truncated EcR or RXR polypeptide comprising a truncation mutation. Specifically, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that affects ligand binding activity or ligand sensitivity.

[0200]Preferably, the truncation mutation results in a truncated EcR polypeptide or a truncated RXR polypeptide comprising a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, the EcR or RXR polypeptide truncation comprises a deletion of at least a partial polypeptide domain. Even more preferably, the EcR or RXR polypeptide truncation comprises a deletion of at least an entire polypeptide domain. In a specific embodiment, the EcR or RXR polypeptide truncation comprises a deletion of at least an A/B-domain deletion, a C-domain deletion, a D-domain deletion, an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion, an AB/1/2-C-domains deletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domains deletion. A combination of several complete and/or partial domain deletions may also be performed.

[0201]In a preferred embodiment, the isolated truncated ecdysone receptor polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6 (DmEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQ ID NO: 9 (DrnEcR-EF), and SEQ ID NO: 10 (DmEcR-DE). In another preferred embodiment, the isolated truncated ecdysone receptor polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), and SEQ ID NO: 20 (DmEcR-DE).

[0202]In a preferred embodiment, the isolated truncated RXR polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 21 (MmRXR-CDEF), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26 (HsRXR-CDEF), SEQ ID NO: 27 (HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF), SEQ ID NO: 29 (HsRXR-truncatedEF) and SEQ ID NO: 30 (HsRXR-E). In another preferred embodiment, the isolated truncated RXR polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF), SEQ ID NO: 39 (HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0203]The present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide, wherein the polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6 (DmEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQ ID NO: 9 (DmEcR-EF), SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21 (MmRXR-CDEF), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26 (HsRXR-CDEF), SEQ ID NO: 27 (HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF), SEQ ID NO: 29 (HsRXR-truncatedEF), and SEQ ID NO: 30 (HsRXR-E).

[0204]Thus, the present invention relates to an isolated truncated EcR or RXR polypeptide comprising a truncation mutation that reduces ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ NO: 12 (CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), SEQ ID NO: 20 (DmEcR-DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF), SEQ ID NO: 39 (HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0205]In a specific embodiment, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR polypeptide, wherein the EcR polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 8 (DmEcR-DEF), or SEQ ID NO: 9 (DmEcR-EF). Accordingly, the present invention also relates to an isolated truncated EcR or RXR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that reduces steroid binding activity or steroid sensitivity of the EcR polypeptide, wherein the EcR polypeptide comprises an amino acid sequence of SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 18 (DmEcR-DEF), or SEQ ID NO: 19 (DmEcR-EF).

[0206]In another specific embodiment, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the EcR polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 4 (CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF). Accordingly, the present invention also relates to an isolated truncated EcR or RXR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that reduces non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the EcR polypeptide comprises an amino acid sequence of SEQ ID NO: 14 (CfEcR-EF) or SEQ ID NO: 19 (DmEcR-EF).

[0207]In particular, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide, wherein the polypeptide is encoded by a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6 (DmEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQ ID NO: 9 (DmEcR-EF), SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21 (MmRXR-CDEF), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26 (HsRXR-CDEF), SEQ ID NO: 27 (HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF), SEQ ID NO: 29 (HsRXR-truncated EF), and SEQ ID NO: 30 (HsRXR-E).

[0208]The present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), SEQ ID NO: 20 (DmEcR-DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 39 (HsRXR-EF), SEQ ID NO: 39 (HsRXR-trancatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0209]The present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances ligand binding activity or ligand sensitivity of the EcR or RXR polypeptide. In a specific embodiment, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. Accordingly, the present invention also relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide.

[0210]In another specific embodiment, the present invention relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the EcR polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF) or SEQ ID NO: 8 (DmEcR-DEF). Accordingly, the present invention also relates to an isolated EcR or RXR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or steroid sensitivity of the EcR or RXR polypeptide. In a preferred embodiment, the present invention relates to an isolated EcR polypeptide comprising a truncation mutation that enhances non-steroid binding activity or non-steroid sensitivity of the EcR polypeptide, wherein the EcR polynucleotide comprises an amino acid sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).

[0211]The present invention also relates to an isolated retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprising the mutated retinoid X receptor polypeptide and a dimerization partner. Preferably, the isolated retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 28 (HsRXR-EF), or SEQ ID NO: 29 (HsRXR-truncatedEF). More preferably, the isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation that increases ligand sensitivity of a heterodimer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 38 (HsRXR-EF), or SEQ ID NO: 39 (HsRXR-truncatedEF).

[0212]In a specific embodiment, the dimerization partner is an ecdysone receptor polypeptide. Preferably, the dimerization partner is a truncated EcR polypeptide. More preferably, the dimerization partner is an EcR polypeptide in which domains A/B/C have been deleted. Even more preferably, the dimerization partner is an EcR polypeptide comprising an amino acid sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).

Method of Modulating Gene Expression of the Invention

[0213]Applicants' invention also relates to methods of modulating gene expression in a host cell using a gene expression modulation system according to the invention. Specifically, Applicants' invention provides a method of modulating the expression of a gene in a host cell comprising the steps of: a) introducing into the host cell a gene expression modulation system according to the invention; and b) introducing into the host cell a ligand that independently combines with the ligand binding domains of the first polypeptide and the second polypeptide of the gene expression modulation system; wherein the gene to be expressed is a component of a gene expression cassette comprising: i) a response element comprising a domain to which the DNA binding domain of the first polypeptide binds; ii) a promoter that is activated by the transactivation domain of the second polypeptide; and iii) a gene whose expression is to be modulated, whereby a complex is formed comprising the ligand, the first polypeptide of the gene expression modulation system and the second polypeptide of the gene expression modulation system, and whereby the complex modulates expression of the gene in the host cell.

[0214]Genes of interest for expression in a host cell using Applicants' methods may be endogenous genes or heterologous genes. Nucleic acid or amino acid sequence information for a desired gene or protein can be located in one of many public access databases, for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications. Thus, those skilled in the art have access to nucleic acid sequence information for virtually all known genes. Such information can then be used to construct the desired constructs for the insertion of the gene of interest within the gene expression cassettes used in Applicants' methods described herein.

[0215]Examples of genes of interest for expression in a host cell using Applicants' methods include, but are not limited to: antigens produced in plants as vaccines, enzymes like alpha-amylase, phytase, glucanes, and xylanse, genes for resistance against insects, nematodes, fungi, bacteria, viruses, and abiotic stresses, nutraceuticals, phaunaceuticals, vitamins, genes for modifying amino acid content, herbicide resistance, cold, drought, and heat tolerance, industrial products, oils, protein, carbohydrates, antioxidants, male sterile plants, flowers, fuels, other output traits, genes encoding therapeutically desirable polypeptides or products, such as genes that can provide, modulate, alleviate, correct and/or restore polypeptides important in treating a condition, a disease, a disorder, a dysfunction, a genetic defect, and the like.

[0216]Acceptable ligands are any that modulate expression of the gene when binding of the DNA binding domain of the two hybrid system to the response element in the presence of the ligand results in activation or suppression of expression of the genes. Preferred ligands include ponasterone, muristerone A, N,N'-diacylhydrazines such as those disclosed in U.S. Pat. No. 6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No. 461,809; N-alkyl-N,N'-diacylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994; N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, and the like.

[0217]Preferably, the ligand for use in Applicants' method of modulating expression of gene is a compound of the formula:

##STR00002##

wherein: [0218]E is a (C₄-C₆)alkyl containing a tertiary carbon or a cyano(C₃-C₅)alkyl containing a tertiary carbon; [0219]R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, SCN, or SCHF₂; [0220]R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH--CN, or joined with R³ and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; [0221]R³ is H, Et, or joined with R² and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; [0222]R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

[0223]Applicants' invention provides for modulation of gene expression in prokaryotic and eukaryotic host cells. Thus, the present invention also relates to a method for modulating gene expression in a host cell selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, and a mammalian cell. Preferably, the host cell is a yeast cell, a plant cell, a murine cell, or a human cell.

[0224]Expression in transgenic host cells may be useful for the expression of various polypeptides of interest including but not limited to therapeutic polypeptides, pathway intermediates; for the modulation of pathways already existing in the host for the synthesis of new products heretofore not possible using the host; cell based assays; and the like. Additionally the gene products may be useful for conferring higher growth yields of the host or for enabling alternative growth mode to be utilized.

Host Cells and Non-Human Organisms of the Invention

[0225]As described above, the gene expression modulation system of the present invention may be used to modulate gene expression in a host cell. Expression in transgenic host cells may be useful for the expression of various genes of interest. Thus, Applicants' invention also provides an isolated host cell comprising a gene expression system according to the invention. The present invention also provides an isolated host cell comprising a gene expression cassette according to the invention. Applicants' invention also provides an isolated host cell comprising a polynucleotide or polypeptide according to the invention. The isolated host cell may be either a prokaryotic or a eukaryotic host cell.

[0226]Preferably, the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, and a mammalian cell. Examples of preferred host cells include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella, plant, animal, and mammalian host cells. More preferably, the host cell is a yeast cell, a plant cell, a murine cell, or a human cell.

[0227]In a specific embodiment, the host cell is a yeast cell selected from the group consisting of a Saccharomyces, a Pichia, and a Candida host cell.

[0228]In another specific embodiment, the host cell is a plant cell selected from the group consisting of an apple, Arabidopsis, bajra, banana, barley, bean, beet, blackgram, chickpea, chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat host cell.

[0229]In another specific embodiment, the host cell is a murine cell.

[0230]In another specific embodiment, the host cell is a human cell.

[0231]Host cell transformation is well known in the art and may be achieved by a variety of methods including but not limited to electroporation, viral infection, plasmid/vector transfection, non-viral vector mediated transfection, Agrobacterium-mediated transformation, particle bombardment, and the like. Expression of desired gene products involves culturing the transformed host cells under suitable conditions and inducing expression of the transformed gene. Culture conditions and gene expression protocols in prokaryotic and eukaryotic cells are well known in the art (see General Methods section of Examples). Cells may be harvested and the gene products isolated according to protocols specific for the gene product.

[0232]In addition, a host cell may be chosen which modulates the expression of the inserted polynucleotide, or modifies and processes the polypeptide product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g., of signal sequence]) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product. However, a polypeptide expressed in bacteria may not be properly folded. Expression in yeast can produce a glycosylated product. Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding of a heterologous protein. Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, the polypeptide's activity. Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.

[0233]Applicants' invention also relates to a non-human organism comprising an isolated host cell according to the invention. Preferably, the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a plant, an animal, and a mammal. More preferably, the non-human organism is a yeast, a plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, or a chimpanzee.

[0234]In a specific embodiment, the non-human organism is a yeast selected from the group consisting of Saccharomyces, Pichia, and Candida.

[0235]In another specific embodiment, the non-human organism is a plant selected from the group consisting of an apple, Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

[0236]In another specific embodiment, the non-human organism is a Mus musculus mouse.

Measuring Gene Expression/Transcription

[0237]One useful measurement of Applicants' methods of the invention is that of the transcriptional state of the cell including the identities and abundances of RNA, preferably mRNA species. Such measurements are conveniently conducted by measuring cDNA abundances by any of several existing gene expression technologies.

[0238]Nucleic acid array technology is a useful technique for determining differential mRNA expression. Such technology includes, for example, oligonucleotide chips and DNA microarrays. These techniques rely on DNA fragments or oligonucleotides which correspond to different genes or cDNAs which are immobilized on a solid support and hybridized to probes prepared from total mRNA pools extracted from cells, tissues, or whole organisms and converted to cDNA. Oligonucleotide chips are arrays of oligonucleotides synthesized on a substrate using photolithographic techniques. Chips have been produced which can analyze for up to 1700 genes. DNA microarrays are arrays of DNA samples, typically PCR products, that are robotically printed onto a microscope slide. Each gene is analyzed by a full or partial-length target DNA sequence. Microarrays with up to 10,000 genes are now routinely prepared commercially. The primary difference between these two techniques is that oligonucleotide chips typically utilize 25-mer oligonucleotides which allow fractionation of short DNA molecules whereas the larger DNA targets of microarrays, approximately 1000 base pairs, may provide more sensitivity in fractionating complex DNA mixtures.

[0239]Another useful measurement of Applicants' methods of the invention is that of determining the translation state of the cell by measuring the abundances of the constituent protein species present in the cell using processes well known in the art.

[0240]Where identification of genes associated with various physiological functions is desired, an assay may be employed in which changes in such functions as cell growth, apoptosis, senescence, differentiation, adhesion, binding to a specific molecules, binding to another cell, cellular organization, organogenesis, intracellular transport, transport facilitation, energy conversion, metabolism, myo genesis, neurogenesis, and/or hematopoiesis is measured.

[0241]In addition, selectable marker or reporter gene expression may be used to measure gene expression modulation using Applicants' invention.

[0242]Other methods to detect the products of gene expression are well known in the art and include Southern blots (DNA detection), dot or slot blots (DNA, RNA), Northern blots (RNA), and RT-PCR (RNA) analyses. Although less preferred, labeled proteins can be used to detect a particular nucleic acid sequence to which it hybidizes.

[0243]In some cases it is necessary to amplify the amount of a nucleic acid sequence. This may be carried out using one or more of a number of suitable methods including, for example, polymerase chain reaction ("PCR"), ligase chain reaction ("LCR"), strand displacement amplification ("SDA"), transcription-based amplification, and the like. PCR is carried out in accordance with known techniques in which, for example, a nucleic acid sample is treated in the presence of a heat stable DNA polymerase, under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer that is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample may be analyzed as described above to assess whether the sequence or sequences to be detected are present.

[0244]The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention.

EXAMPLES

General Methods

[0245]Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

[0246]Methods for plant tissue culture, transformation, plant molecular biology, and plant, general molecular biology may be found in Plant Tissue Culture Concepts and Laboratory Exercises edited by R N Trigiano and D J Gray, 2^nd edition, 2000, CRC press, New York; Agrobacterium Protocols edited by K M A Gartland and M R Davey, 1995, Humana Press, Totowa, N.J.; Methods in Plant Molecular Biology, P. Maliga et al., 1995, Cold Spring Harbor Lab Press, New York; and Molecular Cloning, J. Sambrook et al., 1989, Cold Spring Harbor Lab Press, New York.

[0247]Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of host cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.

[0248]Manipulations of genetic sequences may be accomplished using the suite of programs available from the Genetics Computer Group Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.). Where the GCG program "Pileup" is used the gap creation default value of 12, and the gap extension default value of 4 may be used. Where the CGC "Gap" or "Bestfit" programs is used the default gap creation penalty of 50 and the default gap extension penalty of 3 may be used. In any case where GCG program parameters are not prompted for, in these or any other GCG program, default values may be used.

[0249]The meaning of abbreviations is as follows: "h" means hour(s), "min" means minute(s), "sec" means second(s), "d" means day(s), "μl" means microliter(s), "ml" means milliliter(s), "L" means liter(s), "μM" means micromolar, "mM" means millimolar, "μg" means microgram(s), "mg" means milligram(s), "A" means adenine or adenosine, "T" means thymine or thymidine, "G" means guanine or guanosine, "C" means cytidine or cytosine, "×g" means times gravity, "nt" means nucleotide(s), "aa" means amino acid(s), "bp" means base pair(s), "kb" means kilobase(s), "k" means kilo, "μ" means micro, and "° C." means degrees Celsius.

Example 1

[0250]Applicants' improved EcR-based inducible gene modulation system was developed for use in various applications including gene therapy, expression of proteins of interest in host cells, production of transgenic organisms, and cell-based assays. This Example describes the construction and evaluation of several gene expression cassettes for use in the EcR-based inducible gene expression system of the invention.

