ECDYSTEROID DEPENDENT REGULATION OF GENES IN MAMMALIAN CELLS

Abstract: A method of inducing gene expression in a mammalian cell or an intact mammal comprising contacting an ecdysteroid, such as muristerone A, with an ecdysteroid receptor polypeptide within a mammalian cell, wherein said mammalian cell further contains a DNA binding sequence for said ecdysteroid receptor when in combination with its ligand, such as muristerone A, thereby resulting in gene expression.

Description: ECDYSTEROID DEPENDENT REGULATION OF GENES IN MAMMALIAN CELLS FIELD OF THE INVENTION This invention relates generally to the use of specific ecdysteroids in mammalian cells to induce gene expression of heterologous genes under the control of ecdysteroid receptor binding proteins. BACKGROUND OF THE INVENTION The steroid hormone receptor superfamily represents an evolutionarily conserved group of proteins that influence developmental and metabolic processes primarily by functioning as ligand-dependent transcription factors (for review, see K. R. Yamamoto, Annu. Rev. Genet. 19, 209 [1985]; M. Beato, Cell 56, 335 [1989]; R. M. Evans, Science 240, 889 [1988]). Structural analysis of receptor proteins has identified domains of these proteins that function to bind DNA or ligand and to enhance transcription (S. Rusconi and K. R. Yamamoto, EMBO J. 6, 1309 [1987]; P. J. Godowski, D. Picard and K. R. Yamamoto, Science 241, 812 [1988]; S. M. Hollenberg and R. M. Evans, Ce//55, 899 [1988]; N. J. G. Webster, S. Green, J. R. Jin and P. Chambon, Ce//54, 199 [1988]). Ligand binding is required for the interaction of some receptors with their cognate response elements (P. B. Becker, et al., Nature 324, 686 [1988]) and may also control dimerization and transcriptional activation properties (S. Y. Tsai, etal., Cell 55, 361 [1988); V. Kumar and P. Chambon, Cell 55, 145 [1988]). The ability of transcription factors to function in heterologous species has provided a system to analyze the functional domains of that factor and a novel mechanism to control the expression of heterologous genes (A. J. Courey and R. Tjian, Cell 55, 887 [1988); A. J. Courey, D. A. Holtzman, S. P. Jackson and R. Tjian, Cell 59, 827 [1989]). Mammalian steroid hormone receptors have been shown to function when expressed in yeast (D. Metzger, J. H. White and P. Chambon, Nature 334, 31 [1988); D. Picard, M. Schena and K. R. Yamamoto, Gene 86, 257; M. Schena and K. R. Yamamoto, Science 241 , 965 [1988]) and drosophila cells. The ecdysteroids are steroids whose action is mediated by an intracellular receptor and includes the molting hormones of insects. The ecdysteroid hormone 20-OH ecdysone, also known as β-ecdysone, controls timing of development in many insects. See, generally, Koolman (ed.), Ecdysone: From Chemistry to Mode of Action, Thieme Medical Pub., N.Y. (1989). The generic term "ecdysone" is frequently used as an abbreviation for 20-OH ecdysone. Pulses, or rises and falls, of the ecdysone concentration over a short period of time in insect development are observed at various stages of Drosophila development. These stages include embryogenesis, three larval stages and two pupal stages. The last pupal stage ends with the formation of the adult fly. One studied effect of ecdysone on development is that resulting from a pulse at the end of the third, or last, larval stage. This pulse triggers the beginning of the metamorphosis of the larva to the adult fly. Certain tissues, called imaginal tissues, are induced to begin their formation of adult structures such as eyes, wings and legs. During the larval stages of development, giant polytene chromosomes develop in the non-imaginal larval tissues. These cable-like chromosomes consist of aggregates comprising up to about 2,000 chromosomal copies. These chromosome aggregates are extremely useful because they provide the means whereby the position of a given gene within a chromosome can be determined to a very high degree of resolution, several orders of magnitude higher than is typically possible for normal chromosomes. A "puff" in the polytene chromosomes is a localized expansion or swelling of these cable-like polytene chromosome aggregates that is associated with the transcription of a gene at the puff locus. A puff is, therefore, an indicator of the transcription of a gene located at a particular position in the chromosome. A genetic regulatory model was proposed to explain the temporal sequence of polytene puffs induced by the ecdysone pulse which triggers the larval-to-adult metamorphosis. See, Ashburner et al., "On the Temporal Control of Puffing Activity in Polytene Chromosomes," Cold Spring Harbor Symp. Quant. Biol. 38:655-662 (1974). This model proposed that ecdysone interacts reversibly with a receptor protein, the ecdysone receptor, to form an ecdysone-receptor complex. This complex would directly induce the transcription of a small set of "early" genes responsible for a half dozen immediately induced "early" puffs. These early genes are postulated to encode regulatory proteins that induce the transcription of a second set of "late" genes responsible for the formation of the "late" puffs that appear after the early puffs. The model thus defines a genetic regulatory hierarchy of three ranks, where the ecdysone receptor gene is in the first rank, the early genes in the second rank and the late genes in the third. While this model derived form the puffing pattern observed in a non- imaginal tissue, similar genetic regulatory hierarchies may also determine the metamorphic changes in development of the imaginal tissues that are also targets of ecdysone, as well as the changes in tissue development induced by the pulses of ecdysone that occur at other developmental stages. Various structural data have been derived from vertebrate steroid and other lipophilic receptor proteins. A "superfamiiy" of such receptors has been defined on the basis of their structural similarities. See, Evans, "The Steroid and Thyroid Hormone Receptor Superfamiiy," Science 240:889-895 (1988); Green and Chambon, "Nuclear Receptors Enhance Our Understanding of Transcription Regulation," Trends in Genetics 4:309-314 (1988). Where their functions have been defined, these receptors, complexed with their respective hormones, regulate the transcription of their primary target genes, as proposed for the ecdysone receptor in the above model. Ecdysteroid receptors from Drosophila melanogaster adult females were described by Handler et al. Mol Cell Endo 63:103-9, 1989. Ecdysteroid receptors for the blowfly Calliphora vicina were characterized by Lehmann etal, EurJ. Biochem 181:577-82, 1989. The isolation of 20-hydroxyecdysone from Vitex stricken " (East African herb) is described in JP1135794. The ecdysterone content of culture medium of drosophila salivary glands was described in SU1130605. Muristerone extracted from the seeds of faladana plants and used as a biological insecticide is described in U.S. Patent 3,828,082. The synthesis of ecdysone is described in U.S. Patent 3,354,154. Analogs of ecdysteroids may be produced by plants to disrupt the development of insects. One plant-produced ecdysteroid analog is meristone A. Muristerone or Muristerone A (Figure 7, Compound IV) has a molecular weight of 496.640 daltons, a formula of C27H44O8 and a CHCD name of 2,3,5,11 ,14,20,22-Heptahydroxy-7-cholesten-6-one (Cononica. L et al, Phytochemistry 14: 525, 1975). It is a constitutient of the plant Ipomoea calonyction and shows a high insect moulting activity. It is believed to protect the plant by disrupting insect development in the larval stage (Trematerra et al, Bollettino Zool. Agr. Bachic 18:87-93, [1986]). Muristerone A has been shown to have ecdysteroid activity in insects such as to trigger degeneration of the tick salivary gland (Lindsay et al, J. Insect Physiol 34:351-360, [1988]). Muristerone A-receptor complexes are not as sensitive to dissociation in high salt buffers as other ecdysteroid-receptor complexes and this affinity has allowed the use of radiolabeled muristerone A to follow ecdysteroid receptor during chromatography (Landon et al., J. BiolChem 263:4693-4697, [1988]). Many medically and commercially important proteins can be produced in a usable form by genetically engineered bacteria. However, many expressed proteins are processed incorrectly in bacteria and are preferably produced by genetically engineered eucaryotic cells. Typically, yeast cells or mammalian tissue-culture cells are used. Because it has been observed that protein processing of foreign proteins in yeast cells is also