AU681025B2 - DNA regulatory elements responsive to cytokines - Google Patents

Description

WO 95/28492 PCT/US95/04511 1 DNA REGULATORY ELEMENTS RESPONSIVE TO CYTOKINES Field of the Invention This invention relates to oligonucleotide sequences that bind regulatory proteins that affect transcription in response to various molecules, such as cytokines, to DNA constructs comprising the oligonucleotide sequences, cells transfected with the DNA constructs, and to methods of using the same to provide for the controlled expression of heterologous genes, for the detection and recovery of new regulatory proteins, and for measuring the ability of compounds to act as agonist and antagonists of gene transcription.

Background of the Invention In many cellular systems, extracellular signaling molecules, such as polypeptide ligands, bind to receptors on the surface of the cells, thereby triggering an intracellular signaling pathway that ultimately regulates gene transcription within the cells. For example, cytokines and growth factors, which comprise a large and diverse family of soluble polypeptides that control the growth, differentiation and function of mammalian cells, bind to specific cell surface receptors, that in some way transduce signals that elicit a specific phenotypic response. A. Miyajama et al., 10 Annu. Rev.

Immunol., 295 (1992); M. Aguet et al., 55 Cell, 273 (1988); T. Kishimoto et al., 258 Science, 593 (1992) and A. Ullrich and J. Schlessinger, 61 Cell, 203 (1990). Abundant evidence shows that changes in the transcription rate of specific genes are an important component of this response. This is thought to be a consequence of alterations in the amount or the activity of specific DNA-binding proteins.

In some instances, progress has been made in defining the pathway that leads from a receptor-ligand interaction at the cell surface to changes in the activity of such DNA binding proteins or other nuclear proteins. Ulrich, 61 Cell 203. In this regard, a common response in surface receptor signaling pathways involves the activation of Ras. L.S. Mulcahy et al., 313 Nature, 241 (1985). Activated Ras then ~U~Y ~Dl~h l ~w ~a~aQI~B~B~B% WO 95/28492 PCT/IS95/04511 2 initiates a cascade of serine/threonine phosphorylations through MAP kinases leading to phosphorylation of DNA binding proteins, thereby changing their transcriptional modulatory activity. S.A. Moodie et al., 260 Science, 1658 (1993); C. A. Lange-Carter et al., 260 Science, 315 (1993); C. S. Hill et al., 73 Cell 395 (1993); H. Gille et al., 358 Nature, 414 (1992) and R. H. Chen et al., 12 Mol. Cell. Biol., 915 (1992).

Despite these advances, the signal transduction pathways utilized by many growth factors and cytokines to alter gene expression remain unclear. Thus, although known second messengers have been implicated in signal transduction in response to some of these factors, their role in modulating gene expression remains speculative. Miyajama, 10 Armu. Rev. Immunol., 295 and D.E. Levy and J.E. Darnell, 2 New Biol., 923 (1990). This in turn raises the question of how ligand specific responses are elicited in such cellular systems. Ullrich, 61 Cell, 203; M.V. Chao, 68 Cell, 995 (1992) and Levy, 2 New Biol., 923.

Progress in resolving these issues has been made recently in the interferon (IFN) system. IFNs a and 3 (type I) act as a primary non-specific defense against viral infections. S. Petska and J.A. Langer, 56 Annu. Rev. Biochem., 727 (1987). IFNy (type II) has anti-viral properties but also plays a major role in regulation of the immune response. Id. Type I and type I IFNs bind to distinct cell surface receptors and cause rapid alterations in gene expression. Auget, 55 Cell, 273; Uze, 60 Cell, 225; and G.C.

Sen and P. Lengyel, 267 J. Biol. Chem., 5017 (1992). Specific sequence elements have been identified in the promoters of genes that respond to IFNa, termed interferon-a stimulated response elements (ISREs), that are both necessary and sufficient for regulation by IFNa. Sen, 267 J. Biol. Chem., 5017. Specifically, activation of the IFN a receptors stimulates tyrosine phosphorylation of a family of proteins that serve as DNA binding proteins, and accordingly as transcription regulatory factors via the ISRE.

C. Schitdler et al., 257 Science, 809 (1992); K. Shuai et al., 258 Science, 1808 (1992) and'M. J. Gutch et al., 89 Proc. Natl. Acad. Sci. USA, 11411 (1992). These DNA binding proteins, generically termed "signal transducers and activators of transcription" (STATs), assemble into a multimeric complex, translocate to the nucleus, and bind cisacting enhancer elements in the appropriate regulatory regions. D.E. Levy et al., 3 r WO 95/28492 PCT/US95/04511 3 Genes Dev., 1362 (1989); and D.S. Kessler et al., 4 Genes Dev., 1753 (1990) and Z.

Zhong et al., 264 Science, 95 (1994).