[0251]In various cellular backgrounds, including mammalian cells, insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor (RXR) and, upon binding of ligand, transactivates genes under the control of ecdysone response elements. Applicants constructed several EcR-based gene expression cassettes based on the spruce budworm Choristoneura fumiferana EcR ("CfEcR"; full length polynucleotide and amino acid sequences are set forth in SEQ ID NO: 49 and SEQ ID NO: 50, respectively), C. fumiferana ultraspiracle ("CfUSP"; full length polynucleotide and amino acid sequences are set forth in SEQ ID NO: 51 and SEQ ID NO: 52, respectively), and mouse Mus musculus RXRα (MmRXRα; full length polynucleotide and amino acid sequences are set forth in SEQ ID NO: 53 and SEQ ID NO: 54, respectively). The prepared receptor constructs comprise a ligand binding domain of EcR and of RXR or of USP; a DNA binding domain of GAL4 or of EcR; and an activation domain of VP16. The reporter constructs include a reporter gene, luciferase or LacZ, operably linked to a synthetic promoter construct that comprises either GAL4 or EcR/USP binding sites (response elements). Various combinations of these receptor and reporter constructs were cotransfected into CHO, NIH3T3, CV1 and 293 cells. Gene induction potential (magnitude of induction) and ligand specificity and sensitivity were examined using four different ligands: two steroidal ligands (ponasterone A and muristerone A) and two non-steroidal ligands (N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydra- zine and N-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'-(3,5-dimethylbenzo- yl)-N'-tert-butylhydrazine) in a dose-dependent induction of reporter gene expression in the transfected cells. Reporter gene expression activities were assayed at 24 hr or 48 hr after ligand addition. [0252]Gene Expression Cassettes: Ecdysone receptor-based, chemically inducible gene expression cassettes (switches) were constructed as followed, using standard cloning methods available in the art. The following is brief description of preparation and composition of each switch. [0253]1.1--GAL4EcR/VP16RXR: The D, E, and F domains from spruce budwoiui Choristoneura fumiferana EcR ("CfEcRDEF"; SEQ ID NO: 3) were fused to GAL4 DNA binding domain ("DNABD"; SEQ ID NO: 41) and placed under the control of an SV40e promoter (SEQ ID NO: 55). The DEF domains from mouse (Mus musculus) RXR ("MmRXRDEF"; SEQ ID NO: 22) were fused to the activation domain from VP16 ("VP16AD"; SEQ ID NO: 45) and placed under the control of an SV40e promoter (SEQ ID NO: 55). Five consensus GAL4 binding sites ("5×GAL4RE"; comprising 5, GAL4RE comprising SEQ ID NO: 47) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 56) and placed upstream of the luciferase gene (SEQ ID NO: 57). [0254]1.2--GAL4EcR/VP16USP: This construct was prepared in the same way as in switch 1.1 above except MmRXRDEF was replaced with the D, E and F domains from spruce budworm USP ("CfUSPDEF"; SEQ ID NO: 58). The constructs used in this example are similar to those disclosed in U.S. Pat. No. 5,880,333 except that Choristoneura fumiferana USP rather than Drosophila melanogaster USP was utilized. [0255]1.3--GAL4RXR/VP16CfEcR: MmRXRDEF (SEQ ID NO: 22) was fused to a GAL4DNABD (SEQ ID NO: 41) and CfEcRCDEF (SEQ ID NO: 1) was fused to a VP16AD (SEQ ID NO: 45). [0256]1.4--GAL4RXR/VP16DmEcR: This construct was prepared in the same way as switch 1.3 except CfEcRCDEF was replaced with DmEcRCDEF (SEQ ID NO: 6). [0257]1.5--GAL4USPNP16CfEcR: This construct was prepared in the same way as switch 1.3 except MmRXRDEF was replaced with CfUSPDEF (SEQ ID NO: 58). [0258]1.6--GAL4RXRCfEcRVP16: This construct was prepared so that both the GAL4 DNABD and the VP16AD were placed on the same molecule. GAL4DNABD (SEQ ID NO: 41) and VP16AD (SEQ ID NO: 45) were fused to CfEcRDEF (SEQ ID NO: 3) at N-and C-termini respectively. The fusion was placed under the control of an SV40e promoter (SEQ ID NO: 55). [0259]1.7--VP16CfEcR: This construct was prepared such that CfEcRCDEF (SEQ ID NO: 1) was fused to VP16AD (SEQ ID NO: 45) and placed under the control of an SV40e promoter (SEQ ID NO: 55). Six ecdysone response elements ("EcRE"; SEQ ID NO: 59) from the hsp27 gene were placed upstream of the promoter and a luciferase gene (SEQ ID NO: 57). This switch most probably uses endogenous RXR. [0260]1.8--DmVgRXR: This system was purchased from Invitrogen Corp., Carlsbad, Calif. It comprises a Drosophila melanogaster EcR ("DmEcR") with a modified DNABD fused to VP16AD and placed under the control of a CMV promoter (SEQ ID NO: 60). Full length MmRXR (SEQ ID NO: 53) was placed under the control of the RSV promoter (SEQ ID NO: 61). The reporter, pIND(SP1)LacZ, contains five copies of a modified ecdysone response element ("EcRE", E/GRE), three copies of an SP1 enhancer, and a minimal heat shock promoter, all of which were placed upstream to the LacZ reporter gene. [0261]1.9--CfVgRXR: This example was prepared in the same way as switch 1.8 except DmEcR was replaced with a truncated CfEcR comprising a partial A/B domain and the complete CDEF domains [SEQ ID NO: 62 (polynucleotide) and SEQ ID NO: 63 (polypeptide)]. [0262]1.10--CfVgRXRdel: This example was prepared in the same way as switch 1.9 except MmRXR (SEQ ID NO: 53) was deleted. [0263]Cell lines: Four cell lines: CHO, Chinese hamster Cricetulus griseus ovarian cell line; NIH3T3 (3T3) mouse Mus musculus cell line; 293 human Homo sapiens kidney cell line, and CV1 African green monkey kidney cell line were used in these experiments. Cells were maintained in their respective media and were subcultured when they reached 60% confluency. Standard methods for culture and maintenance of the cells were followed. [0264]Transfections: Several commercially available lipofactors as well as electroporation methods were evaluated and the best conditions for transfection of each cell line were developed. CHO, NIH3T3, 293 and CV1 cells were grown to 60% confluency. DNAs corresponding to the various switch constructs outlined in Examples 1.1 through 1.10 were transfected into CHO cells, NIH3T3 cells, 293 cells, or CV1 cells as follows. [0265]CHO cells: Cells were harvested when they reach 60-80% confluency and plated in 6- or 12- or 24-well plates at 250,000, 100,000, or 50,000 cells in 2.5, 1.0, or 0.5 ml of growth medium containing 10% Fetal bovine serum respectively. The next day, the cells were rinsed with growth medium and transfected for four hours. LipofectAMINE® 2000 (Life Technologies Inc) was found to be the best transfection reagent for these cells. For 12-well plates, 4 μl of LipofectAMINE® 2000 was mixed with 100 μl of growth medium. 1.0 μg of reporter construct and 0.25 μg of receptor construct(s) were added to the transfection mix. A second reporter construct was added [pTKRL (Promega), 0.1 μg/transfection mix] and comprised a Renilla luciferase gene (SEQ ID NO: 64) operably linked and placed under the control of a thymidine kinase (TK) constitutive promoter and was used for normalization. The contents of the transfection mix were mixed in a vortex mixer and let stand at room temperature for 30 min. At the end of incubation, the transfection mix was added to the cells maintained in 400 μl growth medium. The cells were maintained at 37° C. and 5% CO₂ for four hours. At the end of incubation, 500 μl of growth medium containing 20% FBS and either DMSO (control) or a DMSO solution of appropriate ligands were added and the cells were maintained at 37° C. and 5% CO₂ for 24-48 hr. The cells were harvested and reporter activity was assayed. The same procedure was followed for 6 and 24 well plates as well except all the reagents were doubled for 6 well plates and reduced to half for 24-well plates. [0266]NIH3T3 Cells: Superfect® (Qiagen Inc.) was found to be the best transfection reagent for 3T3 cells. The same procedures described for CHO cells were followed for 3T3 cells as well with two modifications. The cells were plated when they reached 50% continency. 125,000 or 50,000 or 25,000 cells were plated per well of 6- or 12- or 24-well plates respectively. The GAl4EcR/VP16RXR and reporter vector DNAs were transfected into NIH3T3 cells, the transfected cells were grown in medium containing PonA, MurA, N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-t-butylhydrazine- , or N-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'-(3,5-dimethylbenzoyl)-- N'-tert-butylhydrazine for 48 hr. The ligand treatments were performed as described in the CHO cell section above. [0267]293 Cells: LipofectAMINE® 2000 (Life Technologies) was found to be the best lipofactor for 293 cells. The same procedures described for CHO were followed for 293 cells except that the cells were plated in biocoated plates to avoid clumping. The ligand treatments were performed as described in the CHO cell section above. [0268]CV1 Cells: LipofectAMINE® plus (Life Technologies) was found to be the best lipofactor for CV1 cells. The same procedures described for NIH3T3 cells were followed for CV1 cells [0269]Ligands: Ponasterone A and Muristerone A were purchased from Sigma Chemical Company. The two non-steroids N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-t-butylhydrazine- , or N-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'-(3,5-dimethylbenzoyl)-- N'-tert-butylhydrazine are synthetic stable ecdysteroids synthesized at Rohm and Haas Company. All ligands were dissolved in DMSO and the final concentration of DMSO was maintained at 0.1% in both controls and treatments. [0270]Reporter Assays: Cells were harvested 24-48 hr after adding ligands. 125, 250, or 500 μl of passive lysis buffer (part of Dual-luciferase® reporter assay system from Promega Corporation) were added to each well of 24- or 12- or 24-well plates respectively. The plates were placed on a rotary shaker for 15 min. Twenty μl of lysate was assayed. Luciferase activity was measured using Dual-luciferase® reporter assay system from Promega Corporation following the manufacturer's instructions. β-Galactosidase was measured using Galacto-Star® assay kit from TROPIX following the manufacturer's instructions. All luciferase and β-galactosidase activities were normalized using Renilla luciferase as a standard. Fold activities were calculated by dividing normalized relative light units ("RLU") in ligand treated cells with normalized RLU in DMSO treated cells (untreated control).

[0271]The results of these experiments are provided in the following tables.

TABLE-US-00001 TABLE 1 Transactivation of reporter genes through various switches in CHO cells Mean Fold Activation with 50 μM N-(2-ethyl-3-methoxybenzoyl)- N'-(3,5-dimethylbenzoyl)- Composition of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 267 pGAL4RELuc 1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 85 pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 312 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 2 pGAL4RELuc 1.6 GAL4CfEcRVP16 9 pGAL4RELuc 1.7 VP16CfEcR 36 pEcRELuc 1.8 DmVgRXR + MmRXR 14 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 27 pIND(SP1)LacZ 1.10 CfVgRXR 29 pIND(SP1)LacZ

TABLE-US-00002 TABLE 2 Transactivation of reporter genes through various switches in 3T3 cells Mean Fold Activation Through N-(2-ethyl-3-methoxybenzoyl)- N'-(3,5-dimethylbenzoyl)- Composition of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 1118 pGAL4RELuc 1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 47 pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 269 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 3 pGAL4RELuc 1.6 GAL4CfEcRVP16 7 pGAL4RELuc 1.7 VP16CfEcR 1 pEcRELuc 1.8 DmVgRXR + MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 19 pIND(SP1)LacZ 1.10 CfVgRXR 2 pIND(SP1)LacZ

TABLE-US-00003 TABLE 3 Transactivation of reporter genes through various switches in 293 cells Mean Fold Activation Through N-(2-ethyl-3-methoxybenzoyl)- N'-(3,5-dimethylbenzoyl)- Composition of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 125 pGAL4RELuc 1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 17 pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 3 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 2 pGAL4RELuc 1.6 GAL4CfEcRVP16 3 pGAL4RELuc 1.7 VP16CfEcR 2 pEcRELuc 1.8 DmVgRXR + MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 12 pIND(SP1)LacZ 1.10 CfVgRXR 3 pIND(SP1)LacZ

TABLE-US-00004 TABLE 4 Transactivation of reporter genes through various switches in CV1 cells Mean Fold Activation Through N-(2-ethyl-3-methoxybenzoyl)- N'-(3,5-dimethylbenzoyl)- Composition of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 279 pGAL4RELuc 1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 25 pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 80 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 3 pGAL4RELuc 1.6 GAL4CfEcRVP16 6 pGAL4RELuc 1.7 VP16CfEcR 1 pEcRELuc 1.8 DmVgRXR + MmRXR 12 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 7 pIND(SP1)LacZ 1.10 CfVgRXR 1 pIND(SP1)LacZ

TABLE-US-00005 TABLE 5 Transactivation of reporter gene GAL4CfEcRDEF/ VP16MmRXRDEF (switch 1.1) through steroids and non-steroids in 3T3 cells. Mean Fold Induction at 1.0 μM Ligand Concentration 1. Ponasterone A 1.0 2. Muristerone A 1.0 3. N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5- 116 dimethylbenzoyl)-N'-tert-butylhydrazine 4. N'-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'- 601 (3,5-dimethylbenzoyl)-N'-tert-butylhydrazine

TABLE-US-00006 TABLE 6 Transactivation of reporter gene GAL4MmRXRDEF/ VP16CfEcRCDEF (switch 1.3) through steroids and non-steroids in 3T3 cells. Mean Fold Induction at 1.0 μM Ligand Concentration 1. Ponasterone A 1.0 2. Muristerone A 1.0 3. N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5- 71 dimethylbenzoyl)-N'-tert-butylhydrazine 4. N'-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'- 54 (3,5-dimethylbenzoyl)-N'-tert-butylhydrazine

[0272]Applicants' results demonstrate that the non-steroidal ecdysone agonists, N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-M-tert-bu- tylhydrazine and N'-(3,4)-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'-(3,5-dimethylbenzoyl)-N'- -tert-butylhydrazine, were more potent activators of CfEcR as compared to Drosophila melanogaster EcR (DmEcR). (see Tables 1-4). Also, in the mammalian cell lines tested, MmRXR performed better than CfUSP as a heterodimeric partner for CfEcR. (see Tables 1-4). Additionally, Applicants' inducible gene expression modulation system performed better when exogenous MmRXR was used than when the system relied only on endogenous RXR levels (see Tables 1-4).

[0273]Applicants' results also show that in a CfEcR-based inducible gene expression system, the non-steroidal ecdysone agonists induced reporter gene expression at a lower concentration (i.e., increased ligand sensitivity) as compared to the steroid ligands, ponasterone A and muristerone A (see Tables 5 and 6).

[0274]Out of 10 EcR based gene switches tested, the GAL4EcR/VP16RXR switch (Switch 1.1) performed better than any other switch in all four cell lines examined and was more sensitive to non-steroids than steroids. The results also demonstrate that placing the activation domain (AD) and DNA binding domain (DNABD) on each of the two partners reduced background when compared to placing both AD and DNABD together on one of the two partners. Therefore, a switch format where the AD and DNABD are separated between two partners, works well for EcR-based gene switch applications.

[0275]In addition, the MmRXR/EcR-based switches performed better than CfUSP/EcR-based switches, which have a higher background activity than the MmRXR/EcR switches in the absence of ligand.

[0276]Finally, the GAL4EcR/VP16RXR switch (Switch 1.1) was more sensitive to non-steroid ligands than to the steroid ligands (see Tables 5 and 6). In particular, steroid ligands initiated transactivation at concentrations of 50 μM, whereas the non-steroid ligands initiated transactivation at less than 1 μM (submicromolar) concentration.

Example 2

[0277]This Example describes Applicants' further analysis of truncated EcR and RXR polypeptides in the improved EcR-based inducible gene expression system of the invention. To identify the best combination and length of two receptors that give a switch with a) maximum induction in the presence of ligand; b) minimum background in the absence of ligand; c) highly sensitive to ligand concentration; and d) minimum cross-talk among ligands and receptors, Applicants made and analyzed several truncation mutations of the CfEcR and MmRXR receptor polypeptides in NIH3T3 cells.

[0278]Briefly, polynucleotides encoding EcR or RXR receptors were truncated at the junctions of A/B, C, D, E and F domains and fused to either a GAL4 DNA binding domain encoding polynucleotide (SEQ ID NO: 41) for CfEcR, or a VP16 activation domain encoding polynucleotide (SEQ ID NO: 45) for MmRXR as described in Example 1. The resulting receptor truncation/fusion polypeptides were assayed in NIH3T3 cells. Plasmid pFRLUC (Stratagene) encoding a luciferase polypeptide was used as a reporter gene construct and pTKRL (Promega) encoding a Renilla luciferase polypeptide under the control of the constitutive TK promoter was used to normalize the transfections as described above. The analysis was performed in triplicates and mean luciferase counts were determined as described above.