frequently inappropriate, mammalian cultured cells have become the central focus for protein production. It is common that the production of large amounts of foreign proteins makes these cells unhealthy, which may affect adversely the yield of the desired protein. This problem may be circumvented, in part, by using an inducible expression system. In such a system, the cells are engineered so that they do not express the foreign protein, and therefore are not unhealthy, until an inducing agent is added to the growth medium. In this way, large quantities of healthy cells can be produced and then induced to produce large amounts of the foreign protein. Unfortunately, in the presently available systems, the inducing agents themselves, such as metal ions or high temperature, adversely affect the cells, thus again lowering the yield of the desired foreign protein the cells produce. A need therefore exists for the development of innocuous inducing factors for efficient production of recombinant proteins. Such innocuous factors could also prove invaluable for human therapy, where the individual suffers from lack of the ability to produce particular proteins. By using methods similar to those for producing proteins in cultured cells, such innocuous factors for inducing the synthesis of the required protein could be used for controlling both the timing and the abundance of the protein produced in the affected individual. Therefore, a need exists for an inducible expression system in both mammalian cell culture and in mammals perse. The hormones that complex with mammalian or other vertebrate members of the steroid receptor superfamiiy are unlikely candidates for such innocuous factors, nor have they been found to satisfy the required properties of such factors, because mammalian cells contain these receptors, or highly homologous proteins, that would alter the expression of many target genes in the presence of the respective hormone, thereby adversely affecting the host cells. For these and other reasons, developing an alternative steroid receptor system has been a goal of researchers. Unfortunately, efforts have been unsuccessful despite significant investment of resources. The absence of information on the structure, function and molecular biology of non-mammalian steroid receptors has significantly hindered the ability to produce such products. Recently, the isolation and characterization of drosophila DHR23α DNA, which contained a partial sequence of the DHR23α polypeptide, a member of the steroid hormone receptor superfamiiy previously identified in the laboratory of Dr. David Hogness (W. Seegraves, thesis, Stanford University [1988]) allows the isolation of the DNA sequence encoding the DHR23α. This isolated DNA sequence allows the construction of expression systems incorporating the DNA encoding the DHR23α ecdysteroid receptor. In summary, the insect steroid hormones and their receptors, such as the ecdysteroids, are a potential source of material for developing innocuous steroid receptor systems for use in mammalian cell culture and in mammals themselves. However, no ecdysteroid has been shown to function to induce activity of an ecdysteroid receptor in a mammalian cell. Therefore, to develop a system based upon an insect hormone-receptor system, there exists a need for an ecdysteroid hormone or hormone analog that induces the ecdysteroid receptor in mammalian cells to express those genes placed under control of the ecdysteroid receptor. SUMMARY OF THE INVENTION I show that DHR23α, a Drosophila steroid receptor homologue, can function in cultured mammalian cells as an ecdysteroid-dependent transcription factor when induced by a specific group of ecdysteroids which includes muristerone A. Muristerone A and related ecdysteroids that lack a 25-hydroxyl group, will induce in mammalian cells the expression of DNA sequences under the transcriptional control of DHR23α. DHR23α inducible activity was not induced by any of the mammalian steroid hormones tested. The DNA-binding and transactivation activities of viral, mammalian or bacterial proteins were rendered ecdysteroid-dependent when fused to the DHR23α ligand-binding domain. This system is useful in selectively regulating the expression of endogenous or heterologous genes in mammalian cells. DESCRIPTION OF THE FIGURES Figure. 1. (A) Nucleotide and derived amino acid sequence of the DHR23α cDNA clone (seq. ID No. 1). Numbers on the left and right indicate nucleotide and amino acid residues, respectively. The conserved amino acids corresponding to the putative DNA-binding domain are underlined. (B) Schematic comparison of the Drosophila DHR23α protein with the human thyroid hormone receptor (hTRβ), the human vitamin D3 receptor (hVDR) and the human retinoic acid receptor (hRARα). The amino acid residues are indicated by numbers above the boxes. The region of DHR23α marked 'DNA' is compared with the DNA-binding domains of the other receptors. The region of DHR23α marked 'Ligand' contains the highest region of homology to the ligand-binding domains of the other receptors. The percent identity in these regions is shown by the numbers within the boxes. The region labeled 'QLQP' is rich in the amino acids glutamine (Q) leucine (L) and proline (P). Figure. 2. Specific ecdysteroids are agonists for the DHR23α receptor in mammalian cells. Human 293 cells were cotransfected with 2.5 μg of the expression plasmid pRSV.DHR23oc or the parental expression plasmid pRSV (Control) and 0.5 μgs of the reporter plasmid pEc4M. 77 CO (EcRE) or PG4M.77CO (GRE). As a control for transfection efficiency, 0.5 μgs of the control plasmid pRSV.hGH was included in the transfection mixture. After transfection, cells were treated without (-) or with alpha-ecdysone (alpha), 20-OH ecdysone (20-OH), polypodine B (ppB), ponasterone A (ponA) or muristeroine A (murA). CAT extracts were harvested 48 hrs after transfection and assayed. The values were normalized to the expression of hGH and the average values of three independent experiments are shown. The "fold induction" represents the level of expression of the reporter gene in cells incubated with hormone divided by the expression of that reporter gene in extracts from cells incubated in the absence of added ligand. Figure. 3. Effect of mammalian hormones on activity of DHR23α. Cells were transfected with the DHR23α expression vector and EC4M.77CO reporter gene and treated without (-) or with the following hormones (1 μM): muristerone A (mur A) dexamethasone (Dex) 17β-estradiol (E2), aldosterone (Aldo), corticosterone (Cort) hydroxycorticosterone (OH-Cort) thyroid hormone (T3), promegestone (Promeg) or 1,25-dihydroxy vitamin D3 (VD3). Reporter gene activity was determined as described in Fig. 2. Figure. 4. Schematic representation and expression of receptor proteins. (A) Receptor constructs are denoted according to a three part nomenclature describing the origin of their N-terminal transactivation, DNA-binding and ligand-binding domains. "G" and "Ec" refer to the glucocorticoid receptor and DHR23α, respectively. '"E"' refers to a derivative of the rat glucocorticoid receptor DNA-binding domain with two amino acid substitutions (G458E, S459G) that convert the DNA-binding specificity to that of the estrogen receptor. "X" (solid box) indicates the DNA-binding domain (amino acidsl -87) of the Escherichia coli LexA protein. "V" (hatched box) denotes a derivative of the GR N-terminal domain in which amino acids 153-406 are replaced by the transcriptional activation domain of the HSV VP16 protein (amino acids 411-490) GGEc was constructed by replacing the ligand-binding domain of GGG (amino acids 528-795) with the ligand-binding domain of DHR23α (amino acids 329-878). Similarly, to construct GXEc, the ligand-binding domain of GXG was replaced with the DHR23α ligand-binding domain. (B) Accumulation of receptor proteins in transfected cells. Whole cell extracts were prepared 48 hrs after transfection with receptor expression plasmids encoding the following fusion proteins. Lane 1, EcEcEc; lane 2, GGG; lane 3, GGEc; lane 4, VGEc; lane 5, G"E"G; lane 6, G"E"Ec; lane 7, GXG; lane 8, GXEc; lane 9, VXEc. The blots were reacted with monoclonal antibody BμgR2 that recognizes an epitope in the N-terminal domain of the rat glucocorticoid receptor and then with a sheep antiserum to mouse antibody coupled to horseradish peroxidase. Positions of the molecular markers are indicated. Figure. 