One example of an IFNa-induced ISRE binding protein complex is ISGF3. T.C. Dale et al., 86 Proc. NatL Acad. Sci., 1203 (1989) and X-Y. Fu et al., 87 Proc. Natl. Acad. Sci., 8555 (1990). ISGF3 is a complex of 4 binding proteins, called p4 8 p84 (STAT1P), p91 (STATla) and p113 (STAT2). Recently, cDNAs encoding the proteins that constitute ISGF3 have been isolated and characterized. X-Y Fu et al., 89 Proc. Natl. Acad. Sci., 7840 (1992); C. Schindler et al., 89 Proc. Natl. Acad. Sci., 7836 (1992) and S.A. Veals et al., 12 Mol. Cell. Biol., 3315 (1992). p48 is the DNA binding component of ISGF3 and has homology to myb. Veals, 12 Mol. Cell. Biol., 3315. p 84 and p91 are probably alternatively spliced products of the same gene and are related to p 1 13. X-Y Fu, 89 Proc. Natl. Acad. Sci., 7840 and Schindler, 89 Proc. Natl.

Acad. Sci., 7836. p84, p 9 1 and p113 are novel proteins that contain SH2 and SH3 domains and are found in the cytoplasm of untreated cells. Schindler, 257 Science, 809 and X.Y. Fu, 70 Cell, 323-335 (1992). Thus, IFNa treatment of cells results in rapid tyrosine pphsphorylation of p84, p 9 1 and p113, causing them to associate and form a heteromeric complex with p48 to form ISGF3, which then translocates to the nucleus and binds to ISREs, stimulating transcription. Id.; Dale, 86 Proc. Natl. Acad. Sci., 1203 and Kessler, 4 Genes Dev., 1753.

Regulation in response to IFNy is conferred by a distinct sequence from the ISRE, the gamma activated sequence (GAS). T. Decker et al., 10 EMBO 927 (1991); K.D. Khan et al., 90 Proc. Natl. Acad. Sci., 6806 (1993) and D.J. Lew et al., 11 Mol. Cell. Biol., 182 (1991), DNA segments containing just a GAS element can confer an IFN7 response to a heterologous promoter when multimerized. Decker et al., 11 Mol. Cell. Biol., 5147-5153 (1991) and Kanno et al., 13 Mol. Cell. Biol., 951-3963 (1993). Treatment of cells with IFNy results in tyrosine phosphorylation of p91 (STATI which then forms homodimers that translocate to the nucleus and bind to the GAS element. Decker, 10 EMBO 927; K. Shuai et al., 258 Science, 1808 (1992) and Shuai et al., 76 Cell, 821-828 (1994). Thus the specificity of binding of either FN 3 0 a or IFNy to their receptors is translated into a specific phosphorylation pattern within a -a WO 95/28492 PCT/US95/04511 4 related family of latent transcription factors DNA binding proteins). This pattern of phosphorylation dictates the association state of the proteins, which determines specificity of binding to either an ISRE or a GAS, and the subsequent transcriptional response.

Yet another cytokine, Interleukin-6 (IL-6) plays a major role in the induction of the acute phase response in hepatocytes. The acute phase response is characterized by the dramatic transcriptional upregulation of a distinct set of genes, termed acute phase response genes. P.C. Heinrich et al, 265 Biochem. 621-636 (1990). Studies of the promoter regions of these genes have identified specific DNA sequences that are required for induction of acute phase response genes by IL-6. See D.R. Kunz et al., 17 Nuc. Acids Res., 1121-1138 (1989); M. Hattori et al., 87 Proc.

Natl. Acad. Sci USA, 2364-2368 (1990); K,A. Won and H. Baumann, 10 Mol. Cell.

Biol, 3965-3978 (1990) and D.R. Wilson et al., 10 Mol. Cell. Biol., 6181-6191 (1990).

These sequences are termed acute phase response elements (APREs). One type of APRE shows many similarities to the GAS elements that confer induction by IFNy.

Several natural GAS elements mediate transcriptional induction by both IFNy and IL-6.

Yuan et al., 14 Mol. Cell. Biol., 1657-1668 (1994); Harroch et al., 13 EMBO J. 1942- 1949 (1994) and Harroch et al., 269 J. Biol. Chem., 26191-26195 (1994). Proteins that bind to this class of APREs have been characterized and purified. U. M. Wegenka, et al., 14 Mol. Cell. Biol., 3186-3196 (1994); U. M. Wegenka et al. 13 Mol. Cell. Biol., 276-288 (1993); T. Ito et al., 17 Nuc. Acids Res., 9425-9435 (1989) and Hattori, 87 Proc. Natl. Acad. Sci. USA, 2364-2368. A cDNA clone encoding the IL-6-induced APRE-binding protein has been isolated (Zhong, 264 Science, 95; Akira et al., 77 Cell 63-71 (1994); Zhong, et al., 91 Proc. Natl. Acad. Sci., 4806-4810 (1994); Raz et al., 269 J. Biol. Chem., 24391-24395 (1994)), and was found to encode a protein that shows considerable homology to p91 (STAT1a). For this reason the protein is termed STAT3. Like STATa, STAT3 is a latent transcription factor that is activated to bind DNA by rapid tyrosine phosphorylation. In some cells, IL-6 also activates STATIa.

In these cells, heterodimers of STATlo and STAT3 form in addition to homodimers of 3 0 STAT1a and homodimers of STAT3. Sadowski et al., 261 Science, 1739-1744 (1993); edl dp~p ~D9sB~B~ WO 95/28492 PCT/US95/04511 Raz et al., 269 J. Biol. Chem., 24391-24395 (1994) and Zhong et al., 264 Science, 98 (1994).