Gene Expression Cassettes Encoding Truncated Ecdysone Receptor Polypeptides

[0279]Gene expression cassettes comprising polynucleotides encoding either full length or truncated CfEcR polypeptides fused to a GAL4 DNA binding domain (SEQ ID NO: 41): GAL4CfEcRA/BCDEF (full length CfEcRA/BCDEF; SEQ ID NO: 49), GAL4CfEcRCDEF (CfEcRCDEF; SEQ ID NO: 1), GAL4CfEcR1/2CDEF (CfEcR1/2CDEF; SEQ ID NO: 2), GAL4CfEcRDEF (CfEcRDEF; SEQ ID NO: 3), GAL4CfEcREF (CfEcREF; SEQ ID NO: 4), and GAL4CfEcRDE (CfEcRDE; SEQ ID NO: 5) were transfected into NIH3T3 cells along with VP16MmRXRDEF (constructed as in Example 1.1; FIG. 11) or VP16MmRXREF [constructed as in Example 1.1 except that MmRXRDEF was replaced with MmRXREF (SEQ ID NO: 23); FIG. 12], and pFRLUc and pTKRL plasmid DNAs. The transfected cells were grown in the presence 0, 1, 5 or 25 uM of N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz- ine or PonA for 48 hr. The cells were harvested, lysed and luciferase reporter activity was measured in the cell lysates. Total fly luciferase relative light units are presented. The number on the top of each bar is the maximum fold induction for that treatment.

[0280]Applicants' results show that the EF domain of MmRXR is sufficient and performs better than DEF domains of this receptor (see FIGS. 11 and 12). Applicants have also shown that, in general, EcR/RXR receptor combinations are insensitive to PonA (see FIGS. 11 and 12). As shown in the FIGS. 11 and 12, the GAL4CfEcRCDEF hybrid polypeptide (SEQ ID NO: 7) performed better than any other CfEcR hybrid polypeptide.

Gene Expression Cassettes Encoding Truncated Retinoid X Receptor Polypeptides

[0281]Gene expression cassettes comprising polynucleotides encoding either full length or truncated MmRXR polypeptides fused to a VP16 transactivation domain (SEQ ID NO: 45): VP16MmRXRA/BCDEF (full length MmRXRA/BCDEF; SEQ ID NO: 53), VP16MmRXRCDEF (MmRXRCDEF; SEQ ID NO: 21), VP16MmRXRDEF (MmRXRDEF; SEQ ID NO: 22), VP16MmRXREF (MmRXREF; SEQ ID NO: 23), VP16MniRXRBam-EF ("MmRXRBam-EF" or "MmRXR-truncatedEF"; SEQ ID NO: 24), and VP16MmRXRAF2del ("MmRXRAF2del" or "MmRXR-E"; SEQ ID NO: 25) constructs were transfected into NIH3T3 cells along with GAL4CfEcRCDEF (constructed as in Example 1.1; FIG. 13) or GAL4CfEcRDEF [constructed as in Example 1.1 except CfEcRCDEF was replaced with CfEcRDEF (SEQ ID NO: 3); FIG. 14], pFRLUc and pTKRL plasmid DNAs as described above. The transfected cells were grown in the presence 0, 1, 5 and 25 uM of N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz- ine or PonA for 48 hr. The cells were harvested and lysed and reporter activity was measured in the cell lysate. Total fly luciferase relative light units are presented. The number on top of each bar is the maximum fold induction in that treatment.

[0282]Of all the truncations of MmRXR tested, Applicants' results show that the MmRXREF receptor was the best partner for CfEcR (FIGS. 13 and 14). CfEcRCDEF showed better induction than CfEcRDEF using MmRXREF. Deleting AF2 (abbreviated "EF-AF2del") or helices 1-3 of the E domain (abbreviated "EF-Bamdel") resulted in an RXR receptor that reduced gene induction and ligand sensitivity when partnered with either CfEcRCDEF (FIG. 13) or CfEcRDEF (FIG. 14) in NIH3T3 cells. In general, the CfEcR/RXR-based switch was much more sensitive to the non-steroid N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz- ine than to the steroid PonA.

Example 3

[0283]This Example describes Applicants' further analysis of gene expression cassettes encoding truncated EcR or RXR receptor polypeptides that affect either ligand binding activity or ligand sensitivity, or both. Briefly, six different combinations of chimeric receptor pairs, constructed as described in Examples 1 and 2, were further analyzed in a single experiment in NIH3T3 cells. These six receptor pair combinations and their corresponding sample numbers are depicted in Table 7.

TABLE-US-00007 TABLE 7 CfEcR + MmRXR Truncation Receptor Combinations in NIH3T3 Cells FIG. 15 EcR Polypeptide RXR Polypeptide X-Axis Sample No. Construct Construct Samples 1 and 2 GAL4CfEcRCDEF VP16RXRA/BCDEF (Full length) Samples 3 and 4 GAL4CfEcRCDEF VP16RXRDEF Samples 5 and 6 GAL4CfEcRCDEF VP16RXREF Samples 7 and 8 GAL4CfEcRDEF VP16RXRA/BCDEF (Full length) Samples 9 and 10 GAL4CfEcRDEF VP16RXRDEF Samples 11 and 12 GAL4CfEcRDEF VP16RXREF

[0284]The above receptor construct pairs, along with the reporter plasmid pFRLuc were transfected into NIH3T3 cells as described above. The six CfEcR truncation receptor combinations were duplicated into two groups and treated with either steroid (odd numbers on x-axis of FIG. 15) or non-steroid (even numbers on x-axis of FIG. 15). In particular, the cells were grown in media containing 0, 1, 5 or 25 uM PonA (steroid) or N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz- ine (non-steroid) ligand. The reporter gene activity was measured and total RLU are shown. The number on top of each bar is the maximum fold induction for that treatment and is the mean of three replicates.

[0285]As shown in FIG. 15, the CfEcRCDEF/MmRXREF receptor combinations were the best switch pairs both in terms of total RLU and fold induction (compare columns 1-6 to columns 7-12). This confirms Applicants' earlier findings as described in Example 2 (FIGS. 11-14). The same gene expression cassettes encoding the truncated EcR and RXR polypeptides were also assayed in a human lung carcinoma cell line A549 (ATCC) and similar results were observed (data not shown).