5. RNase protection analysis of transcripts induced by receptor proteins. Total RNA was prepared from cells 48 hrs after transfection with expression plasmids encoding either DHR23α (EcEcEc), GGG, or GGEc and the reporter gene G4M-77GO. The position of 377 base protected band for G4M.77CO and the 294 base protected band from the internal control gene (expressed from a CMV enhancer/promoter construct) are indicated by the closed and opened arrows, respectively. Figure. 6. Induction of estrogen receptors (EREs) by chimeric receptors. Cells were transfected with expression plasmids encoding either DHR23α (EcEcEc), G"E"G or GΕΕc and E4M-77CO, that contains 4 EREs fused to the MTV promoter. Cells were incubated for 48 hrs with or without (-) or with muristerone A (M) or dexamethasone (D). Figure 7. The chemical structure of the ecdysteroids: (I) ecdysteroid numbering system, RT and R2 are site of electronegative group substitution; (II) ecdysone; (III) 20-OH ecdysone(β-ecdysone); (IV) ponasterone A; (V) muristerone A; (VI) 5-dehydroxy muristerone A; (VII) and 11-dehydroxy muristerone A. DESCRIPTION OF THE PREFERRED EMBODIMENTS Steroid receptors are members of a large family of transcription factors whose activity is tightly regulated by the binding of their cognate steroid ligand. These ligand- dependent transcription factors can be exploited to obtain the regulated expression of heterologous genes in mammalian cells. However, the utility of these systems in transgenic animals is limited by the background of endogenous steroids and their receptors. The present invention demonstrates that an analog of the insect ecdysteroids, muristerone A, will induce gene expression in mammalian cells using the insect steroid receptor DHR23α and its DNA binding sequence. In the present invention, I wished to develop a system to specifically regulate heterologous genes in mammalian cells in tissue culture and in transgenic animals. Such a system provides a powerful method for regulating the synthesis of the products of heterologous genes. Systems that exploit the ability of mammalian steroid hormone receptors to function as ligand-dependent transcription factors have proved useful in regulating the expression of heterologous genes in mammalian cells (D. I. Israel and R. J. Kaufman, Nuc. Acids Res. 17, 4589 [1989]). However, the applications of these systems in cultured cells or transgenic animals is limited by the background of endogenous steroid receptors and their ligands. In the present invention I show that the drosophila DHR23α protein, a member of the steroid hormone receptor superfamiiy previously identified in 1988 by W. Seegraves at Stanford University (W. Seegraves, thesis, Stanford University (1988]), can function as a ligand-dependent transcription factor in mammalian cells when induced by specific ecdysteroids such as DHR23α. The activity of DHR23α is induced upon administration of certain ecdysteroids but not by any of the mammalian hormones tested. Novel target gene specificity was obtained using chimeric receptors containing the DHR23α ligand-binding domains fused to heterologous DNA-binding domains. Finally, the activity of these chimeric proteins could be increased by inclusion of a potent viral transactivation domain. Oligonucleotide probes based on the partial DHR23 sequence (W. Seegraves, thesis, Stanford University (1988]) were used to screen a cDNA library prepared from drosophila early pupal larvae. The deduced amino acid sequence of the DHR23α clone is shown in Fig. 1A. The highest region of homology between DHR23α and other members of the steroid receptor superfamiiy is found in a cysteine-rich region (residues 264-329) that corresponds to the DNA-binding domain (Fig. 1B). The putative ligand-binding domain shares limited homology with members of the retinoic acid and thyroid hormone receptors, and the vitamin D receptor. The N-terminal domain (residues 1-263) does not share significant homology with any steroid receptor superfamiiy member. I also identified a second form of this protein, DHR23β, that appears to arise by differential splicing. DHR23β is identical to DHR23α in the DNA and ligand-binding domains but contains an unrelated N-terminal domain of 234 amino acids. DHR23α has been reported to regulate transcription of genes containing ecdysone response elements (EcREs) in drosophila tissue culture cells treated with 20-OH ecdysone (M. Koelle, Paper presented at a seminar, 2 October 1989, Genentech, Inc., So. San Francisco, CA). I determined whether DHR23α could function in mammalian cells treated with ecdysteroids to enhance the transcription of a reporter gene containing EcREs linked to the murine mammary tumor virus (MTV) promoter and chloramphenicol acetyltransferase (CAT) gene. Human 293 cells were cotransfected with an RSV-based expression vector that encodes DHR23α and the reporter gene EC4M.77CO that contains 4 copies of an ecdysone response element (EcRE) from the Drosophila HSP27 promoter (G. Riddihough and H. R. B. Pelham, EMBO J. 6, 3729 (1987]) linked to an MTV promoter-CAT construct. CAT activity was determined in extracts from cells incubated with or without the ecdysteroids α- or 20-OH ecdysone, polypodine B, ponasterone A, or muristerone A. Neither α-ecdysone, 20- OH ecdysone, nor polypodine B acted as agonists for DHR23α in mammalian cells. In contrast, expression of the reporter gene was markedly increased in cells treated with muristerone A and to a lesser extent with ponasterone A (Fig. 2). This induction was dependent on the presence of an EcRE in the reporter gene because DHR23α did not regulate expression of reporter genes containing binding sites for either the glucocorticoid receptor (GRE, Fig. 2) or for the estrogen receptor (ERE, Fig. 6). Thus, in mammalian cells DHR23α acts in an ecdysteroid-dependent fashion to selectively stimulate expression from an EcRE containing reporter gene. It is unclear why 20-OH ecdysone and polypodine B, which are agonists in drosophila cell lines, fail to activate DHR23α in mammalian cells. Transport failure, inactivation and non-specific binding may account for the lack of activity in mammalian cells However, it has been shown that the specific activities and relative efficacies of ligands for their receptors sometimes differ when that receptor is expressed in a heterologous system. For example, it is known that the activities of glucocorticoid ligands for their receptor differ significantly in yeast and mammalian cells. Surprisingly, only specific derivatives of ecdysone act as agonists, and these share the related feature of lacking the 25-hydroxyl group. The only structural difference between the weak agonist ponasterone A and 20-OH ecdysone, which is inactive, is that the former lacks a hydroxyl group at position 25. Even more surprising and unexpected is that the strong agonist muristerone differs from ponasterone A solely by the addition of hydroxyl groups at positions 5 and 11. Therefore, surprisingly, in mammalian cells ecdysteroid inducers of DHR23α-regulated expression lack a hydroxyl group at position 25. In a preferred embodiment, the ecdysteroid may contain one hydroxyl group at positions 5 or 11. Alternatively, any electronegative group substituted at positions 5 and/or 11 of muristerone would be expected to result in activation of ecdysteroid receptors. Among the electronegative groups that may be substituted at positions 5 and 11 of muristerone are hydroxyl, ketone, sulfhydral, nitrate, nitrite, and halogens, particularly florine, bromine and chlorine. The most active inducer of DHR23α regulated expression is muristerone A, which contains hydroxyl groups at both the 5 and 11 positions. The CHCD name for muristerone A is 2,3,5,11 ,14,20,22-heptahydroxy-7-cholesten-6-one; for 5-dehydroxy muristerone A it is 2,3,11,14,20 I 22-septahydroxy-7-cholesten-6-one; and for 11-dehydroxy muristerone A it is 2,3,5,14,20,22-septahydroxy-7-cholesten-6-one. In the present invention, ecdysteroid receptor is defined as any insect steroid receptor that has a physiologically significant binding affinity for muristerone A, or a derivative of muristerone A which contains an electronegative substitution at the 5 or 11 positions. Among such physiologically significant receptors are the ecdysone receptor and DHR23X. A physiologically significant binding affinity is effective binding at a concentration of 10 *6 molar or less. In the present invention, ecdysteroid is defined as any steroid lacking 25-OH having a physiologically effective affinity for an ecdysteroid receptor. In order to determine if mammalian steroid hormones could act as agonists for DHR23α, I tested representative members of hormones known