Although IFNY and IL-6 activate STAT proteins that can bind to similar sequences (GAS/APREs), they regulate distinct sets of genes. This suggests that there is specificity with respect to the response elements in some of these genes, such that they respond only to one of these cytokines. In addition, many cytokines other than IFN 7 and IL-6 cause the rapid activation of GAS-binding protein complexes. See 0.

Silvennionen et al., 261 Science, 1736 (1993); H.B. Sadowski et al., 261 Science, 1739 (1993); A.C. Lamer et al., 261 Science, 1730 (1993); D. Finbloom et al., 14 Mol. Cell.

Biol., 2113-2118 (1994); D. Meyer et al., 269 J. Biol. Chem., 4701-4704 (1994); Lamb, et al., 83 Blood 2063-2071 (1994); and Tian, et al., 84 Blood 1760-1764 (1994). Some of these complexes contain STATIa and/or STAT3, and some contain uncharacterized proteins. These cytokines also regulate sets of genes distinct'from each other and from those regulated by IFNy and IL-6. It is therefore possible that distinct classes of GASlike sequences exist that are selective in their ability to respond to various cytokines.

Accordingly, specific DNA sequences that show selectivity with respect to their ability to bind cytokine-activated proteins, including STAT proteins, would be usefl tools allowing the responses mediated by different cytokine-activated DNA-binding proteins to be assayed selectively.

The disclosures of the above-cited references are hereby incorporated by reference in their entirety.

Summary of the Invention The present invention is directed to oligonucleotide sequences comprising DNA regulatory elements that bind, either directly or indirectly, to activated transcriptional regulatory proteins, preferably STAT proteins, in response to signaling molecules, including cytokines such as interferon gamma (IFNy), interleukin 4 (JL-4), interleukin 6 and granulocyte-macrophage colony stimulating factor (GM-CSF).

Surprisingly, individual point mutations in the Ly6E GAS regulatory element result in discrete regulatory elements that bind a variety of activated transcriptional regulatory proteins and/or specifically bind a single type or class of activated transcriptional 7 N IdlP WO 95/28492 PCT/US95/04511 6 regulatory protein. Accordingly, the regulatory elements of the present invention can be used in transcriptional assays to discover agonists or antagonists of a signaling molecule, such as IFNy, and its cognate transcriptional regulatory protein, STATla.

In particular, the present invention provides oligonucleotide sequences comprising regulatory elements of the nucleotide sequence TATTCCTGGAAGT (SEQ ID NO. TATTCCGGTAAGT (SEQ ID NO. TCTTCCTGTAAGT (SEQ ID NO.

TATTCTTGTAAGT (SEQ ID NO. TATTCCTGTTAGT (SEQ ID NO. TATTCCCGTAAGT (SEQ ID NO. TATTCCTATAAGT (SEQ ID NO. 7), TATTCCTGTCAGT (SEQ ID NO. TATACCTGTAAGT (SEQ ID NO. 9), TATGCCTGTAAGT (SEQ ID NO. 10), TATTCCTTTAAGT (SEQ ID NO. 11), TATTCCTCTAAGT (SEQ ID NO. 12), TATTCCTGCAAGT (SEQ ID NO. 13), or TATTCCTCGACGT (SEQ ID NO. 14). These oligonucleotide sequences can be double stranded, including their complement.

The present invention also provides a DNA construct comprising the regulatory elements of the oligonucleotide sequences described above operably linked to a promoter, which promoter is operably linked to a heterologous gene, wherein the DNA construct is linked in such a manner that the heterologous gene is under the transcriptional control of the oligonucleotide sequence and promoter. Also provided is a host cell transfected with this DNA construct.

The present invention also provides a method for the controlled expression of a heterologous gene of interest comprising culturing the transfected host cells containing an appropriate transcriptional regulatory protein(s) in the presence of a signaling molecule. Preferably, the signaling molecule in this method comprises a cytokine and the transcriptional regulatory protein comprises a STAT protein.

The present invention further provides a method for detecting the presence of an activated transcriptional regulatory protein, such as a novel STAT protein, in a sample comprising contacting the sample with an oligonucleotide sequence as described above under conditions where the transcriptional regulatory protein is activated and binds with the oligonucleotide sequence to form a complex, and detecting the presence of the complex in the sample. Thereafter, the complex can be separated e -clsrBllr~W~BII WO 95/28492 PCT/US95/04511 7 from the sample, and the transcriptional regulatory protein isolated from the regulatory element.

Further, the present invention provides a method for measuring the ability of a compound to act as an agonist of gene transcription comprising (a) contacting the compound with a transfected host cell as described above under conditions in which the heterologous gene is capable of being expressed in response to the compound, and comparing the level of gene expression in step with the level of gene expression from the host cell in the absence of the compound. Alternatively, the present invention also provides a method for measuring the ability of a compound to act as an antagonist of gene transcription comprising contacting the compound with a transfected host cell as described above in the presence of a predetermined amount of a signaling molecule under conditions in which the heterologous gene is expressed in response to the signaling molecule, and comparing the level of gene expression in step with the level of gene expression from the host cell in the presence of the signaling molecule, but the absence of the compound. In both these methods, the heterologous gene may be any appropriate reporter gene such as the gene for luciferase, chloramphenicol acetyl transferase, green fluorescent protein or (3galactosidase.