Sequence CWU 1

6411288DNAArtificial Sequencemisc_featureNovel Sequence 1aagggccctg cgccccgtca gcaagaggaa ctgtgtctgg tatgcgggga cagagcctcc 60ggataccact acaatgcgct cacgtgtgaa gggtgtaaag ggttcttcag acggagtgtt 120accaaaaatg cggtttatat ttgtaaattc ggtcacgctt gcgaaatgga catgtacatg 180cgacggaaat gccaggagtg ccgcctgaag aagtgcttag ctgtaggcat gaggcctgag 240tgcgtagtac ccgagactca gtgcgccatg aagcggaaag agaagaaagc acagaaggag 300aaggacaaac tgcctgtcag cacgacgacg gtggacgacc acatgccgcc cattatgcag 360tgtgaacctc cacctcctga agcagcaagg attcacgaag tggtcccaag gtttctctcc 420gacaagctgt tggagacaaa ccggcagaaa aacatccccc agttgacagc caaccagcag 480ttccttatcg ccaggctcat ctggtaccag gacgggtacg agcagccttc tgatgaagat 540ttgaagagga ttacgcagac gtggcagcaa gcggacgatg aaaacgaaga gtctgacact 600cccttccgcc agatcacaga gatgactatc ctcacggtcc aacttatcgt ggagttcgcg 660aagggattgc cagggttcgc caagatctcg cagcctgatc aaattacgct gcttaaggct 720tgctcaagtg aggtaatgat gctccgagtc gcgcgacgat acgatgcggc ctcagacagt 780gttctgttcg cgaacaacca agcgtacact cgcgacaact accgcaaggc tggcatggcc 840tacgtcatcg aggatctact gcacttctgc cggtgcatgt actctatggc gttggacaac 900atccattacg cgctgctcac ggctgtcgtc atcttttctg accggccagg gttggagcag 960ccgcaactgg tggaagaaat ccagcggtac tacctgaata cgctccgcat ctatatcctg 1020aaccagctga gcgggtcggc gcgttcgtcc gtcatatacg gcaagatcct ctcaatcctc 1080tctgagctac gcacgctcgg catgcaaaac tccaacatgt gcatctccct caagctcaag 1140aacagaaagc tgccgccttt cctcgaggag atctgggatg tggcggacat gtcgcacacc 1200caaccgccgc ctatcctcga gtcccccacg aatctctagc ccctgcgcgc acgcatcgcc 1260gatgccgcgt ccggccgcgc tgctctga 128821110DNAArtificial Sequencemisc_featureNovel Sequence 2gcggtttata tttgtaaatt cggtcacgct tgcgaaatgg acatgtacat gcgacggaaa 60tgccaggagt gccgcctgaa gaagtgctta gctgtaggca tgaggcctga gtgcgtagta 120cccgagactc agtgcgccat gaagcggaaa gagaagaaag cacagaagga gaaggacaaa 180ctgcctgtca gcacgacgac ggtggacgac cacatgccgc ccattatgca gtgtgaacct 240ccacctcctg aagcagcaag gattcacgaa gtggtcccaa ggtttctctc cgacaagctg 300ttggagacaa accggcagaa aaacatcccc cagttgacag ccaaccagca gttccttatc 360gccaggctca tctggtacca ggacgggtac gagcagcctt ctgatgaaga tttgaagagg 420attacgcaga cgtggcagca agcggacgat gaaaacgaag agtctgacac tcccttccgc 480cagatcacag agatgactat cctcacggtc caacttatcg tggagttcgc gaagggattg 540ccagggttcg ccaagatctc gcagcctgat caaattacgc tgcttaaggc ttgctcaagt 600gaggtaatga tgctccgagt cgcgcgacga tacgatgcgg cctcagacag tgttctgttc 660gcgaacaacc aagcgtacac tcgcgacaac taccgcaagg ctggcatggc ctacgtcatc 720gaggatctac tgcacttctg ccggtgcatg tactctatgg cgttggacaa catccattac 780gcgctgctca cggctgtcgt catcttttct gaccggccag ggttggagca gccgcaactg 840gtggaagaaa tccagcggta ctacctgaat acgctccgca tctatatcct gaaccagctg 900agcgggtcgg cgcgttcgtc cgtcatatac ggcaagatcc tctcaatcct ctctgagcta 960cgcacgctcg gcatgcaaaa ctccaacatg tgcatctccc tcaagctcaa gaacagaaag 1020ctgccgcctt tcctcgagga gatctgggat gtggcggaca tgtcgcacac ccaaccgccg 1080cctatcctcg agtcccccac gaatctctag 111031054DNAArtificial Sequencemisc_featureNovel Sequence 3cctgagtgcg tagtacccga gactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60aaggagaagg acaaactgcc tgtcagcacg acgacggtgg acgaccacat gccgcccatt 120atgcagtgtg aacctccacc tcctgaagca gcaaggattc acgaagtggt cccaaggttt 180ctctccgaca agctgttgga gacaaaccgg cagaaaaaca tcccccagtt gacagccaac 240cagcagttcc ttatcgccag gctcatctgg taccaggacg ggtacgagca gccttctgat 300gaagatttga agaggattac gcagacgtgg cagcaagcgg acgatgaaaa cgaagagtct 360gacactccct tccgccagat cacagagatg actatcctca cggtccaact tatcgtggag 420ttcgcgaagg gattgccagg gttcgccaag atctcgcagc ctgatcaaat tacgctgctt 480aaggcttgct caagtgaggt aatgatgctc cgagtcgcgc gacgatacga tgcggcctca 540gacagtgttc tgttcgcgaa caaccaagcg tacactcgcg acaactaccg caaggctggc 600atggcctacg tcatcgagga tctactgcac ttctgccggt gcatgtactc tatggcgttg 660gacaacatcc attacgcgct gctcacggct gtcgtcatct tttctgaccg gccagggttg 720gagcagccgc aactggtgga agaaatccag cggtactacc tgaatacgct ccgcatctat 780atcctgaacc agctgagcgg gtcggcgcgt tcgtccgtca tatacggcaa gatcctctca 840atcctctctg agctacgcac gctcggcatg caaaactcca acatgtgcat ctccctcaag 900ctcaagaaca gaaagctgcc gcctttcctc gaggagatct gggatgtggc ggacatgtcg 960cacacccaac cgccgcctat cctcgagtcc cccacgaatc tctagcccct gcgcgcacgc 1020atcgccgatg ccgcgtccgg ccgcgctgct ctga 10544735DNAArtificial Sequencemisc_featureNovel Sequence 4taccaggacg ggtacgagca gccttctgat gaagatttga agaggattac gcagacgtgg 60cagcaagcgg acgatgaaaa cgaagagtct gacactccct tccgccagat cacagagatg 120actatcctca cggtccaact tatcgtggag ttcgcgaagg gattgccagg gttcgccaag 180atctcgcagc ctgatcaaat tacgctgctt aaggcttgct caagtgaggt aatgatgctc 240cgagtcgcgc gacgatacga tgcggcctca gacagtgttc tgttcgcgaa caaccaagcg 300tacactcgcg acaactaccg caaggctggc atggcctacg tcatcgagga tctactgcac 360ttctgccggt gcatgtactc tatggcgttg gacaacatcc attacgcgct gctcacggct 420gtcgtcatct tttctgaccg gccagggttg gagcagccgc aactggtgga agaaatccag 480cggtactacc tgaatacgct ccgcatctat atcctgaacc agctgagcgg gtcggcgcgt 540tcgtccgtca tatacggcaa gatcctctca atcctctctg agctacgcac gctcggcatg 600caaaactcca acatgtgcat ctccctcaag ctcaagaaca gaaagctgcc gcctttcctc 660gaggagatct gggatgtggc ggacatgtcg cacacccaac cgccgcctat cctcgagtcc 720cccacgaatc tctag 7355960DNAArtificial Sequencemisc_featureNovel Sequence 5cctgagtgcg tagtacccga gactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60aaggagaagg acaaactgcc tgtcagcacg acgacggtgg acgaccacat gccgcccatt 120atgcagtgtg aacctccacc tcctgaagca gcaaggattc acgaagtggt cccaaggttt 180ctctccgaca agctgttgga gacaaaccgg cagaaaaaca tcccccagtt gacagccaac 240cagcagttcc ttatcgccag gctcatctgg taccaggacg ggtacgagca gccttctgat 300gaagatttga agaggattac gcagacgtgg cagcaagcgg acgatgaaaa cgaagagtct 360gacactccct tccgccagat cacagagatg actatcctca cggtccaact tatcgtggag 420ttcgcgaagg gattgccagg gttcgccaag atctcgcagc ctgatcaaat tacgctgctt 480aaggcttgct caagtgaggt aatgatgctc cgagtcgcgc gacgatacga tgcggcctca 540gacagtgttc tgttcgcgaa caaccaagcg tacactcgcg acaactaccg caaggctggc 600atggcctacg tcatcgagga tctactgcac ttctgccggt gcatgtactc tatggcgttg 660gacaacatcc attacgcgct gctcacggct gtcgtcatct tttctgaccg gccagggttg 720gagcagccgc aactggtgga agaaatccag cggtactacc tgaatacgct ccgcatctat 780atcctgaacc agctgagcgg gtcggcgcgt tcgtccgtca tatacggcaa gatcctctca 840atcctctctg agctacgcac gctcggcatg caaaactcca acatgtgcat ctccctcaag 900ctcaagaaca gaaagctgcc gcctttcctc gaggagatct gggatgtggc ggacatgtcg 96061878DNAArtificial Sequencemisc_featureNovel Sequence 6ggacctgcgc cacgggtgca agaggagctg tgcctggttt gcggcgacag ggcctccggc 60taccactaca acgccctcac ctgtgagggc tgcaaggggt tctttcgacg cagcgttacg 120aagagcgccg tctactgctg caagttcggg cgcgcctgcg aaatggacat gtacatgagg 180cgaaagtgtc aggagtgccg cctgaaaaag tgcctggccg tgggtatgcg gccggaatgc 240gtcgtcccgg agaaccaatg tgcgatgaag cggcgcgaaa agaaggccca gaaggagaag 300gacaaaatga ccacttcgcc gagctctcag catggcggca atggcagctt ggcctctggt 360ggcggccaag actttgttaa gaaggagatt cttgacctta tgacatgcga gccgccccag 420catgccacta ttccgctact acctgatgaa atattggcca agtgtcaagc gcgcaatata 480ccttccttaa cgtacaatca gttggccgtt atatacaagt taatttggta ccaggatggc 540tatgagcagc catctgaaga ggatctcagg cgtataatga gtcaacccga tgagaacgag 600agccaaacgg acgtcagctt tcggcatata accgagataa ccatactcac ggtccagttg 660attgttgagt ttgctaaagg tctaccagcg tttacaaaga taccccagga ggaccagatc 720acgttactaa aggcctgctc gtcggaggtg atgatgctgc gtatggcacg acgctatgac 780cacagctcgg actcaatatt cttcgcgaat aatagatcat atacgcggga ttcttacaaa 840atggccggaa tggctgataa cattgaagac ctgctgcatt tctgccgcca aatgttctcg 900atgaaggtgg acaacgtcga atacgcgctt ctcactgcca ttgtgatctt ctcggaccgg 960ccgggcctgg agaaggccca actagtcgaa gcgatccaga gctactacat cgacacgcta 1020cgcatttata tactcaaccg ccactgcggc gactcaatga gcctcgtctt ctacgcaaag 1080ctgctctcga tcctcaccga gctgcgtacg ctgggcaacc agaacgccga gatgtgtttc 1140tcactaaagc tcaaaaaccg caaactgccc aagttcctcg aggagatctg ggacgttcat 1200gccatcccgc catcggtcca gtcgcacctt cagattaccc aggaggagaa cgagcgtctc 1260gagcgggctg agcgtatgcg ggcatcggtt gggggcgcca ttaccgccgg cattgattgc 1320gactctgcct ccacttcggc ggcggcagcc gcggcccagc atcagcctca gcctcagccc 1380cagccccaac cctcctccct gacccagaac gattcccagc accagacaca gccgcagcta 1440caacctcagc taccacctca gctgcaaggt caactgcaac cccagctcca accacagctt 1500cagacgcaac tccagccaca gattcaacca cagccacagc tccttcccgt ctccgctccc 1560gtgcccgcct ccgtaaccgc acctggttcc ttgtccgcgg tcagtacgag cagcgaatac 1620atgggcggaa gtgcggccat aggacccatc acgccggcaa ccaccagcag tatcacggct 1680gccgttaccg ctagctccac cacatcagcg gtaccgatgg gcaacggagt tggagtcggt 1740gttggggtgg gcggcaacgt cagcatgtat gcgaacgccc agacggcgat ggccttgatg 1800ggtgtagccc tgcattcgca ccaagagcag cttatcgggg gagtggcggt taagtcggag 1860cactcgacga ctgcatag 187871752DNAArtificial Sequencemisc_featureNovel Sequence 7gccgtctact gctgcaagtt cgggcgcgcc tgcgaaatgg acatgtacat gaggcgaaag 60tgtcaggagt gccgcctgaa aaagtgcctg gccgtgggta tgcggccgga atgcgtcgtc 120ccggagaacc aatgtgcgat gaagcggcgc gaaaagaagg cccagaagga gaaggacaaa 180atgaccactt cgccgagctc tcagcatggc ggcaatggca gcttggcctc tggtggcggc 240caagactttg ttaagaagga gattcttgac cttatgacat gcgagccgcc ccagcatgcc 300actattccgc tactacctga tgaaatattg gccaagtgtc aagcgcgcaa tataccttcc 360ttaacgtaca atcagttggc cgttatatac aagttaattt ggtaccagga tggctatgag 420cagccatctg aagaggatct caggcgtata atgagtcaac ccgatgagaa cgagagccaa 480acggacgtca gctttcggca tataaccgag ataaccatac tcacggtcca gttgattgtt 540gagtttgcta aaggtctacc agcgtttaca aagatacccc aggaggacca gatcacgtta 600ctaaaggcct gctcgtcgga ggtgatgatg ctgcgtatgg cacgacgcta tgaccacagc 660tcggactcaa tattcttcgc gaataataga tcatatacgc gggattctta caaaatggcc 720ggaatggctg ataacattga agacctgctg catttctgcc gccaaatgtt ctcgatgaag 780gtggacaacg tcgaatacgc gcttctcact gccattgtga tcttctcgga ccggccgggc 840ctggagaagg cccaactagt cgaagcgatc cagagctact acatcgacac gctacgcatt 900tatatactca accgccactg cggcgactca atgagcctcg tcttctacgc aaagctgctc 960tcgatcctca ccgagctgcg tacgctgggc aaccagaacg ccgagatgtg tttctcacta 1020aagctcaaaa accgcaaact gcccaagttc ctcgaggaga tctgggacgt tcatgccatc 1080ccgccatcgg tccagtcgca ccttcagatt acccaggagg agaacgagcg tctcgagcgg 1140gctgagcgta tgcgggcatc ggttgggggc gccattaccg ccggcattga ttgcgactct 1200gcctccactt cggcggcggc agccgcggcc cagcatcagc ctcagcctca gccccagccc 1260caaccctcct ccctgaccca gaacgattcc cagcaccaga cacagccgca gctacaacct 1320cagctaccac ctcagctgca aggtcaactg caaccccagc tccaaccaca gcttcagacg 1380caactccagc cacagattca accacagcca cagctccttc ccgtctccgc tcccgtgccc 1440gcctccgtaa ccgcacctgg ttccttgtcc gcggtcagta cgagcagcga atacatgggc 1500ggaagtgcgg ccataggacc catcacgccg gcaaccacca gcagtatcac ggctgccgtt 1560accgctagct ccaccacatc agcggtaccg atgggcaacg gagttggagt cggtgttggg 1620gtgggcggca acgtcagcat gtatgcgaac gcccagacgg cgatggcctt gatgggtgta 1680gccctgcatt cgcaccaaga gcagcttatc gggggagtgg cggttaagtc ggagcactcg 1740acgactgcat ag 175281650DNAArtificial Sequencemisc_featureNovel Sequence 8cggccggaat gcgtcgtccc ggagaaccaa tgtgcgatga agcggcgcga aaagaaggcc 60cagaaggaga aggacaaaat gaccacttcg ccgagctctc agcatggcgg caatggcagc 120ttggcctctg gtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc 180gagccgcccc agcatgccac tattccgcta ctacctgatg aaatattggc caagtgtcaa 240gcgcgcaata taccttcctt aacgtacaat cagttggccg ttatatacaa gttaatttgg 300taccaggatg gctatgagca gccatctgaa gaggatctca ggcgtataat gagtcaaccc 360gatgagaacg agagccaaac ggacgtcagc tttcggcata taaccgagat aaccatactc 420acggtccagt tgattgttga gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480gaggaccaga tcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca 540cgacgctatg accacagctc ggactcaata ttcttcgcga ataatagatc atatacgcgg 600gattcttaca aaatggccgg aatggctgat aacattgaag acctgctgca tttctgccgc 660caaatgttct cgatgaaggt ggacaacgtc gaatacgcgc ttctcactgc cattgtgatc 720ttctcggacc ggccgggcct ggagaaggcc caactagtcg aagcgatcca gagctactac 780atcgacacgc tacgcattta tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840ttctacgcaa agctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc 900gagatgtgtt tctcactaaa gctcaaaaac cgcaaactgc ccaagttcct cgaggagatc 960tgggacgttc atgccatccc gccatcggtc cagtcgcacc ttcagattac ccaggaggag 1020aacgagcgtc tcgagcgggc tgagcgtatg cgggcatcgg ttgggggcgc cattaccgcc 1080ggcattgatt gcgactctgc ctccacttcg gcggcggcag ccgcggccca gcatcagcct 1140cagcctcagc cccagcccca accctcctcc ctgacccaga acgattccca gcaccagaca 1200cagccgcagc tacaacctca gctaccacct cagctgcaag gtcaactgca accccagctc 1260caaccacagc ttcagacgca actccagcca cagattcaac cacagccaca gctccttccc 1320gtctccgctc ccgtgcccgc ctccgtaacc gcacctggtt ccttgtccgc ggtcagtacg 1380agcagcgaat acatgggcgg aagtgcggcc ataggaccca tcacgccggc aaccaccagc 1440agtatcacgg ctgccgttac cgctagctcc accacatcag cggtaccgat gggcaacgga 1500gttggagtcg gtgttggggt gggcggcaac gtcagcatgt atgcgaacgc ccagacggcg 1560atggccttga tgggtgtagc cctgcattcg caccaagagc agcttatcgg gggagtggcg 1620gttaagtcgg agcactcgac gactgcatag 165091338DNAArtificial Sequencemisc_featureNovel Sequence 9tatgagcagc catctgaaga ggatctcagg cgtataatga gtcaacccga tgagaacgag 60agccaaacgg acgtcagctt tcggcatata accgagataa ccatactcac ggtccagttg 120attgttgagt ttgctaaagg tctaccagcg tttacaaaga taccccagga ggaccagatc 180acgttactaa aggcctgctc gtcggaggtg atgatgctgc gtatggcacg acgctatgac 240cacagctcgg actcaatatt cttcgcgaat aatagatcat atacgcggga ttcttacaaa 300atggccggaa tggctgataa cattgaagac ctgctgcatt tctgccgcca aatgttctcg 360atgaaggtgg acaacgtcga atacgcgctt ctcactgcca ttgtgatctt ctcggaccgg 420ccgggcctgg agaaggccca actagtcgaa gcgatccaga gctactacat cgacacgcta 480cgcatttata tactcaaccg ccactgcggc gactcaatga gcctcgtctt ctacgcaaag 540ctgctctcga tcctcaccga gctgcgtacg ctgggcaacc agaacgccga gatgtgtttc 600tcactaaagc tcaaaaaccg caaactgccc aagttcctcg aggagatctg ggacgttcat 660gccatcccgc catcggtcca gtcgcacctt cagattaccc aggaggagaa cgagcgtctc 720gagcgggctg agcgtatgcg ggcatcggtt gggggcgcca ttaccgccgg cattgattgc 780gactctgcct ccacttcggc ggcggcagcc gcggcccagc atcagcctca gcctcagccc 840cagccccaac cctcctccct gacccagaac gattcccagc accagacaca gccgcagcta 900caacctcagc taccacctca gctgcaaggt caactgcaac cccagctcca accacagctt 960cagacgcaac tccagccaca gattcaacca cagccacagc tccttcccgt ctccgctccc 1020gtgcccgcct ccgtaaccgc acctggttcc ttgtccgcgg tcagtacgag cagcgaatac 1080atgggcggaa gtgcggccat aggacccatc acgccggcaa ccaccagcag tatcacggct 1140gccgttaccg ctagctccac cacatcagcg gtaccgatgg gcaacggagt tggagtcggt 1200gttggggtgg gcggcaacgt cagcatgtat gcgaacgccc agacggcgat ggccttgatg 1260ggtgtagccc tgcattcgca ccaagagcag cttatcgggg gagtggcggt taagtcggag 1320cactcgacga ctgcatag 133810969DNAArtificial Sequencemisc_featureNovel Sequence 10cggccggaat gcgtcgtccc ggagaaccaa tgtgcgatga agcggcgcga aaagaaggcc 60cagaaggaga aggacaaaat gaccacttcg ccgagctctc agcatggcgg caatggcagc 120ttggcctctg gtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc 180gagccgcccc agcatgccac tattccgcta ctacctgatg aaatattggc caagtgtcaa 240gcgcgcaata taccttcctt aacgtacaat cagttggccg ttatatacaa gttaatttgg 300taccaggatg gctatgagca gccatctgaa gaggatctca ggcgtataat gagtcaaccc 360gatgagaacg agagccaaac ggacgtcagc tttcggcata taaccgagat aaccatactc 420acggtccagt tgattgttga gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480gaggaccaga tcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca 540cgacgctatg accacagctc ggactcaata ttcttcgcga ataatagatc atatacgcgg 600gattcttaca aaatggccgg aatggctgat aacattgaag acctgctgca tttctgccgc 660caaatgttct cgatgaaggt ggacaacgtc gaatacgcgc ttctcactgc cattgtgatc 720ttctcggacc ggccgggcct ggagaaggcc caactagtcg aagcgatcca gagctactac 780atcgacacgc tacgcattta tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840ttctacgcaa agctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc 900gagatgtgtt tctcactaaa gctcaaaaac cgcaaactgc ccaagttcct cgaggagatc 960tgggacgtt 96911412PRTArtificial Sequencemisc_featureNovel Sequence 11Lys Gly Pro Ala Pro Arg Gln Gln Glu Glu Leu Cys Leu Val Cys Gly1 5 10 15Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30Lys Gly Phe Phe Arg Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45Lys Phe Gly His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu65 70 75 80Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys 85 90 95Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp 100 105 110Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala 115 120 125Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu 130 135 140Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln145 150 155 160Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro 165 170 175Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp 180 185 190Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met 195 200 205Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro 210

215 220Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala225 230 235 240Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala 245 250 255Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp 260 265 270Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile Glu Asp Leu Leu His 275 280 285Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln305 310 315 320Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg 325 330 335Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile 340 345 350Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met 355 360 365Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu 370 375 380Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr385 390 395 400Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 405 41012412PRTArtificial Sequencemisc_featureNovel Sequence 12Lys Gly Pro Ala Pro Arg Gln Gln Glu Glu Leu Cys Leu Val Cys Gly1 5 10 15Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30Lys Gly Phe Phe Arg Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45Lys Phe Gly His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu65 70 75 80Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys 85 90 95Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp 100 105 110Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala 115 120 125Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu 130 135 140Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln145 150 155 160Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro 165 170 175Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp 180 185 190Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met 195 200 205Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro 210 215 220Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala225 230 235 240Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala 245 250 255Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp 260 265 270Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile Glu Asp Leu Leu His 275 280 285Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln305 310 315 320Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg 325 330 335Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile 340 345 350Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met 355 360 365Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu 370 375 380Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr385 390 395 400Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 405 41013334PRTArtificial Sequencemisc_featureNovel Sequence 13Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu1 5 10 15Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr 20 25 30Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro 35 40 45Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys 50 55 60Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn65 70 75 80Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu 85 90 95Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln 100 105 110Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr 115 120 125Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly 130 135 140Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu145 150 155 160Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr 165 170 175Asp Ala Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr 180 185 190Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile Glu Asp Leu 195 200 205Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile His 210 215 220Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu225 230 235 240Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr 245 250 255Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser 260 265 270Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu 275 280 285Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg 290 295 300Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser305 310 315 320His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 33014244PRTArtificial Sequencemisc_featureNovel Sequence 14Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile1 5 10 15Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr 20 25 30Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile 35 40 45Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro 50 55 60Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu65 70 75 80Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser Val Leu Phe Ala 85 90 95Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala 100 105 110Tyr Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met 115 120 125Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe 130 135 140Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln145 150 155 160Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser 165 170 175Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu 180 185 190Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser 195 200 205Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp 210 215 220Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser225 230 235 240Pro Thr Asn Leu15320PRTArtificial Sequencemisc_featureNovel Sequence 15Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu1 5 10 15Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr 20 25 30Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro 35 40 45Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys 50 55 60Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn65 70 75 80Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu 85 90 95Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln 100 105 110Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr 115 120 125Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly 130 135 140Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu145 150 155 160Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr 165 170 175Asp Ala Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr 180 185 190Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile Glu Asp Leu 195 200 205Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile His 210 215 220Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu225 230 235 240Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr 245 250 255Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser 260 265 270Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu 275 280 285Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg 290 295 300Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser305 310 315 32016625PRTArtificial Sequencemisc_featureNovel Sequence 16Gly Pro Ala Pro Arg Val Gln Glu Glu Leu Cys Leu Val Cys Gly Asp1 5 10 15Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys Lys 20 25 30Gly Phe Phe Arg Arg Ser Val Thr Lys Ser Ala Val Tyr Cys Cys Lys 35 40 45Phe Gly Arg Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln 50 55 60Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys65 70 75 80Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg Glu Lys Lys Ala 85 90 95Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser Ser Gln His Gly 100 105 110Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gln Asp Phe Val Lys Lys 115 120 125Glu Ile Leu Asp Leu Met Thr Cys Glu Pro Pro Gln His Ala Thr Ile 130 135 140Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln Ala Arg Asn Ile145 150 155 160Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr Lys Leu Ile Trp 165 170 175Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp Leu Arg Arg Ile 180 185 190Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp Val Ser Phe Arg 195 200 205His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe 210 215 220Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile225 230 235 240Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Met Ala 245 250 255Arg Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe Ala Asn Asn Arg 260 265 270Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met Ala Asp Asn Ile 275 280 285Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser Met Lys Val Asp 290 295 300Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg305 310 315 320Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr 325 330 335Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser 340 345 350Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu 355 360 365Arg Thr Leu Gly Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu 370 375 380Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His385 390 395 400Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu Glu 405 410 415Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala Ser Val Gly Gly 420 425 430Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala Ser Thr Ser Ala Ala 435 440 445Ala Ala Ala Ala Gln His Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro 450 455 460Ser Ser Leu Thr Gln Asn Asp Ser Gln His Gln Thr Gln Pro Gln Leu465 470 475 480Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln Leu 485 490 495Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro Gln Ile Gln Pro Gln Pro 500 505 510Gln Leu Leu Pro Val Ser Ala Pro Val Pro Ala Ser Val Thr Ala Pro 515 520 525Gly Ser Leu Ser Ala Val Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser 530 535 540Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala545 550 555 560Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly 565 570 575Val Gly Val Gly Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn 580 585 590Ala Gln Thr Ala Met Ala Leu Met Gly Val Ala Leu His Ser His Gln 595 600 605Glu Gln Leu Ile Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr 610 615 620Ala62517583PRTArtificial Sequencemisc_featureNovel Sequence 17Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met Asp Met Tyr1 5 10 15Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 20 25 30Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys 35 40 45Arg Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser 50 55 60Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly65 70 75 80Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro 85 90 95Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys 100 105 110Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val 115 120 125Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu 130 135 140Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln145 150 155 160Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val 165 170 175Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile 180 185 190Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val 195 200 205Met Met Leu Arg Met Ala Arg Arg Tyr Asp His Ser Ser Asp Ser Ile 210 215 220Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala225 230 235 240Gly Met Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys Arg Gln Met 245 250 255Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile 260 265 270Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu 275 280 285Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn 290 295 300Arg His Cys Gly Asp Ser Met Ser Leu Val