to activate mammalian steroid receptors. None of these hormones could act as agonists for DHR23α (Fig. 3). These results suggest that DHR23 activity is selectively regulated in mammalian cells by ecdysteroids. Therefore, mammalian cells containing genes under the control of DHR23α will be induced by muristerone A, and not by those mammalian steroid hormones tested. Ecdysteroid regulation of mammalian, viral and bacterial DNA binding or transactivation domains. A remarkable feature of steroid hormone receptors is the degree to which individual domains can function when combined with domains of heterologous proteins. The DNA binding specificity of a receptor can be altered. by replacing its DNA-binding domain with those of other steroid receptors (S. Green and P. Chambon, Proc. Natl. Acad. Sci. USA. 325, 75 [1987]) or from bacterial (P. J. Godowski, D. Picard and K. R. Yamamoto, Science 241, 812 (1988)) or yeast (N. J. G. Green, S. Green, J. R. Kin and P. Chambon, Cell 54, 199 [1988]) DNA-binding proteins. In some cases, the activities of heterologous proteins become hormone regulated if that protein is fused to to a steroid receptor ligand-binding domain. For example, the transactivation or transformation activities of E1A, c-myc or c-fos can be brought under hormonal control by fusion of a steroid receptor ligand-binding domain (M. Eilers, D. Picard, K. R. Yamamoto and J. M. Bishop, Nature 340, 66 [1989]; D. Picard, S.J. Salser and K. R. Yamamoto, Cell 54, 1073 [1988]; G. Superti-Furga, G. Bergers, D. Picard and M. Bussiinger, Proc. Natl. Acad. Sci. U.S.A.88, 5114 [1991]). I choose to determine if the ligand-binding domain of DHR23α could be used to regulate the DNA-binding and transactivation domains of the mammalian glucocorticoid receptor for the following reasons. First, in contrast to DHR23α, high affinity DNA-binding sites for the GR have been identified (M. Beato, Cell 56, 335 [1989]) and reporter genes containing these sites are very strongly regulated. Thus, the sensitivity of our assays could be increased. Secondly, these chimeric receptors could be utilized to selectively regulate endogenous genes in mammalian cells in response to ecdysteroids. I constructed a chimeric gene, GGEc, in which the sequences encoding the ligand-binding domain of the GR were replaced with that of DHR23α (Fig. 4A). Western blot analysis indicated that GGEc was expressed in transfected cells, although at considerably lower levels than the intact GR (Fig. 4B). As expected, DHR23α failed to induce the expression of G4M.77CO, a reporter gene that contains 4 copies of a high affinity GRE (Table 1). However, expression of the GRE-containing reporter gene was induced more than 700 fold by GGEc in muristerone-treated cells. The relative efficacies of ecdysteroids as agonists for the chimeric receptor are identical to that as for DHR23α; neither α-ecdysone, 20-OH ecdysone or polypodine B acted as agonists whereas ponasterone A and muristerone A acted as weak and strong agonists respectively. Results of the CAT assays were confirmed and extended by direct analysis of transcripts initiated at the regulated promoter. RNA isolated from transfected ceils was assayed by RNAse mapping experiments using probes complementary to the GRE containing reporter plasmid and a cotransfected control gene expressed from the CMV enhancer and promoter. Figure 5 shows that GGEc acts in a hormone-dependent fashion to stimulate expression from a G RE-MTV promoter construct. I then determined if a DHR23 fusion protein could regulate genes normally responsive to estrogens. I constructed a derivative of GGEc that incorporates a two amino acid change in the first finger of the rat glucocorticoid receptor (G458E, S459G) that has been shown to convert the DNA-binding specificity of the GR to that of the ER (K. Umesono and R. Evans, Cell 57, 1139 [1989]; M. Danielsen, L Hinck, G. Ringold, Cell 57, 1131 [1989]) (Fig. 4A). This fusion protein, G'EΕc, now regulates the ERE containing reporter gene E4M-77CO in a muristerone-dependent fashion (Figure 6). As expected, G"E"Ec failed to induce the expression of reporter genes lacking EREs Chimeric proteins containing the DHR23α ligand-binding domain fused to the DNA- binding domains of mammalian proteins are expected to prove useful in regulating the expression of either native or transgenic endogenous genes in transgenic animals. Such fusion constructs are expected to be useful in regulating the expression of exogenous genes introduced into transgenic mammals or mammalian cells. I next determined if the DHR23α ligand-binding domain could be used to regulate the activity of a DNA-binding domain not normally expressed in mammalian cells. I constructed the chimeric gene GLxEc by replacing the sequences coding for the GR DNA-binding domain in the GGEc fusion with those encoding the DNA-binding domain of the Escherichia coli LexA repressor (J. W. Little and S. A. Hill, Proc. Natl. Acad. Sci. U.S.A. 82, 2301 [1985]) (Fig. 4). A reporter gene, X4C.33CO was constructed that contains 4 copies of a 26-bp lex operator (R. Brent and M. Ptashne, Nature 312, 612 [1984]) at position -33 of the CMV promoter. GLxEc had no effect on the expression of a OC33CO, a reporter gene lacking the lex operator (Fig. 6B). However, transcription of X4C33CO was strongly induced by GLxEc, and this induction was fully hormone-dependent (Fig. 6A). As controls I showed that X4C.33CO was not induced in cells treated with muristerone and cotransfected with either DHR23α, that lacks the lexA DNA-binding domain, or by GLxG (P. J. Godowski, D. Picard and K. R. Yamamoto, Science 241, 812 [1988]) that contains the glucocorticoid receptor ligand-binding domain. Finally, I determined if the activity of DHR23α fusion proteins could be further enhanced by inclusion of a potent viral transactivation domain. I constructed VGEc and VLxEc fusion genes by replacing a portion of the GR N-terminal activation domain in GGEc and GLxEc, respectively, with the herpes virus VP16 acidic activation domain (I. Sadowski et al., Proc. Natl. Acad. Sci. 335, 563 [1988]; D. J. Cousens, R. Greaves, C. R. Coding, and P. O'Hare, EMBO J.8, 2337 [1989]) (Fig. 4A). In transfected cells, these proteins accumulated to similar levels as derivatives lacking the VP16 activation domain (Fig. 4B). Both VGEc and VLxEc acted in an ecdysteroid-dependent fashion to induce activity of the appropriate reporter gene (Tables 1 and 2). However, the activity of VGEc and VLxEc was 5 and 10 fold greater than GGEc and GLxEc, respectively (M. A. Labow, S.B. Bairn, T. Shenk, and A. J. Levine, Mol. and Cell. Biol.10, 3343 [1990]). Thus, the DHR23α ligand-binding domain can be used to regulate the activities of viral, mammalian and bacterial DNA-binding or transactivation domains. The development of a system for regulated expression of endogenous and exogenous genes in eukaryotic cells provides an important method to study the function of those gene products and to develop animals models for disease. Our results demonstrate the feasibility of using nonmammalian steroid hormone receptors to regulate genes in mammalian cells. There are several important features of this system. DHR23α acts as a potent and selective regulator of the transcription of genes containing EcREs', and the activity of DHR23α can be further modified by replacing its DNA-binding or transactivation domains with those from heterologous proteins. Importantly, DHR23α activity is regulated by ecdysteroids, which are not normally expressed in mammalian cells. Although I did not survey all of the mammalian steroids, none of those representing the most abundant of the natural murine steroids acted as agonists for DHR23α. Thus, it is conceivable that the transcriptional regulatory activities of DHR23α or DHR23α fusion proteins will be completely dependent on administration of exogenous ligand. Several reports have demonstrated the feasibility of using the £ coll lac repressor to regulate gene expression in mammalian cells. Both the lac repressor and the steroid receptor based systems can induce or repress (D. M. Omitz, R. W. Moreadith and Leder P., PNAS 88, 698 [1991]) transcription. One attractive feature of a steroid receptor based regulatory system is the remarkable flexibility in the types of activities that can be controlled by the ligand-binding domain. The activities of structurally distinct DNA-binding proteins such as lexA, c-fos, GAL4 and c-myc are rendered hormone-dependent when fused to a steroid receptor ligand-binding domain (P. J. Godowski, D. Picard and K. R. Yamamoto, Science 