Further yet, the present invention provides a method for selectively measuring the ability of a compound to agonize or antagonize the induction of STAT heterodimers comprising contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed in response to the compound, wherein the host cell is transfected with a DNA construct comprising a single copy of an oligonucleotide sequence comprising a regulatory element that that is capable of selectively binding to an activated STAT heterodimer, and comparing the level of gene expression In step with the level of gene expression from the host cell in the absence of the compound.

These and various other advantages and features of novelty which characterize the invention are pointed out with particularity in the claims annexed hereto and forming a part hereof. However, for a better understanding of the invention, its advantages, and objects obtained by its use, reference should be had to the

L_.

WO 95/28492 PCT/US95/04511 8 accompanying drawings and descriptive matter, in which there is illustrated and described preferred embodiments of the invention.

Definitions For the purposes of this invention: "Oligonucleotide" or "DNA" molecule or sequence refers to a molecule comprised of the deoxyribonucleotides adenine guanine thymine and/or cytosine in either single-stranded form or a double-stranded helix, and comprises or includes a "regulatory element" according to the present invention, as that term is defined herein. The exact size, strandedness and orientation 3' to or 5' to will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotides of the present invention. Thus, the term "oligonucleotide" or "DNA" includes double-stranded DNA found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction.

"Regulatory element" refers to a deoxyribonucleotide sequence comprising the whole, or a portion of, an oligonucleotide sequence to which an activated transcriptional regulatory protein, or a complex comprising one or more activated transcriptional regulatory proteins, binds so as to transcriptionally modulate the expression of an associated gene or genes, including heterologous genes.

"Signaling molecule" refers to an extracellular polypeptide, oligosaccaride or other non-peptidyl molecule, in either a free or bound form, that interacts with a receptor at or near the surface of a cell. This interaction in turn triggers an intracellular pathway which includes the activation of one or more transcriptional regulatory proteins that bind to a regulatory element, thereby transcriptionally modulating the expression of an associated gene or genes. As used herein, "signaling molecule" includes naturally occurring molecules, such as cytokines, peptidyl and nonpeptidyl hormones, antibodies, cell-surface antigens, or synthetic mimics of any of 3 0 these signaling molecules, or compounds that mimic the action of any of these signaling molecules.

L

WO 95/28492 PCT/US95/04511 9 "Cytokines" refer to a diverse grouping of soluble polypeptides, including growth factors and hormones, that control the growth, differentiation and function of cells in such a manner as to ultimately elicit a phenotypic response in an organism. Preferred cytokines useful with the regulatory elements and associated methods of the present invention include IFN, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, EL-9, IL-11, IL-12, IL-13, L-14, IL-15, Epo, Tpo, GM-CSF, Oncostatin M, growth hormone, G-CSF, LIF, EGF, CNTF and PDGF.

"Transcriptional regulatory protein" refers to cytoplasmic or nuclear proteins that, when activated, bind the regulatory elements/oligonucleotide sequences of the present invention either directly, or indirectly through a complex of transcriptional regulatory proteins or other adapter proteins, to transcriptionally modulate the activity of an associated gene or genes. Thus, transcriptional regulatory proteins can bind directly to the DNA regulatory elements of the present invention, or can bind indirectly to the regulatory elements by binding to another protein, which in turn binds to or is bound to a DNA regulatory element of the present invention. See S.A. Veals et al., 13 Molec. Cell. Biol., 196-206 (1993). As used herein, transcriptional regulatory proteins, include, but are not limited to, those proteins referred to in the art as STAT proteins Zhong et al., 264 Science, 95) STF proteins Schindler et al., 13 EMBO 1350 (1994)), Mammary Gland-Specific Nuclear Factor Schmidt-Ney et al., 6 Mol. Endochronol., 1988 (1992); Wakao, et al., 13 EMBO J. 2182-2191 (1994); and H. Wakao et al., 267 J. Biol. Chem., 16365 (1992)), APRF (Wegenka, 13 Mol. Cell Bio., 276), GHIF (Mayer, 269 J. Biol. Chem., 4701); IL-4 STAT (Hou, et al., 265 Science 1701-1706 (1994)), GHSF and EPOSF (Finbloom, 14 Mol. Cell Bio., 2113), as well as to all substantially homologous analogs and allelic variations thereof.

"Transcriptionally modulate the expression of an associated gene or genes" means to change the rate of transcription of such gene or genes.

"STAT protein" refers to those transcriptional regulatory proteins designated as "Signal Transducers and Activators of Transcription" (STAT) by Dr. J.E.