Phe Tyr Ala Lys Leu Leu305 310 315 320Ser Ile Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met 325 330 335Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu 340 345 350Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val Gln Ser His Leu 355 360 365Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala Glu Arg Met 370 375 380Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser385 390 395 400Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln Pro Gln Pro 405 410 415Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp Ser Gln His 420 425 430Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly 435 440 445Gln Leu Gln Pro Gln Leu Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro 450 455 460Gln Ile Gln Pro Gln Pro Gln Leu Leu Pro Val Ser Ala Pro Val Pro465 470 475 480Ala Ser Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser 485 490 495Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr 500 505 510Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala 515 520 525Val Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn 530 535 540Val Ser Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu Met Gly Val545 550 555 560Ala Leu His Ser His Gln Glu Gln Leu Ile Gly Gly Val Ala Val Lys 565 570 575Ser Glu His Ser Thr Thr Ala 58018549PRTArtificial Sequencemisc_featureNovel Sequence 18Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg1 5 10 15Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser 20 25 30Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gln Asp 35 40 45Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro Pro Gln 50 55 60His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln65 70 75 80Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr 85 90 95Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp 100 105 110Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp 115 120 125Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln Leu 130 135 140Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln145 150 155 160Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met 165 170 175Leu Arg Met Ala Arg Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe 180 185 190Ala Asn Asn Arg Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met 195 200 205Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser 210 215 220Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile225 230 235 240Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile 245 250 255Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His 260 265 270Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser Ile 275 280 285Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met Cys Phe 290 295 300Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile305 310 315 320Trp Asp Val His Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile 325 330 335Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala 340 345 350Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala Ser 355 360 365Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln Pro Gln Pro Gln Pro 370 375 380Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp Ser Gln His Gln Thr385 390 395 400Gln Pro Gln Leu Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly Gln Leu 405 410 415Gln Pro Gln Leu Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro Gln Ile 420 425 430Gln Pro Gln Pro Gln Leu Leu Pro Val Ser Ala Pro Val Pro Ala Ser 435 440 445Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser Glu Tyr 450 455 460Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser465 470 475 480Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val Pro 485 490 495Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn Val Ser 500 505 510Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu Met Gly Val Ala Leu 515 520 525His Ser His Gln Glu Gln Leu Ile Gly Gly Val Ala Val Lys Ser Glu 530 535 540His Ser Thr Thr Ala54519445PRTArtificial Sequencemisc_featureNovel Sequence 19Tyr Glu Gln Pro Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro1 5 10 15Asp Glu Asn Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu 20 25 30Ile Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu 35 40 45Pro Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys 50 55 60Ala Cys Ser Ser Glu Val Met Met Leu Arg Met Ala Arg Arg Tyr Asp65 70 75 80His Ser Ser Asp Ser Ile Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg 85 90 95Asp Ser Tyr Lys Met Ala Gly Met Ala Asp Asn Ile Glu Asp Leu Leu 100 105 110His Phe Cys Arg Gln Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr 115 120 125Ala Leu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu 130 135 140Lys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu145 150 155 160Arg Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val 165 170 175Phe Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg Thr Leu Gly 180 185 190Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys 195 200 205Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His Ala Ile Pro Pro 210 215 220Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu225 230 235 240Glu Arg Ala Glu Arg Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala 245 250 255Gly Ile Asp Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala 260 265 270Gln His Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr 275 280 285Gln Asn Asp Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu 290 295 300Pro Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln Pro Gln Leu305 310 315 320Gln Thr Gln Leu Gln Pro Gln Ile Gln Pro Gln Pro Gln Leu Leu Pro 325 330 335Val Ser Ala Pro Val Pro Ala Ser Val Thr Ala Pro Gly Ser Leu Ser 340 345 350Ala Val Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly 355 360 365Pro Ile Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala 370 375 380Ser Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly385 390 395 400Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala 405 410 415Met Ala Leu Met Gly Val Ala Leu His Ser His Gln Glu Gln Leu Ile 420 425 430Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr Ala 435 440 44520323PRTArtificial Sequencemisc_featureNovel Sequence 20Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg1 5 10 15Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser 20 25 30Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gln Asp 35 40 45Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro Pro Gln 50 55 60His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln65 70 75 80Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr 85 90 95Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp 100 105 110Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp 115 120 125Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln Leu 130 135 140Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln145 150 155 160Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met 165 170 175Leu Arg Met Ala Arg Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe 180 185 190Ala Asn Asn Arg Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met 195 200 205Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser 210 215 220Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile225 230 235 240Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile 245 250 255Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His 260 265 270Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser Ile 275 280 285Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met Cys Phe 290 295 300Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile305 310 315 320Trp Asp Val21987DNAArtificial Sequencemisc_featureNovel Sequence 21tgtgctatct gtggggaccg ctcctcaggc aaacactatg gggtatacag ttgtgagggc 60tgcaagggct tcttcaagag gacagtacgc aaagacctga cctacacctg ccgagacaac 120aaggactgcc tgatcgacaa gagacagcgg aaccggtgtc agtactgccg ctaccagaag 180tgcctggcca tgggcatgaa gcgggaagct gtgcaggagg agcggcagcg gggcaaggac 240cggaatgaga acgaggtgga gtccaccagc agtgccaacg aggacatgcc tgtagagaag 300attctggaag ccgagcttgc tgtcgagccc aagactgaga catacgtgga ggcaaacatg 360gggctgaacc ccagctcacc aaatgaccct gttaccaaca tctgtcaagc agcagacaag 420cagctcttca ctcttgtgga gtgggccaag aggatcccac acttttctga gctgccccta 480gacgaccagg tcatcctgct acgggcaggc tggaacgagc tgctgatcgc ctccttctcc 540caccgctcca tagctgtgaa agatgggatt ctcctggcca ccggcctgca cgtacaccgg 600aacagcgctc acagtgctgg ggtgggcgcc atctttgaca gggtgctaac agagctggtg 660tctaagatgc gtgacatgca gatggacaag acggagctgg gctgcctgcg agccattgtc 720ctgttcaacc ctgactctaa ggggctctca aaccctgctg aggtggaggc gttgagggag 780aaggtgtatg cgtcactaga agcgtactgc aaacacaagt accctgagca gccgggcagg 840tttgccaagc tgctgctccg cctgcctgca ctgcgttcca tcgggctcaa gtgcctggag 900cacctgttct tcttcaagct catcggggac acgcccatcg acaccttcct catggagatg 960ctggaggcac cacatcaagc cacctag 98722789DNAArtificial Sequencemisc_featureNovel Sequence 22aagcgggaag ctgtgcagga ggagcggcag cggggcaagg accggaatga gaacgaggtg 60gagtccacca gcagtgccaa cgaggacatg cctgtagaga agattctgga agccgagctt 120gctgtcgagc ccaagactga gacatacgtg gaggcaaaca tggggctgaa ccccagctca 180ccaaatgacc ctgttaccaa catctgtcaa gcagcagaca agcagctctt cactcttgtg 240gagtgggcca agaggatccc acacttttct gagctgcccc tagacgacca ggtcatcctg 300ctacgggcag gctggaacga gctgctgatc gcctccttct cccaccgctc catagctgtg 360aaagatggga ttctcctggc caccggcctg cacgtacacc ggaacagcgc tcacagtgct 420ggggtgggcg ccatctttga cagggtgcta acagagctgg tgtctaagat gcgtgacatg 480cagatggaca agacggagct gggctgcctg cgagccattg tcctgttcaa ccctgactct 540aaggggctct caaaccctgc tgaggtggag gcgttgaggg agaaggtgta tgcgtcacta 600gaagcgtact gcaaacacaa gtaccctgag cagccgggca ggtttgccaa gctgctgctc 660cgcctgcctg cactgcgttc catcgggctc aagtgcctgg agcacctgtt cttcttcaag 720ctcatcgggg acacgcccat cgacaccttc ctcatggaga tgctggaggc accacatcaa 780gccacctag 78923714DNAArtificial Sequencemisc_featureNovel Sequence 23gccaacgagg acatgcctgt agagaagatt ctggaagccg agcttgctgt cgagcccaag 60actgagacat acgtggaggc aaacatgggg ctgaacccca gctcaccaaa tgaccctgtt 120accaacatct gtcaagcagc agacaagcag ctcttcactc ttgtggagtg ggccaagagg 180atcccacact tttctgagct gcccctagac gaccaggtca tcctgctacg ggcaggctgg 240aacgagctgc tgatcgcctc cttctcccac cgctccatag ctgtgaaaga tgggattctc 300ctggccaccg gcctgcacgt acaccggaac agcgctcaca gtgctggggt gggcgccatc 360tttgacaggg tgctaacaga gctggtgtct aagatgcgtg acatgcagat ggacaagacg 420gagctgggct gcctgcgagc cattgtcctg ttcaaccctg actctaaggg gctctcaaac 480cctgctgagg tggaggcgtt gagggagaag gtgtatgcgt cactagaagc gtactgcaaa 540cacaagtacc ctgagcagcc gggcaggttt gccaagctgc tgctccgcct gcctgcactg 600cgttccatcg ggctcaagtg cctggagcac ctgttcttct tcaagctcat cggggacacg 660cccatcgaca ccttcctcat ggagatgctg gaggcaccac atcaagccac ctag 71424536DNAArtificial Sequencemisc_featureNovel Sequence 24ggatcccaca cttttctgag ctgcccctag acgaccaggt catcctgcta cgggcaggct 60ggaacgagct gctgatcgcc tccttctccc accgctccat agctgtgaaa gatgggattc 120tcctggccac cggcctgcac gtacaccgga acagcgctca cagtgctggg gtgggcgcca 180tctttgacag ggtgctaaca gagctggtgt ctaagatgcg tgacatgcag atggacaaga 240cggagctggg ctgcctgcga gccattgtcc tgttcaaccc tgactctaag gggctctcaa 300accctgctga ggtggaggcg ttgagggaga aggtgtatgc gtcactagaa gcgtactgca 360aacacaagta ccctgagcag ccgggcaggt ttgccaagct gctgctccgc ctgcctgcac 420tgcgttccat cgggctcaag tgcctggagc acctgttctt cttcaagctc atcggggaca 480cgcccatcga caccttcctc atggagatgc tggaggcacc acatcaagcc acctag 53625672DNAArtificial Sequencemisc_featureNovel Sequence 25gccaacgagg acatgcctgt agagaagatt ctggaagccg agcttgctgt cgagcccaag 60actgagacat acgtggaggc aaacatgggg ctgaacccca gctcaccaaa tgaccctgtt 120accaacatct gtcaagcagc agacaagcag ctcttcactc ttgtggagtg ggccaagagg 180atcccacact tttctgagct gcccctagac gaccaggtca tcctgctacg ggcaggctgg 240aacgagctgc tgatcgcctc cttctcccac cgctccatag ctgtgaaaga tgggattctc 300ctggccaccg gcctgcacgt acaccggaac agcgctcaca gtgctggggt gggcgccatc 360tttgacaggg tgctaacaga gctggtgtct aagatgcgtg acatgcagat ggacaagacg 420gagctgggct gcctgcgagc cattgtcctg ttcaaccctg actctaaggg gctctcaaac 480cctgctgagg tggaggcgtt gagggagaag gtgtatgcgt cactagaagc gtactgcaaa 540cacaagtacc ctgagcagcc gggcaggttt gccaagctgc tgctccgcct gcctgcactg 600cgttccatcg ggctcaagtg cctggagcac ctgttcttct tcaagctcat cggggacacg 660cccatcgaca cc 672261123DNAArtificial Sequencemisc_featureNovel Sequence 26tgcgccatct gcggggaccg ctcctcaggc aagcactatg gagtgtacag ctgcgagggg 60tgcaagggct tcttcaagcg gacggtgcgc aaggacctga cctacacctg ccgcgacaac 120aaggactgcc tgattgacaa gcggcagcgg aaccggtgcc agtactgccg ctaccagaag 180tgcctggcca tgggcatgaa gcgggaagcc gtgcaggagg agcggcagcg tggcaaggac 240cggaacgaga atgaggtgga gtcgaccagc agcgccaacg aggacatgcc ggtggagagg 300atcctggagg ctgagctggc cgtggagccc aagaccgaga cctacgtgga ggcaaacatg 360gggctgaacc ccagctcgcc gaacgaccct gtcaccaaca tttgccaagc agccgacaaa 420cagcttttca ccctggtgga gtgggccaag cggatcccac acttctcaga gctgcccctg 480gacgaccagg tcatcctgct gcgggcaggc tggaatgagc tgctcatcgc ctccttctcc 540caccgctcca tcgccgtgaa ggacgggatc ctcctggcca ccgggctgca cgtccaccgg 600aacagcgccc acagcgcagg ggtgggcgcc atctttgaca gggtgctgac ggagcttgtg 660tccaagatgc gggacatgca gatggacaag acggagctgg gctgcctgcg cgccatcgtc 720ctctttaacc ctgactccaa ggggctctcg aacccggccg aggtggaggc gctgagggag 780aaggtctatg cgtccttgga ggcctactgc aagcacaagt acccagagca gccgggaagg 840ttcgctaagc tcttgctccg cctgccggct ctgcgctcca tcgggctcaa atgcctggaa 900catctcttct tcttcaagct catcggggac acacccattg acaccttcct tatggagatg 960ctggaggcgc cgcaccaaat gacttaggcc tgcgggccca tcctttgtgc ccacccgttc 1020tggccaccct gcctggacgc cagctgttct tctcagcctg agccctgtcc ctgcccttct 1080ctgcctggcc