241 , 812 [1988]; N. J. G. Green, S. Green, J. R. Kin and P. Chambon, Cell 54, 199 [1988]; M. Eilers, D. Picard, K. R. Yamamoto and J. M. Bishop, Nature 340, 66 [1989]; D. Picard, S. J. Salser and K. R. Yamamoto, Cell 54, 1073 [1988]; G. Superti-Furga, G. Bergers, D. Picard and M. Busslinger, Proc. Natl. Acad. Sci. U.S.A. 88, 5114 [1991]). Chimeric proteins can be further modified by addition or subtraction of transactivation domains. Thus, it is expected that DHR23α fusion genes can be constructed to regulate the expression of virtually any gene for which a cis-acting regulatory sequence and its cognate DNA-binding domain have been identified. Among the genes that are anticipated for use in the method of the present invention are those encoding cytokines, hormones, structural proteins, enzymes and nucleic acids that are are present in less than therapeutically needed concentrations in mammals. Among the cytokines and hormones are polypeptides such as: growth hormone, insulin-like growth factors, interleukins, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone FSH), thyroid stimulating hormone (TSH), parathyroid hormone, and leutinizing hormone (LH), hemopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-alpha and -beta, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, glucagon, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, erythropoietin, nerve growth factors such as NGF-β, platelet-growth factor, hemopoietic growth factor, tissue factor protein, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, latency associated peptide, erythropoietin, osteoinductive factors, osteoinductive factors, interferons such as interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF, interleukins (ILs) such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 and other polypeptide factors. Among the enzymes are tissue plasminogen activase (TPA), urokinase, elastase, adenosine deaminase, bombesin, factor IX, factor VIII, thrombin, enkephalinase, β-lactamase, superoxide dismutase, reverse transcriptase, RNase, DNase, enzymes involved in glycolysis, Kreb's cycle, gluconeogenesis, urea cycle, oxidative phosphorylation, the synthesis or degradation of purines, pyrimidines, nucleic acid polymers, cholesterol, protein, glycogen, fatty acids, carbohydrate, phospholipids, glycoproteins or lipids. Among the structural proteins are lung surfactant, albumin, antibodies, carrier proteins, glycoprotein hormone receptors, CD-4, insulin-like growth factor binding proteins, calcitonin, factor VIII, an antibody, protein A or D, rheumatoid factors, viral antigens, HIV envelope proteins GP120 and GP140, immunoglobulins, serum proteins and any protein present in a suboptimal amount. Among the nucleic acids are DNA and RNA, both sense and antisense strands. The RNA may be any type, including ribosomal RNA, messenger RNA, transfer RNA, small nuclear RNA and RNA which has an enzymatic function or participates in an enzymatic function. The methods of the present invention have application to the production of nucleic acids which act as antisense or suppressor nucleic acids and inhibit the activity of a naturally occurring mammalian nucleic acid. Such nucleic acids have particular therapeutic efficacy in treating viral diseases, cancers and excessive production of a protein or other metabolic product. Ornitz, etal. (D. M. Omitz, R. W. Moreadith and Leder P., PNASβS, 698 ([1991]) have described a binary system for regulating expression of heterologous genes in transgenic mice. In this system, a "transactivator" strain expressing the yeast GAL4 protein is crossed with "target" strains containing a transcriptionally silent transgene controlled by UAS sequences. The bigenic progeny of this cross express both transgenes in the same tissue. Bigenic systems incorporating DHR23α or DHR23α gene-fusions will provide a general method to control the abundance, time course and/or tissue specific expression of endogenous or exogenous genes. For example, tissue specific and developmental^ regulated expression of transgenes controlled by EcREs is expected to be achieved by targeting the expression DHR23α with appropriate tissue specific enhancers. The expression of the EcRE containing "target" gene is expected to be induced at appropriate times by "turning-on" its activator with ecdysteroids. This development of a system that allows tissue and developmental control of genes in transgenic animals provides an important approach that complements strategies based on "gene-knockout" technology. Mammalian cells suitable for the present method include those contained in an intact mammal. They include both differentiated and undifferentiated cells. Also included within the term mammalian cells are established cell lines and primary cell cultures derived from mammalian tissue. Among the preferred mammalian cell lines are 293 cell line and CHO cell line. Therapeutic Compositions and Administration of Ecdysteroid Ecdysteroids may be administered to transgenic mammals or mammalian cells that contain a gene under the transcriptional control of an ecdysteroid. For example, gene therapy to enable a mammal to express a polypeptide previously produced in insufficient quantities. Examples of such polypeptides are described above. Therapeutic formulations of ecdysteroids are prepared for storage by mixing ecdysteroid subunit having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers {Remington's Pharmaceutical Sciences, supra), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). The ecdysteroid to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The ecdysteroid ordinarily will be stored in lyophilized form or in solution. Therapeutic ecdysteroid compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of ecdysteroid administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems as noted below. The ecdysteroid is administered continuously by infusion or by bolus injection. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the ecdysteroid, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poIy(2-hydroxyethyl-methacrylate) as described by Langer et al., Biomed. Mater. Res. 15: 167-277 (1981) and Langer, Chem. Tech. 12: 98-105 (1982) or poly(vinylalcohol), polylactides (U.S. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman etal., Biopolymers 22: 547-556 [1983]), non-degradable elhylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable micropheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release ecdysteroid compositions also include liposomally entrapped ecdysteroid. Liposomes containing ecdysteroid are prepared by methods known per se: DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci.82: 3688-3692 (1985); Hwang etal., Proc. Natl. Acad. Sci.77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal ecdysteroid therapy. An effective amount of ecdysteroid to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, the activity of the polypeptide induced by the ecdysteroid and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer the ecdysteroid until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays for the presence or activity of the induced polypeptide. Ecdysteroid compositions are administered to transgenic mammals, or to transgenic mammalian cell culture, when there is within the target cell population an available ecdysteroid receptor and an ecdysteroid DNA-binding domain positioned to promote the expression of a DNA sequence encoding a desired polypeptide or nucleic acid. The ecdysteroid receptor, such as DHR23α, acts as a potent and selective regulator of the transcription of genes containing ecdysteroid receptor DNA-binding sites. The activity of ecdysteroids, such as DHR23α, can be further modified by replacing its DNA-binding or transactivation domains ' with those from other naturally occurring genes, thereby facilitating ecdysteroid control over the other naturally occurring genes and the products they produce. The chimeric ecdysteroid receptors still bind the same ligand, however, they induce expression of the gene specified by the heterologous DNA binding domain specified by fused transactivation domain. Transformation of mammalian cells is accomplished using standard methods. Vectors suitable for transforming mammalian cells include viral and mammalian DNA capable of expression in mammalian cells. The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1 CLONING OF DNA ENCODING DHR23α Oligonucleotide probes based on the partial DHR23 sequence (W. Seegraves, thesis, Stanford University [1988]) were used to screen a cDNA library prepared