Darnell of Rockefeller University. See Zhong, 264 Science 95. As used herein, STAT proteins include the p91 (STATIc), p84 (STAT13), p113 (STAT2) proteins and the WO 95/28492 PCT/US95/04511 STAT-associated p48 family of proteins. S.A. Veals et al., 12 Mol. Cell. Biol., 3315 (1992). Further, STAT proteins also include a binding protein designated as STAT3 (Zhong, et al., 91 Proc. Natl. Acad. Sci., 4806-4810 (1994); Zhong, 264 Science and a binding protein designated as STAT4 In addition, MGF is now renamed STAT 5 (Gouilleux et al., 13 EMBO J. 4361-4369 (1994)), and IL-4-STAT is referred to as STAT6 by some investigators (Ihle et al., 11 Trends Genet., 69 (1995)). Also included are substantially homologous analogs and allelic variations of all of the above STAT proteins.

"Activate", "activated", "activation" or derivatives thereof, means that one or more transcriptional regulatory proteins within a cell are modified posttranslationally, or are constituitively active, such that they can bind directly or indirectly to DNA regulatory elements/oligonucleotide sequences of the present invention in a sequence-specific manner. This modification will typically comprises phosphorylation of the. transcriptional regulatory proteins via a variety of mechanisms, including, but not limited to activation by various protein kinases. See, eg, (Shuai, 258 Science, 1808 and P. Cohen, 17 TIBS, 408 (1992)).

"DNA construct" refers to any genetic element, including, but not limited to, plasmids, vectors, chromosomes and viruses, that incorporate the oligonucleotide sequences of the present invention. For example, the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.

"Promoter" refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of the present invention, the promoter is bounded at its 3' terminus by the transcription initiation site and extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" WO 95/28492 PCT/US95/04511 11 boxes and "CCAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

"Gene" refers to a nucleic acid molecule, the sequence of which includes all the information required for the normal regulated production of a particular protein.

A "heterologous" region of a DNA construct a heterologous gene) is an identifiable segment of DNA within a larger DNA construct that is not found in association with the other genetic components of the construct in nature. Thus, when the heterologous gene encodes a mammalian gene, the gene will usually be flanked by a promoter that does not flank the structural genomic DNA in the genome of the source organism.

A promoter of a DNA construct, including an oligonucleotide sequence according to the present invention, is "operably linked" to a heterologous gene when tile presence of the promoter influences transcription from the heterologous gene, including genes for reporter sequences such as luciferase, chloramphenicol acetyl transferase, A3-galactosidase and secreted placental alkaline phosphatase. Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript. Thus, two sequences, such as a promoter and a "reporter sequence" are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another.

A host cell has been "transfected" by exogenous or heterologous DNA a DNA construct) when such DNA has been introduced inside the cell. The transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stablely transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA.

"Host cell" refers to a cell line that expresses, either normally or after transfection of the requisite cDNAs, the relevant receptor components for a given WO 95/28492 PCTIUS95/04511 12 signaling molecule, signaling kinase) proteins, transcriptional regulatory proteins, and accessory factors such that, upon binding of the signaling molecule to the cell surface, transcriptional regulatory protein-mediated gene transcription is affected.

Preferably, the host cell line is responsive to cytokines, such that the host cell line expresses, either normally or after transfection of the requisite cDNAs, the relevant cytokine receptor components, JAK proteins, STAT proteins, and accessory factors such that, upon cytokine binding to the cell surface, STAT-mediated gene transcription is affected.

Brief Description of the Drawings The invention may be further illustrated by reference to the accompanying Drawings wherein: FIG. 1 is a summary of the oligonucleotide competition data for complexes containing transcriptional regulatory proteins activated by IFN7, IL-6, GM- CSF and IL-4. The name of each oligonucleotide, followed by its sequence, is shown on the left. The mutated nucleotide is underlined and shown in bold type. The ability of each sequence to compete with the Ly6E GAS element (termed the "wild type, WT" sequence) for binding to transcriptional regulatory proteins activated by either IFN-7, IL-6, GM-CSF or IL-4 is shown by the number in the appropriate column; FIG. 2 is an example of an oligonucleotide competition experiment.

Nuclear extracts from cells treated with either IFN-y, IL-6, IL-4 or GM-CSF were incubated with the indicated amounts of oligonucleotide 8T (SEQ ID NO. 11) prior to the addition of radiolabeled Ly6E GAS element probe. Complexes were then separated on a non-denaturing polyacrylamide gel, which was then exposed to X-ray film. A reproduction of the resulting autoradiogram is shown. A and B indicate the positions of the complexes of transcriptional regulatory proteins that correspond to STAT1a and STAT3 respectively; FIG. 3 is a graph showing transcriptional induction mediated by a single Ly6E GAS element and selected regulatory element-containing oligonucleotide sequences of the present invention in response to cytokines. Cells were transfected with reporter plasmids containing a single copy of the inventive regulatory elements or