tgtttggact ttggggcaca gcctgtcact gct 112327925DNAArtificial Sequencemisc_featureNovel Sequence 27aagcgggaag ccgtgcagga ggagcggcag cgtggcaagg accggaacga gaatgaggtg 60gagtcgacca gcagcgccaa cgaggacatg ccggtggaga ggatcctgga ggctgagctg 120gccgtggagc ccaagaccga gacctacgtg gaggcaaaca tggggctgaa ccccagctcg 180ccgaacgacc ctgtcaccaa catttgccaa gcagccgaca aacagctttt caccctggtg 240gagtgggcca agcggatccc acacttctca gagctgcccc tggacgacca ggtcatcctg 300ctgcgggcag gctggaatga gctgctcatc gcctccttct cccaccgctc catcgccgtg 360aaggacggga tcctcctggc caccgggctg cacgtccacc ggaacagcgc ccacagcgca 420ggggtgggcg ccatctttga cagggtgctg acggagcttg tgtccaagat gcgggacatg 480cagatggaca agacggagct gggctgcctg cgcgccatcg tcctctttaa ccctgactcc 540aaggggctct cgaacccggc cgaggtggag gcgctgaggg agaaggtcta tgcgtccttg 600gaggcctact gcaagcacaa gtacccagag cagccgggaa ggttcgctaa gctcttgctc 660cgcctgccgg ctctgcgctc catcgggctc aaatgcctgg aacatctctt cttcttcaag 720ctcatcgggg acacacccat tgacaccttc cttatggaga tgctggaggc gccgcaccaa 780atgacttagg cctgcgggcc catcctttgt gcccacccgt tctggccacc ctgcctggac 840gccagctgtt cttctcagcc tgagccctgt ccctgccctt ctctgcctgg cctgtttgga 900ctttggggca cagcctgtca ctgct 92528850DNAArtificial Sequencemisc_featureNovel Sequence 28gccaacgagg acatgccggt ggagaggatc ctggaggctg agctggccgt ggagcccaag 60accgagacct acgtggaggc aaacatgggg ctgaacccca gctcgccgaa cgaccctgtc 120accaacattt gccaagcagc cgacaaacag cttttcaccc tggtggagtg ggccaagcgg 180atcccacact tctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 240aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc 300ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 360tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 420gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac 480ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc ctactgcaag 540cacaagtacc cagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 600cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca 660cccattgaca ccttccttat ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 720gggcccatcc tttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 780cagcctgagc cctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc 840tgtcactgct 85029670DNAArtificial Sequencemisc_featureNovel Sequence 29atcccacact tctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 60aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc 120ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 180tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 240gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac 300ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc ctactgcaag 360cacaagtacc cagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 420cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca 480cccattgaca ccttccttat ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 540gggcccatcc tttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 600cagcctgagc cctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc 660tgtcactgct 67030672DNAArtificial Sequencemisc_featureNovel Sequence 30gccaacgagg acatgccggt ggagaggatc ctggaggctg agctggccgt ggagcccaag 60accgagacct acgtggaggc aaacatgggg ctgaacccca gctcgccgaa cgaccctgtc 120accaacattt gccaagcagc cgacaaacag cttttcaccc tggtggagtg ggccaagcgg 180atcccacact tctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 240aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc 300ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 360tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 420gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac 480ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc ctactgcaag 540cacaagtacc cagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 600cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca 660cccattgaca cc 67231328PRTArtificial Sequencemisc_featureNovel Sequence 31Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His Tyr Gly Val Tyr1 5 10 15Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr Val Arg Lys Asp 20 25 30Leu Thr Tyr Thr Cys Arg Asp Asn Lys Asp Cys Leu Ile Asp Lys Arg 35 40 45Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln Lys Cys Leu Ala Met 50 55 60Gly Met Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp65 70 75 80Arg Asn Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met 85 90 95Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr 100 105 110Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn 115 120 125Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr 130 135 140Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu145 150 155 160Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185 190Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val 195 200 205Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg 210 215 220Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val225 230 235 240Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu 245 250 255Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His 260 265 270Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu 275 280 285Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe 290 295 300Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met305 310 315 320Leu Glu Ala Pro His Gln Ala Thr 32532262PRTArtificial Sequencemisc_featureNovel Sequence 32Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn1 5 10 15Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val 20 25 30Glu Lys Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr 35 40 45Tyr Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55 60Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val65 70 75 80Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp 85 90 95Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser 100 105 110Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr 115 120 125Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala 130 135 140Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met145 150 155 160Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe 165 170 175Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu 180 185 190Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr 195 200 205Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala 210 215 220Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys225 230 235 240Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu 245 250 255Ala Pro His Gln Ala Thr 26033237PRTArtificial Sequencemisc_featureNovel Sequence 33Ala Asn Glu Asp Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala1 5 10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp65 70 75 80Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu 165 170 175Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys 180 185 190Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu 195 200 205Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr 210 215 220Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Ala Thr225 230 23534177PRTArtificial Sequencemisc_featureNovel Sequence 34Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu1 5 10 15Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser 20 25 30Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His 35 40 45Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val 50 55 60Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr65 70 75 80Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys 85 90 95Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr 100 105 110Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly 115 120 125Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly 130 135 140Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr145 150 155 160Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Ala 165 170 175Thr35224PRTArtificial Sequencemisc_featureNovel Sequence 35Ala Asn Glu Asp Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala1 5 10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp65 70 75 80Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu 165 170 175Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys 180 185 190Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu 195 200 205Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr 210 215 22036328PRTArtificial Sequencemisc_featureNovel Sequence 36Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His Tyr Gly Val Tyr1 5 10 15Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys Arg Thr Val Arg Lys Asp 20 25 30Leu Thr Tyr Thr Cys Arg Asp Asn Lys Asp Cys Leu Ile Asp Lys Arg 35 40 45Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln Lys Cys Leu Ala Met 50 55 60Gly Met Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp65 70 75 80Arg Asn Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met 85 90 95Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr 100 105 110Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn 115 120 125Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr 130 135 140Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu145 150 155 160Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185 190Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val 195 200 205Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg 210 215 220Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val225 230 235 240Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu 245 250 255Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His 260 265 270Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu 275 280 285Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe 290 295 300Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met305 310 315 320Leu Glu Ala Pro His Gln Met Thr 32537262PRTArtificial Sequencemisc_featureNovel Sequence 37Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn1 5 10 15Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val 20 25 30Glu Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr 35 40 45Tyr Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55 60Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val65 70 75 80Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp 85 90 95Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser 100 105 110Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr 115 120 125Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala 130 135 140Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met145 150 155 160Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe 165 170 175Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu 180 185 190Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr 195 200 205Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala 210 215 220Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys225 230 235 240Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu 245 250 255Ala Pro His Gln Met Thr 26038237PRTArtificial Sequencemisc_featureNovel Sequence 38Ala Asn Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala1 5

10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp65 70 75 80Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu 165 170 175Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys 180 185 190Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu 195 200 205Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr 210 215 220Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met Thr225 230 23539177PRTArtificial Sequencemisc_featureNovel Sequence 39Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu1 5 10 15Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser 20 25 30Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His 35 40 45Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val 50 55 60Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr65 70 75 80Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys 85 90 95Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr 100 105 110Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly 115 120 125Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly 130 135 140Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr145 150 155 160Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met 165 170 175Thr40224PRTArtificial Sequencemisc_featureNovel Sequence 40Ala Asn Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala1 5 10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp65 70 75 80Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu 165 170 175Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys 180 185 190Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu 195 200 205Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr 210 215 22041441DNAArtificial Sequencemisc_featureNovel Sequence 41atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg 180ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt 240ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat 300aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta 360acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420caaagacagt tgactgtatc g 44142147PRTArtificial Sequencemisc_featureNovel Sequence 42Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu1 5 10 15Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile65 70 75 80Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140Thr Val Ser14543606DNAArtificial Sequencemisc_featureNovel Sequence 43atgaaagcgt taacggccag gcaacaagag gtgtttgatc tcatccgtga tcacatcagc 60cagacaggta tgccgccgac gcgtgcggaa atcgcgcagc gtttggggtt ccgttcccca 120aacgcggctg aagaacatct gaaggcgctg gcacgcaaag gcgttattga aattgtttcc 180ggcgcatcac gcgggattcg tctgttgcag gaagaggaag aagggttgcc gctggtaggt 240cgtgtggctg ccggtgaacc acttctggcg caacagcata ttgaaggtca ttatcaggtc 300gatccttcct tattcaagcc gaatgctgat ttcctgctgc gcgtcagcgg gatgtcgatg 360aaagatatcg gcattatgga tggtgacttg ctggcagtgc ataaaactca ggatgtacgt 420aacggtcagg tcgttgtcgc acgtattgat gacgaagtta ccgttaagcg cctgaaaaaa 480cagggcaata aagtcgaact gttgccagaa aatagcgagt ttaaaccaat tgtcgtagat 540cttcgtcagc agagcttcac cattgaaggg ctggcggttg gggttattcg caacggcgac 600tggctg 60644202PRTArtificial Sequencemisc_featureNovel Sequence 44Met Lys Ala Leu Thr Ala Arg Gln Gln Glu Val Phe Asp Leu Ile Arg1 5 10 15Asp His Ile Ser Gln Thr Gly Met Pro Pro Thr Arg Ala Glu Ile Ala 20 25 30Gln Arg Leu Gly Phe Arg Ser Pro Asn Ala Ala Glu Glu His Leu Lys 35 40 45Ala Leu Ala Arg Lys Gly Val Ile Glu Ile Val Ser Gly Ala Ser Arg 50 55 60Gly Ile Arg Leu Leu Gln Glu Glu Glu Glu Gly Leu Pro Leu Val Gly65 70 75 80Arg Val Ala Ala Gly Glu Pro Leu Leu Ala Gln Gln His Ile Glu Gly 85 90 95His Tyr Gln Val Asp Pro Ser Leu Phe Lys Pro Asn Ala Asp Phe Leu 100 105 110Leu Arg Val Ser Gly Met Ser Met Lys Asp Ile Gly Ile Met Asp Gly 115 120 125Asp Leu Leu Ala Val His Lys Thr Gln Asp Val Arg Asn Gly Gln Val 130 135 140Val Val Ala Arg Ile Asp Asp Glu Val Thr Val Lys Arg Leu Lys Lys145 150 155 160Gln Gly Asn Lys Val Glu Leu Leu Pro Glu Asn Ser Glu Phe Lys Pro 165 170 175Ile Val Val Asp Leu Arg Gln Gln Ser Phe Thr Ile Glu Gly Leu Ala 180 185 190Val Gly Val Ile Arg Asn Gly Asp Trp Leu 195 20045271DNAArtificial Sequencemisc_featureNovel Sequence 45atgggcccta aaaagaagcg taaagtcgcc cccccgaccg atgtcagcct gggggacgag 60ctccacttag acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat 120ctggacatgt tgggggacgg ggattccccg gggccgggat ttacccccca cgactccgcc 180ccctacggcg ctctggatat ggccgacttc gagtttgagc agatgtttac cgatgccctt 240ggaattgacg agtacggtgg ggaattcccg g 2714690PRTArtificial Sequencemisc_featureNovel Sequence 46Met Gly Pro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser1 5 10 15Leu Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His 20 25 30Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp 35 40 45Ser Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala 50 55 60Leu Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu65 70 75 80Gly Ile Asp Glu Tyr Gly Gly Glu Phe Pro 85 904719DNAArtificial Sequencemisc_featureNovel Sequence 47ggagtactgt cctccgagc 1948666DNAArtificial Sequencemisc_featureNovel Sequence 48ggatccccag cttggaattc gacaggttat cagcaacaac acagtcatat ccattctcaa 60ttagctctac cacagtgtgt gaaccaatgt atccagcacc acctgtaacc aaaacaattt 120tagaagtact ttcactttgt aactgagctg tcatttatat tgaattttca aaaattctta 180cttttttttt ggatggacgc aaagaagttt aataatcata ttacatggca ttaccaccat 240atacatatcc atatacatat ccatatctaa tcttacctcg actgctgtat ataaaaccag 300tggttatatg tacagtactg ctgtatataa aaccagtggt tatatgtaca gtacgtcgac 360tgctgtatat aaaaccagtg gttatatgta cagtactgct gtatataaaa ccagtggtta 420tatgtacagt acgtcgaggg atgataatgc gattagtttt ttagccttat ttctggggta 480attaatcagc gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca 540taaccacttt aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta 600ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa 660actata 666491542DNAArtificial Sequencemisc_featureNovel Sequence 49ctggacctga aacacgaagt ggcttaccga ggggtgctcc caggccaggt gaaggccgaa 60ccgggggtcc acaacggcca ggtcaacggc cacgtgaggg actggatggc aggcggcgct 120ggtgccaatt cgccgtctcc gggagcggtg gctcaacccc agcctaacaa tgggtattcg 180tcgccactct cctcgggaag ctacgggccc tacagtccaa atgggaaaat aggccgtgag 240gaactgtcgc cagcttcaag tataaatggg tgcagtacag atggcgaggc acgacgtcag 300aagaagggcc ctgcgccccg tcagcaagag gaactgtgtc tggtatgcgg ggacagagcc 360tccggatacc actacaatgc gctcacgtgt gaagggtgta aagggttctt cagacggagt 420gttaccaaaa atgcggttta tatttgtaaa ttcggtcacg cttgcgaaat ggacatgtac 480atgcgacgga aatgccagga gtgccgcctg aagaagtgct tagctgtagg catgaggcct 540gagtgcgtag tacccgagac tcagtgcgcc atgaagcgga aagagaagaa agcacagaag 600gagaaggaca aactgcctgt cagcacgacg acggtggacg accacatgcc gcccattatg 660cagtgtgaac ctccacctcc tgaagcagca aggattcacg aagtggtccc aaggtttctc 720tccgacaagc tgttggagac aaaccggcag aaaaacatcc cccagttgac agccaaccag 780cagttcctta tcgccaggct catctggtac caggacgggt acgagcagcc ttctgatgaa 840gatttgaaga ggattacgca gacgtggcag caagcggacg atgaaaacga agagtctgac 900actcccttcc gccagatcac agagatgact atcctcacgg tccaacttat cgtggagttc 960gcgaagggat tgccagggtt cgccaagatc tcgcagcctg atcaaattac gctgcttaag 1020gcttgctcaa gtgaggtaat gatgctccga gtcgcgcgac gatacgatgc ggcctcagac 1080agtgttctgt tcgcgaacaa ccaagcgtac actcgcgaca actaccgcaa ggctggcatg 1140gcctacgtca tcgaggatct actgcacttc tgccggtgca tgtactctat ggcgttggac 1200aacatccatt acgcgctgct cacggctgtc gtcatctttt ctgaccggcc agggttggag 1260cagccgcaac tggtggaaga aatccagcgg tactacctga atacgctccg catctatatc 1320ctgaaccagc tgagcgggtc ggcgcgttcg tccgtcatat acggcaagat cctctcaatc 1380ctctctgagc tacgcacgct cggcatgcaa aactccaaca tgtgcatctc cctcaagctc 1440aagaacagaa agctgccgcc tttcctcgag gagatctggg atgtggcgga catgtcgcac 1500acccaaccgc cgcctatcct cgagtccccc acgaatctct ag 154250513PRTArtificial Sequencemisc_featureNovel Sequence 50Leu Asp Leu Lys His Glu Val Ala Tyr Arg Gly Val Leu Pro Gly Gln1 5 10 15Val Lys Ala Glu Pro Gly Val His Asn Gly Gln Val Asn Gly His Val 20 25 30Arg Asp Trp Met Ala Gly Gly Ala Gly Ala Asn Ser Pro Ser Pro Gly 35 40 45Ala Val Ala Gln Pro Gln Pro Asn Asn Gly Tyr Ser Ser Pro Leu Ser 50 55 60Ser Gly Ser Tyr Gly Pro Tyr Ser Pro Asn Gly Lys Ile Gly Arg Glu65 70 75 80Glu Leu Ser Pro Ala Ser Ser Ile Asn Gly Cys Ser Thr Asp Gly Glu 85 90 95Ala Arg Arg Gln Lys Lys Gly Pro Ala Pro Arg Gln Gln Glu Glu Leu 100 105 110Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu 115 120 125Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Thr Lys Asn 130 135 140Ala Val Tyr Ile Cys Lys Phe Gly His Ala Cys Glu Met Asp Met Tyr145 150 155 160Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 165 170 175Gly Met Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys 180 185 190Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser 195 200 205Thr Thr Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro 210 215 220Pro Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu225 230 235 240Ser Asp Lys Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu 245 250 255Thr Ala Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp 260 265 270Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr 275 280 285Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg 290 295 300Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe305 310 315 320Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile 325 330 335Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala 340 345 350Arg Arg Tyr Asp Ala Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln 355 360 365Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile 370 375 380Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp385 390 395 400Asn Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg 405 410 415Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr 420 425 430Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala 435 440 445Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu 450 455 460Arg Thr Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu465 470 475 480Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala 485 490 495Asp Met Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn 500 505 510Leu514375DNAArtificial Sequencemisc_featureNovel Sequence 51tgtaattttg atgggcgccg tgatgcaccg tgtgccatat tgccatccag tcgaatagaa 60aaaaaaaaaa aaaaaaaaat atcagttgtt ttgtccctcg ctcgctttcg agtgtattcg 120gaatattaga cgtcataatt cacgagtgtc ttttaaattt atatagcgat tagcggggcc 180gtttgttgga cgtgcgcttg cgtttagtgg agtgcaggga tagtgaggcg agtatggtag 240ttcgtggtca tgtcaagtgt ggcgaagaaa gacaagccga cgatgtcggt gacggcgctg 300atcaactggg cgcggccggc gccgccaggc ccgccgcagc cgcagtcagc gtcgcctgcg 360ccggcagcca tgctgcagca gctcccgacg cagtcaatgc agtcgttaaa ccacatccca 420actgtcgatt gctcgctcga tatgcagtgg cttaatttag aacctggatt catgtcgcct 480atgtcacctc ctgagatgaa accagacacc gccatgcttg atgggctacg agacgacgcc 540acttcgccgc ctaacttcaa gaactacccg cctaatcacc ccctgagtgg ctccaaacac 600ctatgctcta tatgcggcga cagggcgtct gggaagcact atggggtgta cagttgcgaa 660ggatgcaagg gtttcttcaa gcggaccgtc cggaaggacc tgtcgtacgc ttgccgggag 720gagcggaact gcatcataga caagcgacaa aggaaccgat gccagtactg ccgctatcaa 780aagtgtttgg cttgcggtat gaagcgagag gcggtgcaag aggagcgcca gaggaatgct 840cgcggcgcgg aggatgcgca cccgagtagc tcggtgcagg taagcgatga gctgtcaatc 900gagcgcctaa cggagatgga gtctttggtg gcagatccca gcgaggagtt ccagttcctc 960cgcgtggggc ctgacagcaa cgtgcctcca cgttaccgcg cgcccgtctc ctccctctgc 1020caaataggca acaagcaaat agcggcgttg