from drosophila early pupal larvae. The deduced amino acid sequence of the DHR23α clone is shown in Fig. 1A. The highest region of homology between DHR23α and other members of the steroid receptor superfamiiy is found in a cysteine-rich region (residues 264-329) that corresponds to the DNA-binding domain (Fig. 1B). The putative ligand-binding domain shares limited homology with members of the retinoic acid and thyroid hormone receptors, and the vitamin D receptor. The N-terminal domain (residues 1-263) does not share significant homology with any steroid receptor superfamiiy member. I also identified a second form of this protein, DHR23β, that appears to arise by differential splicing. DHR23β is identical to DHR23α in the DNA and ligand-binding domains but contains an unrelated N-terminal domain of 234 amino acids. Figure 1 A illustrates the nucleotide and derived amino acid sequence of the DHR23α cDNA clone. Numbers on the left and right indicate nucleotide and amino acid residues, respectively. The first ATG following an upstream, in-frame stop codon is underlined and was chosen as the initiating codon. The initiator methionine conforms to the Drosophila consensus sequence for translation initiation. The conserved amino acids corresponding to the putative DNA-binding domain are underlined. To isolate the full-length DHR23α clones, -600,000 phage from a Drosophila third instar larval library were screened with two 50-mer oligonucleotide probes corresponding to nucleotides 109-158 and 1820-1869 of the partial DHR23α sequence in Seegraves, Stanford University Thesis, 1988. Eight positive clones were isolated, further characterized by PCR analysis and the nucleotide sequence of the two largest inserts was determined. The results of the sequencing are shown in figure 1A. EXAMPLE 2 EXPRESSION AND ACTIVITY OF DHR23α POLYPEPTIDE IN MAMMALIAN CELLS I determined whether DHR23α could function in mammalian cells treated with ecdysteroids to enhance the transcription of a reporter gene containing EcREs linked to the murine mammary tumor virus (MTV) promoter and chloramphenicol acetyltransferase (CAT) gene. Human 293 cells were cotransfected with an RSV-based expression vector that encodes DHR23α and the reporter gene EC4M.77CO that contains 4 copies of an ecdysone response element (EcRE) from the Drosophila HSP27 promoter (G. Riddihough and H. R. B. Pelham, EMBO J. 6, 3729 [1987]) linked to an MTV promoter-CAT construct. CAT activity was determined in extracts from cells incubated with or without the ecdysteroids - or 20-OH ecdysone, polypodine B, ponasterone A, or muristerone A. Neither α-ecdysone, 20- OH ecdysone, nor polypodine B acted as agonists for DHR23α in mammalian cells. In contrast, expression of the reporter gene was markedly increased in cells treated with muristerone A and to a lesser extent with ponasterone A (Fig. 2). This induction was dependent on the presence of an EcRE in the reporter gene because DHR23α did not regulate expression of reporter genes containing binding sites for either the glucocorticoid receptor (GRE, Fig. 2) or for the estrogen receptor (ERE, Fig. 6). Thus, in mammalian cells DHR23α acts in an ecdysteroid-dependent fashion to selectively stimulate expression from an EcRE containing reporter gene. Specific ecdysteroids are agonists for the DHR23α receptor in mammalian cells. Human 293 cells were cotransfected with 2.5 μg of the expression plasmid pRSV.DHR23α or the parental expression plasmid pRSV (Control) and 0.5 μgs of the reporter plasmid PEC4M. 77CO (EcRE) or PG4M-77CO (GRE). As a control for transfection efficiency, 0.5 μgs of the control plasmid pRSV.hGH was included in the transfection mixture. After transfection, cells were treated without (-) or with alpha-ecdysone (alpha), 20-OH ecdysone (20-OH), polypodine B (ppB), ponasterone A (ponA) or muristeroine A (murA). All candidate ligands were added at a concentration of 1 μM. CAT extracts were harvested 48 hrs after transfection, and assays were performed as described (D. R. Cavener, Nucl. Acids Res. 15, 1353-1361 [1987]). The values were normalized to the expression of hGH and the average values of three independent experiments are shown in Figure 2. The "fold induction" represents the level of expression of the reporter gene in cells incubated with hormone divided by the expression of that reporter gene in extracts from cells incubated in the absence of added ligand. The effect of mammalian hormones on activity of DHR23α is shown in Figure 3. Cells were transfected with the DHR23α expression vector and EC4M.77CO reporter gene and treated without (-) or with the following hormones (1 μM): muristerone A (mur A) dexamethasone (Dex) 17β-estradioi (E2), aldosterone (Aldo), corticosterone (Cort) hydroxycorticosterone (OH-Cort) thyroid hormone (T3), promegestone (Promeg) or 1,25- dihydroxy vitamin D3 (VD3). Reporter gene activity was determined as described above for Figure 2. Retinoic Acid also did not act as an agonist for DHR23α. A schematic representation of chimeric genetic constructs and a Western blot of the resulting expressed receptor proteins are illustrated in figures 4A and 1B. In Figure 4A receptor constructs are denoted according to a three-part nomenclature describing the origin of their N-terminal transactivation, DNA-binding and ligand-binding domains. "G" and "Ec" refer to the glucocorticoid receptor and DHR23α, respectively. "E" refers to a derivative of the rat glucocorticoid receptor DNA-binding domain with two amino acid substitutions (G458E, S459G) that convert the DNA-binding specificity to that of the estrogen receptor (K. Umesono and R. Evans, Cell 57, 1139 [1989]; M. Danielsen, L. Hinck, G. Ringold, Cell 57, 1131 [1989]). "X" (solid box) indicates the DNA-binding domain (amino acids 1-87) of the Escherichia co// LexA protein. The "V" (hatched box) denotes a derivative of the GR N-terrhinal domain in which amino acids 153-406 are replaced by the transcriptional activation domain of the HSV VP16 protein (amino acids 411-490) The construct GGEc was constructed by replacing the ligand-binding domain of GGG (amino acids 528-795) with the ligand-binding domain of DHR23α (amino acids 329-878). Similarly, to construct GXEc, the ligand-binding domain of GXG (referred to as NLxC in P. J. Godowski, D. Picard and K. R. Yamamoto, Science 241 , 812 [1988]) was replaced with the DHR23α ligand-binding domain. In figure 4B, the accumulation of receptor proteins in transfected cells are shown in a Western blot detected with enzyme-linked antibodies. Whole cell extracts were prepared 48 hrs after transfection with receptor expression plasmids encoding the following fusion proteins. Lane 1, EcEcEc; lane 2, GGG; lane 3, GGEc; lane 4, VGEc; lane 5, G"E"G; lane 6, G"E"Ec; lane 7, GXG; lane 8, GXEc; lane 9, VXEc. The blots were reacted with monoclonal antibody BuGR2, that recognizes an epitope in the N-terminal domain of the rat glucocorticoid receptor (30) and then with a sheep antiserum to mouse antibody coupled to horseradish peroxidase. Positions of the molecular markers are indicated. RNase protection analysis of transcripts induced by receptor proteins is shown in Figure 5. Total RNA was prepared from cells 48 hrs after transfection with expression plasmids encoding either DHR23α (EcEcEc), GGG, or GGEc and the reporter gene G4M. 77GO. This reporter gene contains 4 GREs fused at position -77 of an MTV promoter human growth hormone gene construct. Assays used 50 μgs of total RNA. The position of 377 base protected band for G4M.77CO and the 294 base protected band from the internal control gene (expressed from a CMV enhancer/promoter construct) are indicated by the closed and opened arrows, respectively. The induction of EREs by chimeric receptors is shown in Figure 6. Cells were transfected with expression plasmids encoding either DHR23α (EcEcEc), G"E"G or G"E"Ec and E4M-77CO, that contains 4 EREs fused to the MTV promoter. Cells were incubated for 48 hrs with or without (-) or with muristerone A (M) or dexamethasone (D). Reporter gene activity was determined as described above for Figure 2. Illustrated in Table 1 below is the induction of a glucocorticoid receptor (GRE) responsive gene by chimeric receptors. 