I

WO 95/28492 PCT/US95/04511 13 the wild type Ly6E GAS element driving expression of luciferase. Cells were treated with either IFN-y, IL-6, LIF or OSM as indicated, and induction of transcription over untreated cells determined as described in the Examples. The TK luc control reporter lacks a regulatory element; FIG. 4 is a graph showing transcriptional induction mediated by multiple regulatory elements in response to cytokines. Cells were transfected with reporter plasmids containing four copies of either the Ly6E GAS element or the 7C or 8A oligonucleotides containing regulatory elements of the present invention, treated with the indicated cytokines, and fold inductions calculated as described in the Examples; FIG. 5 is a comparison of the in vitro binding affinities of the Ly6E GAS element and the 7C, 8A, 4G, and 8T oligonucleotides containing regulatory elements of the present invention for STATla, with the transcriptional induction in response to IFN-y from constructs containing 4 copies of the same elements. The first column lists the regulatory elements studied. The next column indicates the ability of each element to tiid to STATIa in vitro (data taken from FIG. The last column shows the fold induction of luciferase activity in response to IFN-y in cells transfected with reporter plasmids containing four copies of the listed regulatory elements; and FIG. 6 is a series of gel panels showing tyrosine phosphorylation of STAT@a and STAT3 in response to cytokines. Cells were treated with either IFN-y, LIF, IL-6 or OSM for the indicated time, and lysates prepared. Lysates were immunoprecipitated with either a STAT1 antisera (top panels) or a STAT3 antisera (bottom panels). After resolution on SDS-polyacrylamide gels and blotting, proteins were detected with antisera directed against either phosphotyrosine, STAT1 or STAT3 as indicated at the left of FIG 6.

11~"1)1 __111 WO 95/28492 PCT/US95/04511 14 Detailed Description of Embodiments of the Invention The present inventors have discovered a series of DNA regulatory elements response elements) that in response to various signaling molecules, bind, either directly or indirectly, to activated transcriptional regulatory proteins, and accordingly, transcriptionally modulate the expression of one or more genes operably linked with such regulatory elements. In this regard, the inventors have surprisingly discovered that selected point mutations in the Ly6E GAS response element can transform an element from one that binds with a variety of cytokine-induced, activated transcriptional regulatory proteins to a regulatory element that selectively binds just one type or class of cytokine-induced, activated transcriptional regulatory proteins. For example, the sequence TATACCTGTAAGT (SEQ ID NO. 9) yields a regulatory element that is selective for a STATIa transcriptional regulatory protein, while the sequence TATTCCCGTAAGT (SEQ ID NO. 6) yields a regulatory element that is selective for the STAT1a and STAT3 transcriptional regulatory proteins. On the other hand, nucleotide sequences such as TATTCCTGGAAGT (SEQ ID NO. 1) yield a regulatory element that can mediate transcriptional induction in response to a variety of different cytokine-induced transcriptional regulatory proteins, including the STAT1a and STAT3 proteins, as well as those transcriptional regulatory protein(s) that are activated by the IL-4, IL-13 and GM-CSF cytokines.

The regulatory elements according to the present invention are selected from the nucleotide sequences TATTCCTGGAAGT (SEQ ID NO. 1), TATTCCGGTAAGT (SEQ ID NO. TCTTCCTGTAAGT (SEQ ID NO. 3), TATTCTTGTAAGT (SEQ ID NO. TATTCCTGTTAGT (SEQ ID NO. TATTCCCGTAAGT (SEQ ID NO. TATICCTATAAGT (SEQ ID NO. 7), TATTCCTGTCAGT (SEQ ID NO. TATACCTGTAAGT (SEQ ID NO. 9), TATGCCTGTAAGT (SEQ ID NO. 10), TATTCCT AAGT (SEQ ID NO. 11), TATTCCTCTAAGT (SEQ ID NO. 12), TATTCCTGCAAGT (SEQ ID NO. 13), or TATTCCTGTACGT (SEQ ID NO. 14). In this regard, SEQ ID NOs 9-14 comprise STATIa protein selective regulatory elements, SEQ ID NOs 1-5 comprise regulatory elements that bind a variety of transcriptional regulatory proteins, and SEQ ID NOs 6-8 WO 95/28492 PCT/US95/04511 comprise regulatory elements that selectively bind the STAT 1 and STAT3 transcriptional regulatory proteins. Furthermore, these regulatory elements alone, or with additional flanking nucleotide sequences, can form various oligonucleotide sequences according to the present invention. In this regard, it is preferable that such nucleotide sequences comprise between 13 and 200 nucleotides, including the regulatory elements of the present invention. However, sequences in excess of 200 nucleotides that contain the regulatory elements of the present invention, that are capable of binding activated transcriptional regulatory proteins, and of transcriptionally modulating the expression of one or more genes thereby, are considered to be within the scope of the present invention.

The oligonucleotide sequences of the present invention can also comprise multimers of two or more "units" of the basic regulatory elements. In this regard, such multimer oligonucleotide sequences can, as a practical matter, contain from about 2 to 15 units of the same or different regulatory elements according to the present invention. However, theoretically, there is no limit to the number of regulatory elements within such a multimer oligonucleotide sequence. Such multimeric oligonucleotide sequences are useful as probes for detecting, isolating and/or purifying transcriptional regulatory proteins. Further, when used in a DNA construct, including a promoter and heterologous gene, according to the present invention, a multimer of the 2 0 regulatory elements can enhance the expression of the gene from the DNA construct in response to various cytokines or other signaling molecules.