gtggtatggg cgcgcgacat ccctcatttc 1080gggcagctgg agctggacga tcaagtggta ctcatcaagg cctcctggaa tgagctgcta 1140ctcttcgcca tcgcctggcg ctctatggag tatttggaag atgagaggga gaacggggac 1200ggaacgcgga gcaccactca gccacaactg atgtgtctca tgcctggcat gacgttgcac 1260cgcaactcgg cgcagcaggc gggcgtgggc gccatcttcg accgcgtgct gtccgagctc 1320agtctgaaga tgcgcacctt gcgcatggac caggccgagt acgtcgcgct caaagccatc 1380gtgctgctca accctgatgt gaaaggactg aagaatcggc aagaagttga cgttttgcga 1440gaaaaaatgt tctcttgcct ggacgactac tgccggcggt cgcgaagcaa cgaggaaggc 1500cggtttgcgt ccttgctgct gcggctgcca gctctccgct ccatctcgct caagagcttc 1560gaacacctct acttcttcca cctcgtggcc gaaggctcca tcagcggata catacgagag 1620gcgctccgaa accacgcgcc tccgatcgac gtcaatgcca tgatgtaaag tgcgatacac 1680gccctgccga tgtgagaaga actatggcta atagaagcga aactgaatac atctagggtg 1740ggacttaact tgggactatc attaaagtat cacgcaaatt atgcgtagtc agaaagtcgc 1800gtcgatcaaa cttttttata aacgaattga gtttctaacg actgcaacac agcggagttt 1860tgcttctgat agtttttatt ctaatggtta agatgcttta cacgggcatt attgacattc 1920aagtgtaagt ggaagttgac aaccttgaca tttatatcac gtttgtaatt ggttaaataa 1980attaattaat cacaagtaag actaacatca acgtcacgat actaacgcca tttagtgata 2040tttttcatgt caagaaactc attgttttga taaaatattt ttctaattac tccagtgaac 2100tcatccaaat gtgacccagt ttcccgcaga gttgcccgtg taaaatcatc tttagggaca 2160tatcccccgc tatctcatga aattccaagg atcagtaggg gccaattccc ccgatgtgtt 2220gggaggcaga attttcgata atctacgact attgttagcc tacgaattag ttgaattttt 2280tgaaattatt tttattaagt cgccactttc caaacacatc agcagggtat atgtgcaatt 2340ttgtaacgat aactctattc atttctgata tttatcgaaa ttttatctta cataacatgc 2400tggctggtcc aggtgtttgg tagttacata tgtatctacg gtttgtttta aattatagct 2460tttttattgt aatctgtata aaattgagtt atcttacttc acactacgat cgagtaaacc 2520catcgtcagc tacgaaaaac taatcgtata aggcgtaaga gtaaataact aattgacaac 2580cagcaacgag gaccacctca gtcctcgtgc ttacattgtg ccgtagctta atatgatgga 2640agctgtcgtc gttacgacat tagataaagt gcatgaatac caaaaatgta ccatcccgta 2700ctgatctctc atgctctcgc tgcgtgggac ccgtgtcgag tgtcgtaagg actgactaat 2760attttagact aggcgtctat gcttcagtaa ttccttatac atattataag tcatccaaat 2820aacgagtaag gcggcatgtt gagatcagca ttccgagagt caaagagccc ctaacgtgac 2880tgagaagtag agacaataca ctgattttct gagatgaacg caaccgagat tgacactaaa 2940aatctattta tggatttcaa aatggcgatg cttgattgtc tgcggcgtgg atagactgaa 3000atgggtttgc ttaacactgg atattgtttt tattagttaa tagtcttaca ttgcaagttg 3060gtaattcggt gctaatatcg accggtttgt taactatcta acggttccca gtgtcaggca 3120cacatctttc ccaagcagac aacgcaagag tgtacaaaat gtacatgtta caaaataagg 3180aacattcgtc ggataagtgt aacagttgat aggtaaagaa aatggggccg cctctttatt 3240attacgtagc cgtaaaatta ttaacgtatt tagtttagat gttcagctaa ttaggataat 3300tctatttgtc gagtacctag atgtccatag tgaattaata taataattag actgttacgc 3360gtaggtaatt ataaagttta ccaaatctct cttcaaagca aaaactttgt acacttccgt 3420actgagacgt cgtagcttat tctgattcac gaaatatttg gatcacattg ttacaaggcg 3480accgtcacgt agtatatgat tatttacaaa tgacacgtat gtatcaatgc tataagtgtt 3540ttcgttacat atgtcggtgc tttaacgtgc atttcgatgt gcagattaaa aatagcaaga 3600aatcttgaaa ttgttttaga aaatatttga tttccttatt gaaagttatt tttaaatgta 3660aatatttcgt aatcataata attatgtatt gtgtagttat ttcaccttta cggttgggat 3720attatttaat ggtggcctac gaaagtgatt ataaccatcc gcgtcctcaa aaaggccagt 3780ttatttttgt acctcataca tactaattac gtaagtaata tcaggcgaat ggttgactaa 3840caactaacca gtattaaaaa ttaaaagact tcgtcctaat aaaatgtaat atctatgtat 3900aaaaatgaaa aatctggcgt ataataggta aaattaaact agattgttaa tgaatgtgat 3960gtctcataaa cgtttagttt ttaatgagaa acatgtttag tcgcctacta taagacgaga 4020cggcaagctc accgagttaa ctcgtaaaca ggaatgttga aaaagatgac acaatttata 4080tttggtattg aaattatgac taaccatgcg ctctatcgtt tgttatggat gcatagtatt 4140gctgttgaaa ataatggaat taggtaatta ctgcattaat gttgaaaact tgatattatt 4200ctatggttgg gtatgaattc tatgttggaa gtgttgcagc ggttgtaaag atgatttata 4260atgatgttca ctaaatatct gactaaatgt aagttatttt tttttgtata gacatagctt 4320taagatgaag gtgattaaac tttatcctta tcacaataaa aaaaaaaaaa aaaaa 437552472PRTArtificial Sequencemisc_featureNovel Sequence 52Met Ser Ser Val Ala Lys Lys Asp Lys Pro Thr Met Ser Val Thr Ala1 5 10 15Leu Ile Asn Trp Ala Arg Pro Ala Pro Pro Gly Pro Pro Gln Pro Gln 20 25 30Ser Ala Ser Pro Ala Pro Ala Ala Met Leu Gln Gln Leu Pro Thr Gln 35 40 45Ser Met Gln Ser Leu Asn His Ile Pro Thr Val Asp Cys Ser Leu Asp 50 55 60Met Gln Trp Leu Asn Leu Glu Pro Gly Phe Met Ser Pro Met Ser Pro65 70 75 80Pro Glu Met Lys Pro Asp Thr Ala Met Leu Asp Gly Leu Arg Asp Asp 85 90 95Ala Thr Ser Pro Pro Asn Phe Lys Asn Tyr Pro Pro Asn His Pro Leu 100 105 110Ser Gly Ser Lys His Leu Cys Ser Ile Cys Gly Asp Arg Ala Ser Gly 115 120 125Lys His Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys 130 135 140Arg Thr Val Arg Lys Asp Leu Ser Tyr Ala Cys Arg Glu Glu Arg Asn145 150 155 160Cys Ile Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr 165 170 175Gln Lys Cys Leu Ala Cys Gly Met Lys Arg Glu Ala Val Gln Glu Glu 180 185 190Arg Gln Arg Asn Ala Arg Gly Ala Glu Asp Ala His Pro Ser Ser Ser 195 200 205Val Gln Val Ser Asp Glu Leu Ser Ile Glu Arg Leu Thr Glu Met Glu 210 215 220Ser Leu Val Ala Asp Pro Ser Glu Glu Phe Gln Phe Leu Arg Val Gly225 230 235 240Pro Asp Ser Asn Val Pro Pro Arg Tyr Arg Ala Pro Val Ser Ser Leu 245 250 255Cys Gln Ile Gly Asn Lys Gln Ile Ala Ala Leu Val Val Trp Ala Arg 260 265 270Asp Ile Pro His Phe Gly Gln Leu Glu Leu Asp Asp Gln Val Val Leu 275 280 285Ile Lys Ala Ser Trp Asn Glu Leu Leu Leu Phe Ala Ile Ala Trp Arg 290 295 300Ser Met Glu Tyr Leu Glu Asp Glu Arg Glu Asn Gly Asp Gly Thr Arg305 310 315 320Ser Thr Thr Gln Pro Gln Leu Met Cys Leu Met Pro Gly Met Thr Leu 325 330 335His Arg Asn Ser Ala Gln Gln Ala Gly Val Gly Ala Ile Phe Asp Arg 340 345 350Val Leu Ser Glu Leu Ser Leu Lys Met Arg Thr Leu Arg Met Asp Gln 355 360 365Ala Glu Tyr Val Ala Leu Lys Ala Ile Val Leu Leu Asn Pro Asp Val 370 375 380Lys Gly Leu Lys Asn Arg Gln Glu Val Asp Val Leu Arg Glu Lys Met385 390 395 400Phe Ser Cys Leu Asp Asp Tyr Cys Arg Arg Ser Arg Ser Asn Glu Glu 405 410 415Gly Arg Phe Ala Ser Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile 420 425 430Ser Leu Lys Ser Phe Glu His Leu Tyr Phe Phe His Leu Val Ala Glu 435 440 445Gly Ser Ile Ser Gly Tyr Ile Arg Glu Ala Leu Arg Asn His Ala Pro 450 455 460Pro Ile Asp Val Asn Ala Met Met465 470531404DNAArtificial Sequencemisc_featureNovel Sequence 53atggacacca aacatttcct gccgctcgac ttctctaccc aggtgaactc ttcgtccctc 60aactctccaa cgggtcgagg ctccatggct gtcccctcgc tgcacccctc cttgggtccg 120ggaatcggct ctccactggg ctcgcctggg cagctgcact ctcctatcag caccctgagc 180tcccccatca atggcatggg tccgcccttc tctgtcatca gctcccccat gggcccgcac 240tccatgtcgg tacccaccac acccacattg ggcttcggga ctggtagccc ccagctcaat 300tcacccatga accctgtgag cagcactgag gatatcaagc cgccactagg cctcaatggc 360gtcctcaagg ttcctgccca tccctcagga aatatggcct ccttcaccaa gcacatctgt 420gctatctgtg gggaccgctc ctcaggcaaa cactatgggg tatacagttg tgagggctgc 480aagggcttct tcaagaggac agtacgcaaa gacctgacct acacctgccg agacaacaag 540gactgcctga tcgacaagag acagcggaac cggtgtcagt actgccgcta ccagaagtgc 600ctggccatgg gcatgaagcg ggaagctgtg caggaggagc ggcagcgggg caaggaccgg 660aatgagaacg aggtggagtc caccagcagt gccaacgagg acatgcctgt agagaagatt 720ctggaagccg agcttgctgt cgagcccaag actgagacat acgtggaggc aaacatgggg 780ctgaacccca gctcaccaaa tgaccctgtt accaacatct gtcaagcagc agacaagcag 840ctcttcactc ttgtggagtg ggccaagagg atcccacact tttctgagct gcccctagac 900gaccaggtca tcctgctacg ggcaggctgg aacgagctgc tgatcgcctc cttctcccac 960cgctccatag ctgtgaaaga tgggattctc ctggccaccg gcctgcacgt acaccggaac 1020agcgctcaca gtgctggggt gggcgccatc tttgacaggg tgctaacaga gctggtgtct 1080aagatgcgtg acatgcagat ggacaagacg gagctgggct gcctgcgagc cattgtcctg 1140ttcaaccctg actctaaggg gctctcaaac cctgctgagg tggaggcgtt gagggagaag 1200gtgtatgcgt cactagaagc gtactgcaaa cacaagtacc ctgagcagcc gggcaggttt 1260gccaagctgc tgctccgcct gcctgcactg cgttccatcg ggctcaagtg cctggagcac 1320ctgttcttct tcaagctcat cggggacacg cccatcgaca ccttcctcat ggagatgctg 1380gaggcaccac atcaagccac ctag 140454467PRTArtificial Sequencemisc_featureNovel Sequence 54Met Asp Thr Lys His Phe Leu Pro Leu Asp Phe Ser Thr Gln Val Asn1 5 10 15Ser Ser Ser Leu Asn Ser Pro Thr Gly Arg Gly Ser Met Ala Val Pro 20 25 30Ser Leu His Pro Ser Leu Gly Pro Gly Ile Gly Ser Pro Leu Gly Ser 35 40 45Pro Gly Gln Leu His Ser Pro Ile Ser Thr Leu Ser Ser Pro Ile Asn 50 55 60Gly Met Gly Pro Pro Phe Ser Val Ile Ser Ser Pro Met Gly Pro His65 70 75 80Ser Met Ser Val Pro Thr Thr Pro Thr Leu Gly Phe Gly Thr Gly Ser 85 90 95Pro Gln Leu Asn Ser Pro Met Asn Pro Val Ser Ser Thr Glu Asp Ile 100 105 110Lys Pro Pro Leu Gly Leu Asn Gly Val Leu Lys Val Pro Ala His Pro 115 120 125Ser Gly Asn Met Ala Ser Phe Thr Lys His Ile Cys Ala Ile Cys Gly 130 135 140Asp Arg Ser Ser Gly Lys His Tyr Gly Val Tyr Ser Cys Glu Gly Cys145 150 155 160Lys Gly Phe Phe Lys Arg Thr Val Arg Lys Asp Leu Thr Tyr Thr Cys 165 170 175Arg Asp Asn Lys Asp Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys 180 185 190Gln Tyr Cys Arg Tyr Gln Lys Cys Leu Ala Met Gly Met Lys Arg Glu 195 200 205Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn Glu Asn Glu 210 215 220Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val Glu Lys Ile225 230 235 240Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr Tyr Val Glu 245 250 255Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro Val Thr Asn 260 265 270Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val Glu Trp Ala 275 280 285Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp Gln Val Ile 290 295 300Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser Phe Ser His305 310 315 320Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His 325 330 335Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp 340 345 350Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp 355 360 365Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp 370 375 380Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys385 390 395 400Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln 405 410 415Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser 420 425 430Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly 435 440 445Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala Pro His 450 455 460Gln Ala Thr46555309DNAArtificial Sequencemisc_featureNovel Sequence 55ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 120tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 180ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 240aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 300gcctaggct 3095624DNAArtificial Sequencemisc_featureNovel Sequence 56tatataatgg atccccgggt accg 24571653DNAArtificial Sequencemisc_featureNovel Sequence 57atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 60accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ttcctctgga 600tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 1380caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620aaggccaaga agggcggaaa gtccaaattg taa 165358867DNAArtificial Sequencemisc_featureNovel Sequence 58aagcgagagg cggtgcaaga ggagcgccag aggaatgctc gcggcgcgga ggatgcgcac 60ccgagtagct cggtgcaggt aagcgatgag ctgtcaatcg agcgcctaac ggagatggag 120tctttggtgg cagatcccag cgaggagttc cagttcctcc gcgtggggcc tgacagcaac 180gtgcctccac gttaccgcgc gcccgtctcc tccctctgcc aaataggcaa caagcaaata 240gcggcgttgg tggtatgggc gcgcgacatc cctcatttcg ggcagctgga gctggacgat 300caagtggtac tcatcaaggc ctcctggaat gagctgctac tcttcgccat cgcctggcgc 360tctatggagt atttggaaga tgagagggag aacggggacg gaacgcggag caccactcag 420ccacaactga tgtgtctcat gcctggcatg acgttgcacc gcaactcggc gcagcaggcg 480ggcgtgggcg ccatcttcga ccgcgtgctg tccgagctca gtctgaagat gcgcaccttg 540cgcatggacc aggccgagta cgtcgcgctc aaagccatcg tgctgctcaa ccctgatgtg 600aaaggactga agaatcggca agaagttgac gttttgcgag aaaaaatgtt ctcttgcctg 660gacgactact gccggcggtc gcgaagcaac gaggaaggcc ggtttgcgtc cttgctgctg 720cggctgccag ctctccgctc catctcgctc aagagcttcg aacacctcta cttcttccac 780ctcgtggccg aaggctccat cagcggatac atacgagagg cgctccgaaa ccacgcgcct 840ccgatcgacg tcaatgccat gatgtaa 86759225DNAArtificial Sequencemisc_featureNovel Sequence 59tcgacattgg acaagtgcat tgaacccttg tctctcgaga gacaaggggg ttcaatgcac 60ttgtccaatg tcgagagaca agggggttca atgcacttgt ccaatgtcga gagacaaggg 120ggttcaatgc acttgtccaa tgtcgagaga caagggggtt caatgcactt gtccaatgtc 180gagagacaag ggggttcaat gcacttgtcc aatgtcgact ctaga 22560619DNAArtificial Sequencemisc_featureNovel Sequence 60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 540ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg 600ggaacggtgc attggaacg 61961262DNAArtificial Sequencemisc_featureNovel Sequence 61atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat 60gccttacaag gagagaaaaa gcaccgtgca tgccgatagg tggaagtaag gtggtacgat 120cgtgccttat taggaaggca acagacgggt