293 cells were transfected with effector plasmids encoding the indicated receptor proteins and the reporter gene G4M.77CO. These transfected cells were incubated either without added hormone (-), with muristerone A (M) or with dex (D). The reporter gene activity was determined as described above for Fig. 2. The "fold induction" represents the level of expression of the reporter gene in cells incubated with hormone divided by the expression of that reporter gene in extracts from cells incubated in the absence of added ligand. Table 1 Induction of Glucocorticoid Receptor By Chimeric receptors Illustrated in Table 2 is the transcriptional activation by receptor-LexA fusion proteins. Effector plasmids encoding the indicated receptor proteins were transfected with a reporter gene either containing (X4C33CO) or lacking (OC.33CO) lex-operators. "Control" indicates the cells were transfected with an "effector" plasmid which does not contain a receptor cDNA. The cells were incubated either with (+) or without (-) muristerone A. The fold induction was determined as described previously; the standard deviations are shown in parenthesis. Table 2 Transcriptional Activation By Chimeric Receptors X4C33CO OC33CO Receptor + While the invention has necessarily been described- in conjunction with preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc. (ii) TITLE OF INVENTION: Ecdysteroid Dependent Regulation of Genes In Mammalian Cells (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE : California (E) COUNTRY: USA (F) ZIP: 94.080 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 5.25 inch, 360 Kb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: patin (Genentech) (vii) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 03-AUG-1992 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Fitts, Renee A. (B) REGISTRATION NUMBER: 35,136 (C) REFERENCE/DOCKET NUMBER: 607 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415/225-1489 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2970 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single- CD) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAGCTTCTTG TCCCCAGCCG ACGCTAAGTG AACGGAAAAC GGCCACAAAA 50 CGGCGACTAT CGGCTGCCAG AGG ATG AAG CGG CGC TGG TCG 91 Met Lys Arg Arg Trp Ser 1 5 AAC AAC GGC GGC TTC ATG -CGC CTA CCG GAG GAG TCG TCC 130 Asn Asn Gly Gly Phe Met Arg Leu Pro Glu Glu Ser Ser 10 15 TCG GAG GTC ACG TCC TCC TCG AAC GGG CTC GTC CTG CCC 169 Ser Glu Val Thr Ser Ser Ser Asn Gly Leu Val Leu Pro 20 25 30 TCG GGG GTG AAC ATG TCG CCC TCG TCG CTG GAC TCG CAC 208 Ser Gly Val Asn Met Ser Pro Ser Ser Leu Asp Ser His 35 40 45 GAC TAT TGC GAT CAG GAC CTT TGG CTC TGC GGC AAC GAG 247 Asp Tyr Cys Asp Gin Asp Leu Trp Leu Cys Gly Asn Glu 50 55 TCC GGT TCG TTT GGC GGC TCC AAC GGC CAT GGC CTA AAT 286 Ser Gly Ser Phe Gly Gly Ser Asn Gly His Gly Leu Asn 60 65 70 CAG CAG CAG CAG AGC GTC ATC ACG CTG GCC ATG CAC GGG 325 Gin Gin Gin Gin Ser Val He Thr Leu Ala Met His Gly 75 80 TGC TCC AGC ACT CTG CCC GCG CAG ACA ACC ATC ATT CCG 364 Cys Ser Ser Thr Leu Pro Ala Gin Thr Thr He He Pro 85 90 95 ATC AAC GGC AAC GCG AAT GGG AAT GGA GGC TCC ACC AAT 403 He Asn Gly Asn Ala Asn Gly Asn Gly Gly Ser Thr Asn 100 105 110 GGC CAA TAT GTG CCG GGT GCC ACC AAT CTG GGA GCG TTG 442 Gly Gin Tyr Val Pro Gly Ala Thr Asn Leu Gly Ala Leu 115 120 GCC AAC GGG ATG CTC AAT GGG GGC CTC AAT GGA ATG CAG 481 Ala Asn Gly Met Leu Asn Gly Gly Leu Asn Gly Met Gin 125 130 135 CAA CAG ATT CAG AAT GGC CAC GGC CTC ATC AAC TCC ACA 520 Gin Gin He Gin Asn Gly His Gly Leu He Asn Ser Thr 140 145 ACG CCC TCA ACG CCG ACC ACC CCG CTC CAC CTT CAG CAG 559 Thr Pro Ser Thr Pro Thr Thr Pro Leu His Leu Gin Gin 150 155 160 AAC CTG GGG GGC GCG GGC GGC GGC GGT ATC GGG GGA ATG 598 Asn Leu Gly Gly Ala Gly Gly Gly Gly He Gly Gly Met 165 170 175 GGT ATT CTT CAC CAC GCG AAT GGC ACC CCA AAT GGC CTT 637 Gly He Leu His His Ala Asn Gly Thr Pro Asn Gly Leu 180 185 ATC GGA GTT GTG GGA GGC GGC GGC GGA GTA GGT CTT GGA 676 He Gly Val Val Gly Gly Gly Gly Gly Val Gly Leu Gly 190 195 200 GTA GGC GGA GGC GGA GTG GGA GGC CTG GGA ATG CAG CAC 715 Val Gly Gly Gly Gly Val Gly Gly Leu Gly Met Gin His 205 210 ACA CCC CGA AGC GAT TCG-GTG AAT TCT ATA TCT TCA GGT 754 Thr Pro Arg Ser Asp Ser Val Asn Ser He Ser Ser Gly 215 220 225 CGC GAT GAT CTC TCG CCT TCG AGC AGC TTG AAC GGA TAC 793 Arg Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr 230 235 240 TCG GCG AAC GAA AGC TGC GAT GCG AAG AAG AGC AAG AAG 832 Ser Ala Asn Glu Ser Cys Asp Ala Lys Lys Ser Lys Lys 245 250 GGA CCT GCG CCA CGG GTG CAA GAG GAG CTG TGC CTG GTT 871 Gly Pro Ala Pro Arg Val Gin Glu Glu Leu Cys Leu Val 255 260 265 TGC GGC GAC AGG GCC TCC GGC TAC CAC TAC AAC GCC CTC 910 Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu 270 275 ACC TGT GAG GGC TGC AAG GGG TTC TTT CGA CGC AGC GTT 949 Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Val 280 285 290 ACG AAG AGC GCC GTC TAC TGC TGC AAG TTC GGG CGC GCC 988 Thr Lys Ser Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala 295 300 305 TGC GAA ATG GAC ATG TAC ATG AGG CGA AAG TGT CAG GAG 1027 Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gin Glu 310 315 TGC CGC CTG AAA AAG TGC CTG GCC GTG GGT ATG CGG CCG 1066 Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro 320 325 330 GAA TGC GTC GTC CCG GAG AAC CAA TGT GCG ATG AAG CGG 1105 Glu Cys Val Val Pro Glu Asn Gin Cys Ala Met Lys Arg 335 340 CGC GAA AAG AAG GCC CAG AAG GAG AAG GAC AAA ATG ACC 114 Arg Glu Lys Lys Ala Gin Lys Glu Lys Asp Lys Met Thr 345 350 355 ACT TCG CCG AGC TCT CAG CAT GGC GGC AAT GGC AGC TTG 1183 Thr Ser Pro Ser Ser Gin His " Gly Gly Asn Gly Ser Leu 360 365 370 GCC TCT GGT GGC GGC CAA GAC TTT GTT AAG AAG GAG ATT 1222 Ala Ser Gly Gly Gly Gin Asp Phe Val Lys Lys Glu He 375 380 CTT GAC CTT ATG ACA TGC GAG CCG CCC CAG CAT GCC ACT 1261 Leu Asp Leu Met Thr Cys Glu Pro Pro Gin His Ala Thr 385 390 395 ATT CCG CTA CTA CCT GAT GAA ATA TTG GCC AAG TGT CAA 1300 He Pro Leu Leu Pro Asp Glu He Leu Ala Lys Cys Gin 400 405 GCG CGC AAT ATA CCT TCC TTA ACG TAC AAT CAG TTG GCC 1339 Ala Arg Asn He Pro Ser Leu Thr Tyr Asn Gin Leu Ala 410 415 420 GTT ATA TAC AAG TTA ATT-TGG TAC CAG GAT GGC TAT GAG 1378 Val He Tyr Lys Leu He Trp Tyr Gin Asp Gly Tyr Glu 425 430 435 CAG CCA TCT GAA GAG GAT CTC AGG CGT ATA ATG AGT CAA 1417 Gin Pro Ser Glu Glu Asp Leu Arg Arg He Met Ser Gin 440 445 CCC GAT GAG AAC GAG AGC CAA ACG GAC GTC AGC TTT CGG 1456 Pro Asp Glu Asn Glu Ser Gin Thr Asp Val Ser Phe Arg 450 455 460 CAT ATA ACC GAG ATA ACC ATA CTC ACG GTC CAG TTG ATT 1495 His He Thr Glu He Thr He Leu Thr Val Gin Leu He 465 470 GTT GAG TTT GCT AAA GGT CTA CCA GCG TTT ACA AAG ATA 1534 Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys He 475 480 485 CCC CAG GAG GAC CAG ATC ACG TTA CTA AAG GCC TGC TCG 1573 Pro Gin Glu Asp Gin He Thr Leu Leu Lys Ala Cys Ser 490 495 500 TCG GAG GTG ATG ATG CTG CGT ATG GC CGA CGC TAT GAC 1612 Ser Glu Val Met Met Leu Arg Met Ala Arg Arg Tyr Asp 505 510 CAC AGC TCG GAC TCA ATA TTC TTC GCG AAT AAT AGA TCA 1651 His Ser Ser Asp Ser He Phe Phe Ala Asn Asn Arg Ser 515 520 525 TAT ACG CGG GAT TCT TAC AAA ATG GCC GGA ATG GCT GAT 1690 Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met Ala Asp 530 535 AAC ATT GAA GAC CTG CTG CAT TTC TGC CGC CAA ATG TTC 1729 • Asn He Glu Asp Leu Leu His Phe Cys Arg Gin Met Phe 540 545 550 TCG ATG AAG GTG GAC AAC GTC GAA TAC GCG CTT CTC ACT 1768 Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr 555 560 565 GCC ATT GTG ATC TTC TCG GAC CGG CCG GGC CTG GAG AAG 1807 Ala He Val He Phe Ser Asp Arg Pro Gly Leu Glu Lys 570 575 GCC CAA CTA GTC GAA GCG ATC CAG AGC TAC TAC ATC GAC 1.846 Ala Gin Leu Val Glu Ala He Gin Ser Tyr Tyr He Asp 580 585 590 ACG CTA CGC ATT TAT ATA CTC AAC CGC CAC TGC GGC GAC 1885 Thr Leu Arg He Tyr He Leu Asn Arg His Cys Gly Asp 595 600 TCA ATG AGC CTC GTC TTC TAC GCA AAG CTG CTC TCG ATC 1924 Ser Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser He 605 610 615 CTC ACC GAG CTG CGT ACG CTG GGC AAC CAG AAC GCC GAG 1963 Leu Thr Glu Leu Arg Thr Leu Gly Asn Gin Asn Ala Glu 620 625 630 ATG TGT TTC TCA CTA AAG CTC AAA AAC CGC AAA CTG CCC 2002 Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro 635 640 AAG TTC CTC GAG GAG ATC TGG GAC GTT CAT GCC ATC CCG 2041 Lys Phe Leu Glu Glu He Trp Asp Val His Ala He Pro 645 650 655 CCA TCG GTC CAG TCG CAC CTT CAG ATT ACC CAG GAG GAG 2080 Pro Ser Val Gin Ser His Leu Gin He Thr Gin Glu Glu 660 665 AAC GAG CGT CTC GAG CGG GCT GAG CGT ATG CGG GCA TCG 2119 Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala Ser 670 675 680 GTT GGG GGC GCC ATT ACC GCC GGC ATT GAT TGC GAC TCT 2158 Val Gly Gly Ala He Thr Ala Gly He Asp Cys Asp Ser 685 690 695 GCC TCC ACT TCG GCG GCG GCA GCC GCG GCC CAG CAT CAG 2197 Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gin His Gin 700 705 CCT CAG CCT CAG CCC CAG CCC CAA CCC TCC TCC CTG ACC 2236 Pro Gin Pro Gin Pro Gin Pro Gin Pro Ser Ser Leu Thr 710 715 720 CAG AAC GAT TCC CAG CAC CAG ACA CAG CCG CAG CTA CAA 2275 Gin Asn Asp Ser Gin His Gin Thr Gin Pro