A variety of signaling molecules activate transcriptional regulatory proteins that bind directly or indirectly to the regulatory elements/oligonucleotide sequences of the present invention. Nonlimiting examples of such signaling molecules include polypeptides such as cytokines and antibodies, and cell-surface antigens, oligosaccarides typically found at or near the surface of cell, non-peptidyl molecules such as TUBag4 Constant et al., 264 Science, 267 (1994)) and synthetic mimics any of these molecules, in both their free and bound forms. Thus, the present invention includes cell to cell or cell to substrate transcriptional regulatory protein activation via 3 0 signaling molecules bound to or near the surface of a cell or other substrate.

i ra~--rru WO 95/28492 PCT/US95/04511 16 Preferably, the signaling molecules according to the present invention comprise cytokines that activate transcriptional regulatory proteins that bind to the regulatory elements/oligonucleotide sequences of the present invention. Examples of such cytokines include, but are not limited to, Interleukins 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13 and 15 (IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, LL-12, IL-13 and Lgranulocyte-macrophage colony stimulating factor (GM-CSF), granuloctyte colony stimulating factor (G-CSF), colony stimulating factor 1 (CSF-1), interferons alpha, beta, and gamma (IFNa, IFN3, IFNy), epidermal growth factor (EGF), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF), Oncostatin-M, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), erythropoietin (Epo), thrombopoietin (Tpo), growth hormone and prolactin.

Particularly preferred cytokines according to the present invention include, but are not limited to, IFNy, IL-4, IL-6, GM-CSF, Oncostatin-M, G-CSF, LIF,, EGF, PDGF, Epo, Tpo and CNTF.

The regulatory elements and/or oligonucleotide sequences of the present invention will also prove useful in detecting, isolating and purifying new transcriptional regulatory proteins that display binding specificity to the regulatory elements/ oligonucleotide sequences of the present invention. Further, it is contemplated that these regulatory elements/oligonucleotide sequences will prove particularly useful in the discovery of novel STAT proteins or STAT-related transcriptional regulatory proteins. In this regard, detection of such novel transcriptional regulatory proteins can be accomplished with the following technique. Cells, such as HepG2 cells, are treated with an appropriate cytokine, for example, with IFNy for 15 minutes to induce the activation of one or more transcriptional regulatory proteins. Extracts of the nucleus and cytoplasm of these cells are then prepared using conventional methods and tested for binding to the regulatory elements/oligonucleotide sequences by an electrophoretic mobility shift assay, in comparison with untreated cells that will show little or no specific binding as described in Levy, 3 Genes Dev., 1362 and Kessler, 4 Genes Dev., 1753, the disclosures of which are herein incorporated by reference. Furthermore, DNA 3 0 regulatory element binding activity may also be stimulated in vitro by treating a

-I

WO 95/28492 PCTIUS95/04511 17 cytoplasmic extract, supplemented with cell membranes, with a signaling molecule, such as cytokine.

If an antibody specific for a transcriptional regulatory protein is available, it can be used to specifically interfere with the binding of the regulatory element of the present invention to the activated transcriptional regulatory protein, thereby assisting in the identification of the transcriptional regulatory protein.

Furthermore, an unknown transcriptional regulatory protein identified or purified using the regulatory elements/oligonucleotide sequences of the present invention can be used Sto immunize animals to prepare an antibody specific for the transcriptional regulatory protein using methods well known in the art. See, E. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the disclosure of which is herein incorporated by reference.

Thus, the regulatory elements/nucleotide sequences of the present invention thus can serve as a "probe", similar to those used in a variety of nucleic acid detection systems well known in the art, except that the probes of the present invention are used to detect proteins, rather than a nucleic acid sequences, which specifically bind to the regulatory elements/oligonucleotide sequences of the present invention.

The sensitivity of such a nucleic acid detection assay can be increased by altering the manner in which a signal is detected by an observer. For example, assay sensitivity can be increased through the use of labeled oligonucleotide sequences using a wide variety of detectable labels, including, without limitation, enzyme labels, radioisotopic labels, fluorescent labels, and modified bases. See, U.S. Patent Nos.

4,581,333, 4,358,535,4,446,237, 4,582,789, and 4,563,417, as well as European Patent Application Nos. EP 144914 and EP 119448, the disclosures of which are herein incorporated by reference. Thus, DNA probes according to the present invention preferably include the regulatory elements alone, or as part of a longer oligonucleotide sequence of the present invention, labeled with a detectable label, such as a radioisotope, an enzyme, a fluorescent label, a chemical label, or a modified base.

Thus, the present invention provides a method for detecting the presence of novel transcriptional ,pulatory proteins in a sample. Such samples are preferably biological samples, inc: a wqg, but not limited to, cells, cell culture supematant, cell or L -e m WO 95/28492 PCT/US95/04511 18 tissue extracts, or particular fractions thereof, and other biological fluids such as blood, sera, urine, saliva, etc. Binding of the probe containing the regulatory elements/ oligonucleotide sequences of the present invention to a transcriptional regulatory protein in the sample may be detected by any appropriate means known in the art. For example, direct or indirect, or competitive binding assays may be used. In such assays, association of the labeled probe with the proteinaceous material of the sample is then detected. In a preferred embodiment, the oligonucleotide sequence is modified by the incorporation of a radioactivity labeled nucleotide.