ctgacatgga ttggacgaac cactgaattc 180cgcattgcag agatattgta tttaagtgcc tagctcgata caataaacgc catttgacca 240ttcaccacat tggagtgcac ct 262621247DNAArtificial Sequencemisc_featureNovel Sequence 62tctatttcct caggccgtga ggaactgtcg ccagcttcaa gtataaatgg gtgcagtaca 60gatggcgagg cacgacgtca gaagaagggc cctgcgcccc gtcagcaaga ggaactgtgt 120ctggtatgcg gggacagagc ctccggatac cactacaatg cgctcacgtg tgaagggtgt 180aaagggttct tcagacggag tgttaccaaa aatgcggttt atatttgtaa attcggtcac 240gcttgcgaaa tggacatgta catgcgacgg aaatgccagg agtgccgcct gaagaagtgc 300ttagctgtag gcatgaggcc tgagtgcgta gtacccgaga ctcagtgcgc catgaagcgg 360aaagagaaga aagcacagaa ggagaaggac aaactgcctg tcagcacgac gacggtggac 420gaccacatgc cgcccattat gcagtgtgaa cctccacctc ctgaagcagc aaggattcac 480gaagtggtcc caaggtttct ctccgacaag ctgttggaga caaaccggca gaaaaacatc 540ccccagttga cagccaacca gcagttcctt atcgccaggc tcatctggta ccaggacggg 600tacgagcagc cttctgatga agatttgaag aggattacgc agacgtggca gcaagcggac 660gatgaaaacg aagagtctga cactcccttc cgccagatca cagagatgac tatcctcacg 720gtccaactta tcgtggagtt cgcgaaggga ttgccagggt tcgccaagat ctcgcagcct 780gatcaaatta cgctgcttaa ggcttgctca agtgaggtaa tgatgctccg agtcgcgcga 840cgatacgatg cggcctcaga cagtgttctg ttcgcgaaca accaagcgta cactcgcgac 900aactaccgca aggctggcat ggcctacgtc atcgaggatc tactgcactt ctgccggtgc 960atgtactcta tggcgttgga caacatccat tacgcgctgc tcacggctgt cgtcatcttt 1020tctgaccggc cagggttgga gcagccgcaa ctggtggaag aaatccagcg gtactacctg 1080aatacgctcc gcatctatat cctgaaccag ctgagcgggt cggcgcgttc gtccgtcata 1140tacggcaaga tcctctcaat cctctctgag ctacgcacgc tcggcatgca aaactccaac 1200atgtgcatct ccctcaagct caagaacaga aagctgccgc ctttcct 124763440PRTArtificial Sequencemisc_featureNovel Sequence 63Ser Ile Ser Ser Gly Arg Glu Glu Leu Ser Pro Ala Ser Ser Ile Asn1 5 10 15Gly Cys Ser Thr Asp Gly Glu Ala Arg Arg Gln Lys Lys Gly Pro Ala 20 25 30Pro Arg Gln Gln Glu Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser 35 40 45Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe 50 55 60Arg Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys Lys Phe Gly His65 70 75 80Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg 85 90 95Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro 100 105 110Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu 115 120 125Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met Pro 130 135 140Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His145 150 155 160Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Glu Thr Asn Arg 165 170 175Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala 180 185 190Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp 195 200 205Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu 210 215 220Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr225 230 235 240Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys 245 250 255Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu 260 265 270Val Met Met Leu Arg Val Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser 275 280 285Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys 290 295 300Ala Gly Met Ala Tyr Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys305 310 315 320Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala 325 330 335Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val 340 345 350Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu 355 360 365Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile 370 375 380Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn385 390 395 400Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu 405 410 415Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro 420 425 430Ile Leu Glu Ser Pro Thr Asn Leu 435 44064943DNAArtificial Sequencemisc_featureNovel Sequence 64atgacttcga aagtttatga tccagaacaa aggaaacgga tgataactgg tccgcagtgg 60tgggccagat gtaaacaaat gaatgttctt gattcattta ttaattatta tgattcagaa 120aaacatgcag aaaatgctgt tattttttta catggtaacg cggcctcttc ttatttatgg 180cgacatgttg tgccacatat tgagccagta gcgcggtgta ttataccaga ccttattggt 240atgggcaaat caggcaaatc tggtaatggt tcttataggt tacttgatca ttacaaatat 300cttactgcat ggtttgaact tcttaattta ccaaagaaga tcatttttgt cggccatgat 360tggggtgctt gtttggcatt tcattatagc tatgagcatc aagataagat caaagcaata 420gttcacgctg aaagtgtagt agatgtgatt gaatcatggg atgaatggcc tgatattgaa 480gaagatattg cgttgatcaa atctgaagaa ggagaaaaaa tggttttgga gaataacttc 540ttcgtggaaa ccatgttgcc atcaaaaatc atgagaaagt tagaaccaga agaatttgca 600gcatatcttg aaccattcaa agagaaaggt gaagttcgtc gtccaacatt atcatggcct 660cgtgaaatcc cgttagtaaa aggtggtaaa cctgacgttg tacaaattgt taggaattat 720aatgcttatc tacgtgcaag tgatgattta ccaaaaatgt ttattgaatc ggacccagga 780ttcttttcca atgctattgt tgaaggtgcc aagaagtttc ctaatactga atttgtcaaa 840gtaaaaggtc ttcatttttc gcaagaagat gcacctgatg aaatgggaaa atatatcaaa 900tcgttcgttg agcgagttct caaaaatgaa caataattct aga 943

Claims

1-71. (canceled)

72. A method of modulating the expression of a gene in a host cell comprising the gene to be modulated, the method comprising:(a) introducing into the host cell a gene expression modulation system comprising(i) a first gene expression cassette comprising a polynucleotide sequence that encodes a first polypeptide comprising an ecdysone receptor ligand binding domain; and(ii) a second gene expression cassette comprising a polynucleotide sequence that encodes a second polypeptide comprising a nuclear receptor ligand binding domain that is not an ultraspiracle ligand binding domain; and(b) introducing into the host cell a ligand that binds to the Group H nuclear receptor ligand binding domain,wherein one of the first gene expression cassette or the second gene expression cassette comprises a DNA-binding domain that recognizes a response element associated with a gene of interest,wherein the first gene expression cassette or the second gene expression cassette that does not comprise the DNA-binding domain comprises a transactivation domain that is not an ecdysone receptor transactivation domain, a retinoid X receptor transactivation domain, or an ultraspiracle receptor transactivation domain,wherein the ligand binding domain in the first polypeptide and the ligand binding domain in the second polypeptide are different and dimerize, andwherein the gene that is expressed is a component of a chimeric gene comprising:(i) a response element to which the DNA-binding binds;(ii) a promoter that is activated by the transactivation domain; and(iii) the gene that is expressed.

73. The method of claim 72, wherein the gene expression modulation system further comprises:(iii) a third gene expression cassette comprising(A) a response element to which the DNA-binding domain of the first polypeptide binds;(B) a promoter that is activated by the transactivation domain of the second polypeptide; and(C) the gene that is expressed.

74. The method of claim 72, wherein the ligand is a diacylhydrazine.

75. The method of claim 74, wherein the ligand is a compound of the formula: ##STR00003## wherein:E is a (C₄-C₆)alkyl containing a tertiary carbon or a cyano(C₃-C₅) alkyl containing a tertiary carbon;R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, SCN, or SCHF₂;R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH--CN, or joined with R³ and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;R³ is H, Et, or joined with R² and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

76. The method of claim 72, wherein the ligand binding domain of the first polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

77. The method of claim 72, wherein the ligand binding domain of the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

78. The method of claim 72, wherein the ligand binding domain of the second polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

79. The method of claim 72, wherein the ligand binding domain of the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.

80. The method of claim 72, wherein in the gene expression modulation system is contained in a vector.

81. The method of claim 80, wherein the vector is a plasmid.

82. The method of claim 80, wherein the vector is an expression vector.

83. The method of claim 80, wherein the vector is a viral vector.

84. The method of claim 83, wherein the viral vector is an adenovirus vector.

85. The method of claim 72, wherein the ecdysone receptor ligand binding domain is selected from the group consisting of a Lepidopteran ecdysone receptor ligand binding domain, a Dipteran ecdysone receptor ligand binding domain, an Arthropod ecdysone receptor ligand binding domain, a Homopteran ecdysone receptor ligand binding domain, a spruce budworm Choristoneura fumiferana ecdysone receptor ligand binding domain, a Tenebrio molitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain, a silk moth Bombyx mori ecdysone receptor ligand binding domain, a fruit fly Drosophila melanogaster ecdysone receptor ligand binding domain, a mosquito Aedes aegypti ecdysone receptor ligand binding domain, a blowfly Lucilia capitata ecdysone receptor ligand binding domain, a Mediterranean fruit fly Ceratitis capitata ecdysone receptor ligand binding domain, a locust Locusta migratoria ecdysone receptor ligand binding domain, an aphid Myzus persicae ecdysone receptor ligand binding domain, a fiddler crab Uca pugilator ecdysone receptor ligand binding domain, and an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain.

86. The method of claim 85, wherein the ecdysone receptor is Choristoneura fumiferana ecdysone receptor ligand binding domain.

87. The method of claim 72, wherein the expression of the gene is tissue-specific expression.

88. The method of claim 72, wherein the first polypeptide does not contain the A and B domains of the ecdysone receptor.

89. The method of claim 72, wherein the second polypeptide does not contain the A and B domains of the nuclear receptor.

90. The method of claim 72, wherein the first polypeptide does not contain the A and B domains of the Group H nuclear receptor, andwherein the second polypeptide does not contain the A and B domains of the nuclear receptor.

91. The method of claim 72, wherein the gene expression modulation system is more sensitive to a diacylhydrazine ligand than to a steroid ligand.

92. The method of claim 91, wherein the gene expression modulation system is more sensitive to a diacylhydrazine ligand than to a steroid ligand when expressed in a mammalian cell.

93. The method of claim 72, wherein the DNA binding domain is selected from the group consisting of a GAL4 DNA binding domain, a LexA DNA binding domain, a transcription factor DNA binding domain, a steroid/thyroid hormone nuclear receptor superfamily member DNA binding domain and a bacterial LacZ DNA binding domain.

94. The method of claim 72, wherein the transactivation domain is selected from the group consisting of a steroid/thyroid hormone nuclear receptor transactivation domain, a polyglutamine transactivation domain, a basic or acidic amino acid transactivation domain, a VP16 transactivation domain, a GAL4 transactivation domain, an NF-.kappa.B transactivation domain and a BP64 transactivation domain.

95. The method of claim 72, wherein the nuclear receptor ligand binding domain of the second polypeptide is a retinoic X receptor ligand binding domain.

96. The method of claim 95, wherein the retinoic X receptor ligand binding domain of the second polypeptide is selected from the group consisting of a mouse Mus musculus retinoic X receptor ligand binding domain, a human Homo sapiens retinoic X receptor ligand binding domain.

97. The method of claim 95, wherein the retinoic X receptor ligand binding domain of the second polypeptide is selected from the group consisting of an RXRα ligand binding domain, an RXRβ ligand binding domain and an RXRγ ligand binding domain.

98. The method of claim 72, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, a mammalian cell, a mouse cell, and a human cell.

99. The method of claim 98, wherein the host cell is selected from the group consisting of an Aspergillus cell, a Trichoderma cell, a Saccharomyces cell, a Pichia cell, a Candida cell, and a Hansenula cell.

100. The method of claim 98, wherein the host cell is selected from the group consisting of a Synechocystis cell, a Synechococcus cell, a Salmonella cell, a Bacillus cell, an Acinetobacter cell, a Rhodococcus cell, a Streptomyces cell, an Escherichia cell, a Pseudomonas cell, a Methylomonas cell, a Methylobacter cell, an Alcaligenes cell, a Synechocystis cell, an Anabaena cell, a Thiobacillus cell, a Methanobacterium cell and a Klebsiella cell.

101. The method of claim 98, wherein the host cell is a plant cell.

102. The method of claim 101, wherein the plant cell is selected from the group consisting of an apple cell, an Arabidopsis cell, a bajra cell, a banana cell, a barley cell, a bean cell, a beet cell, a blackgram cell, a chickpea cell, a chili cell, a cucumber cell, an eggplant cell, a favabean cell, a maize cell, a melon cell, a millet cell, a mungbean cell, an oat cell, an okra cell, a Panicum cell, a papaya cell, a peanut cell, a pea cell, a pepper cell, a pigeonpea cell, a pineapple cell, a Phaseolus cell, a potato cell, a pumpkin cell, a rice cell, a sorghum cell, a soybean cell, a squash cell, a sugarcane cell, a sugarbeet cell, a sunflower cell, a sweet potato cell, a tea cell, a tomato cell, a tobacco cell, a watermelon cell, and a wheat cell.

103. The method of claim 98, wherein host cell is a mammalian cell.

104. The method of claim 103, wherein the mammalian cell is selected from the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell, a chimpanzee cell, and a human cell.

105. The method of claim 98, wherein the mammalian cell is a human cell.

106. The method of claim 72, wherein the first polypeptide comprises a DNA binding domain and the second polypeptide comprises a transactivation domain.

107. The method of claim 72, wherein the first polypeptide comprises a transactivation domain and the second polypeptide comprises a DNA binding domain.