Gin Leu Gin 725 730 CCT CAG CTA CCA CCT CAA CTG CAA GGT CAA CTG CAA CCC 2314 Pro Gin Leu Pro Pro Gin-Leu Gin Gly Gin Leu Gin Pro 735 740 745 CAG CTC CAA CCA CAG CTT CAG ACG CAA CTC CAG CCA CAG 2353 Gin Leu Gin Pro Gin Leu Gin Thr Gin Leu Gin Pro Gin 750 755 760 ATT CAA CCA CAG CCA CAG CTC CTT CCC GTC TCC GCT CCC 2392 He Gin Pro Gin Pro Gin Leu Leu Pro Val Ser Ala Pro 765 770 GTG CCC GCC TCC GTA ACC GCA CCT GGT TCC TTG TCC GCG 2431 Val Pro Ala Ser Val Thr Ala Pro Gly Ser Leu Ser Ala 775 780 785 GTC AGT ACG AGC AGC GAA TAC ATG GGC GGA AGT GCG GCC 2470 Val Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser Ala Ala 790 795 ATA GGA CCC ATC ACG CCG GCA ACC ACC AGC AGT ATC ACG 2509 He Gly Pro.He Thr Pro Ala Thr Thr Ser Ser He Thr 800 805 810 GCT GCC GTT ACC GCT AGC TCC ACC ACA TCA GCG GTA CCG 2548 Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val Pro 815 820 825 ATG GGC AAC GGA GTT GGA GTC GGT GTT GGG GTG GGC GGC 2587 Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly 830 835 AAC GTC AGC ATG TAT GCG AAG CCC CAG ACG GCG ATG GCC 2626 Asn Val Ser Met Tyr Ala Lys Pro Gin Thr Ala Met Ala 840 845 850 TTG ATG GGT GTA GCC CTG CAT TCG CAC CAA GAG CAG CTT 2665 Leu Met Gly Val Ala Leu His Ser His Gin Glu Gin Leu 855 860 ATC GGG GGA GTG GCG GTT AAG TCG GAG CAC TCG ACG ACT 2704 He Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr 865 870 875 GCA TAG CAGGCGCAGA GTCAGCTCCA CCAAC TCAC CACC CAAC 2750 Ala 878 TCGACGTCCT GCTGGAGTAG AAAGCGCAGC TGAACCCACA CAGACATAGG 2800 GGAAATGGGG AAGTTCTCTC CAGAGAGTTC GAGCCGAACT AAATAGTAAA 2850 AAGTGAATAA TTAATGGACA AGCGTAAAAT GCAGTTATTT AGTCTTAAGC 2900 CTGCAAATAT TACCTATTAT TCATACAAAT TAACATATAA TACAGCCTAT 2950 TAACAATTAC GCTAAAGCTT 2970

Claims: Claims: 1. A method of inducing gene expression in a mammalian cell comprising contacting an ecdysteroid with an ecdysteroid receptor polypeptide within a mammalian cell, wherein said mammalian cell further contains a DNA binding sequence for said ecdysteroid receptor when in combination with its ligand ecdysteroid, wherein formation of a receptor-ligand-DNA binding sequence complex induces gene expression. 2 The method of claim 1 wherein said ecdysteroid does not contain a 25 hydroxyl group. 3. The method of claim 2 wherein said ecdysteroid contains an electron-withdrawing group at position 5. 4. The method of claim 2 wherein said ecdysteroid contains an electron-withdrawing group at position 11. 5. The method of claim 3 wherein said electron-withdrawing group is selected from the following: hydroxyl, ketone, sulfhydral, nitrate, nitrite, florine, bromine, iodine and chlorine. 6. The method of claim 4 wherein said electron-withdrawing group is selected from the following: hydroxyl, ketone, sulfhydral, nitrate, nitrite, florine, bromine, iodine and chlorine 7. The method of claim 2 wherein said ecdysteroid is selected from the following: muristerone A, 5-dehydroxy muristerone A, 11-dehydroxy muristerone A and ponasterone. 8. The method of claim 1 wherein said DNA binding sequence further contains a DNA sequence encoding a eukaryotic gene. 9. The method of claim 8 wherein said heterologous eukaryotic gene encodes a polypeptide or nucleic acid selected from the following: cytokine, enzyme, structural polypeptide, DNA and RNA. 10. The method of claim 8 wherein said cytokine is selected from the following: growth hormone, insulin-like growth factors, interleukins, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxϊn, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), parathyroid hormone, and leutinizing hormone (LH), hemopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-alpha and -beta, mullerian-inhibiting substance, mouse gonadotropin- associated peptide, glucagon, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, erythropoietin, nerve growth factors such as NGF-β, platelet-growth factor, hemopoietic growth factor, tissue factor protein, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-l and -II, latency associated peptide, erythropoietin, osteoinductive factors, osteoinductive factors, interferon-alpha, -beta, and -gamma, the colony stimulating factor (CSF) M-CSF, GM-CSF, and G-CSF, interleukins (ILs) IL-1, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8 and other polypeptide factors. 11. The method of claim 8 wherein said enzyme is selected from the following: tissue plasminogen activase (TPA), urokinase, elastase, adenosine deaminase, bombesin, factor IX, factor VIII, thrombin, enkephalinase, β-lactamase, superoxide dismutase, reverse transcriptase, RNase, DNase, enzymes catalyzing glycolysis, Kreb's cycle, gluconeogenesis, urea cycle, oxidative phosphorylation, the synthesis or degredation of purines, pyrimidines, nucleic acid polymers, cholesterol, protein, glycogen, fatty acids, carbohydrate, phospholipids, glycoproteins or lipids. 12 The method of claim 8 wherein said structural polypeptide is selected from the following: lung surfactant, albumin, antibodies, carrier proteins, transport proteins, glycoprotein hormone receptors, CD-4, insulin-like growth factor binding proteins, calcitonin, factor VIII, an antibody, protein A or D, rheumatoid factors, viral antigens, HIV envelope proteins GP120 and GP140, immunoglobulins, serum proteins and any cytoskeletal polypeptide. 13. The method of claim 8 wherein said nucleic acid is DNA and RNA. 14. The method of claim 1 wherein said mammalian cell is within a mammal. 15. The method of claim 15 wherein said mammal is a human. 16. A method of producing a desired protein comprising: a) contacting an ecdysteroid with an ecdysteroid receptor polypeptide within a mammalian cell, wherein said mammalian cell further contains a chimeric DNA sequence comprising 1) a first DNA binding sequence having binding specificity for said ecdysteroid receptor when in combination with its ligand ecdysteroid and 2) a second DNA sequence encoding a desired protein heterologous to said DNA binding sequence and under the transcriptional control of the ecdysteroid receptor-ligand complex; and b) incubating said cell and producing said desired protein. 17. The method of claim 16 wherein said mammalian cell is in a mammal. 18. The method of claim 16 wherein said desired protein is selected from the following: cytokines, enzymes and structural polypeptides. 19. The method of claim 16 wherein said ecdysteroid is selected from the following: muristerone A, 5-dehydroxy muristerone A, 11-dehydroxy muristerone A and ponasterone. 20. A method of regulating endogenous or heterologous genes in transgenic mammals comprising: a) contacting an ecdysteroid with an ecdysteroid receptor polypeptide within a mammalian cell, wherein said mammalian cell further contains a chimeric DNA sequence comprising 1) a first DNA binding sequence having binding specificity for said ecdysteroid receptor when in combination with its ligand ecdysteroid, and 2) a second DNA sequence encoding a desired gene product heterologous to said DNA binding sequence and under the transcriptional control of the ecdysteroid receptor-ligand complex; and b) incubating said cell and producing said desired gene product. 21. The method of claim 20 wherein said ecdysteroid receptor is one having binding affinity for muristerone A. 22. The method of claim 20 wherein said ecdysteroid is one having no hydroxyl group at position 25. 23. The method of claim 22 wherein said ecdysteroid is selected from the following: muristerone A, 5-dehydroxy muristerone A, 11-dehydroxy muristerone A and ponasterone. 24. A method of regulating gene expression in a mammalian cell containing a DNA sequence encoding a polypeptide heterologous to said mammalian cell and under the transcriptional control of an ecdysteroid receptor comprising: (a) transforming said mammalian cell to express a ecdysteroid receptor fusion polypeptide wherein said fusion polypeptide contains a heterologous transactivation domain; and (b) contacting said transformed mammalian cell with an ecdysteroid that activates said ecdysteroid receptor. 25. The method of claim 24 wherein said transactivation domain is selected from the following: herpes virus VP16 acidic activation domain, lexA, c-fos, GAL4 and c-rnyc. 26. The method of claim 24 wherein said ecdysteroid is selected from the following: muristerone A, 5-dehydroxy muristerone A, 11-dehydroxy muristerone A and ponasterone. 27. The method of claim 24 wherein said ecdysteroid receptor is DHR23 .

Inventors: GODOWSKI PAUL J, GODOWSKI, PAUL, J.
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1992
Main classification: C12N15
IPC codes: A01K67, A61K38, C07K14, C12N5, C12N15, C12P21