Once detected, the novel transcriptional regulatory protein can be separated and purified from the probe-protein complex by any of a variety of techniques well known to those of skill in the art. For example, such isolation and purification can be based on affinity chromatography, which relies on the interaction of the protein to be purified with an immobilized ligand. In the present invention, a regulatory element and/or oligonucleotide sequence of the present invention immobilized on a support would serve as the immobilized ligand, which in turn would be used to isolate and purify a novel transcriptional regulatory protein from a sample.

In a preferred embodiment, the regulatory element/oligonucleotide sequence of the present invention is immobilized on a solid support or carrier. As used herein "solid phase carrier or support" refers to any support capable of binding the oligonucleotide sequences/DNA regulatory elements of the present invention. Well known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. Methods for coupling nucleic acids to the solid phase, the solid phase substances useful in these methods, and the means for elution of the proteins from the bound ligand, are well known to those of skill in the art.

In addition to the specific methods described above, purification steps prior to affinity separation may also include one or more additional methods, such as ammonium sulfate precipitation, size exclusion chromatography (gel filtration), ion exchange chromatography, differential precipitation and the like, all well known in the art. Also useful is the method known as hydrophobic interaction chromatography (HIC) which is based on the interaction between the solute and the gel that is hydrophobic.

WO 95/28492 PCT/US95/04511 19 Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene glycol. General principles of HIC are described in U.S. Patents 3,917,527 and 4,000,098. Purification of specific proteins using HIC is described in the U.S. Patent Nos.: 4,332,717; 4,771,128; 4,743,680; 4,894,439; 4,908,434; and 4,920,196, the disclosures of which are herein incorporated by reference.

The regulatory elements/oligonucleotide sequences of the present invention may be included in a recombinant DNA construct which contains a regulatory element/oligonucleotide sequence operably linked to a promoter and a heterologous gene. Typically the heterologous gene comprises a reporter sequence, such as the gene for luciferase. In this regard, a recombinant DNA construct, such as a reporter plasmid according to the present invention, can be constructed using conventicir molecular biology, microbiology, and recombinant DNA techniques well knowo to we of skill in the art. Such techniques are explained fully in the literature, including Maniatis, Fritsch Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II Glover ed. 1985); "Oligonucleotide Synthesis" Gait ed. 1984); "Nucleic Acid Hybridization" [B.D.

Hames S.J. Higgins, eds. (1984)]; "Animal Cell Culture" Freshney, ed. (1986)]; "Immobilized Cells and Enzymes" [IRL Press, (1986)] and B. Perbal, "A Practical Guide to Molecular Cloning" (1984), the disclosures of which are herein incorporated by reference.

Promoter sequences useful in DNA constructs according to the present invention include all prokaryotic, eukaryotic or viral promoters capable of driving transcription of a heterologous gene of interest in combination with a regulatory element of the present invention, when transfected into an appropriate host cell.

Suitable prokaryotic promoters include, but are not limited to, promoters recognized by the T4, T3, Sp6, and T7 polymerases, the PR and PL promoters of bacteriophage X, the transcriptional regulatory protein, recA, he t shock, and lacZ promoters of E. coli, the a -amylase and the a-28-specific promoters of B. subtilis, the promoters of the c s WO 95/28492 PCT/US95/04511 bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage X, the bla promoter of the P-lactamase gene of pBR322 and the CAT promoter of the chloramphenicol acetyl transferase gene ofpPR325. See, B.R. Glick, 1 J. Ind.

Microbiol., 277-282 (1987); Y. Cenatiempo, 68 Biochimie, 505-516 (1986); J.D.

Watson et al., In: Molecular Biology of the Gene, Fourth Edition, Benjamin Cummins, Menlo Park, CA (1987) and S. Gottesman, 18 Ann. Rev. Genet., 415-442 (1984), the disclosures of which are herein incorporated by reference. Preferred eukaryotic promoters include the yeast cyc- promoter, the promoter of the mouse metallothionein I gene, the thymidine kinase promoter of the Herpes simplex virus, the SV40 early promoter, and the yeast gal-4 gene promoter. See Guarante et al., 78 Proc. Natl. Acad.

Sci. USA, 2199-2203 (1981), D. Hamer et al., 1 J. Mol. Appl. Gen., 273-288 (1982), S.

McKnight, 31 Cell, 355-365 (1982), C. Benoist et al., 290 Nature (London), 304-310 (1981), S.A. Johnston et al., 79 Proc Natl. Acad. Sci. (USA), 6971-6975 (1982) and P.A. Silver et al., 81 Proc. Natl. Acad. Sci. (USA), 5951-5955 (1984), the disclosures of which are herein incorporated by reference herein. Preferably, a DNA construct according to the present invention utilizes the thymidine kinase gene promoter of the Herpes simplex virus.

The third component of the recombinant DNA or construct molecules of the present invention is a "heterologous gene" which may be composed of any set of nucleotides regardless of sequence. Nonlimiting examples of such heterologous genes include the structural genes for luciferase, P-galactosidase, chloramphenicol acetyl transferase, secreted placental alkaline phosphatase, human growth hormone, tPA and interferon. For a more extensive list of heterologous genes usable in the constructs and methods of the present invention, see Beaudet, 37 Am. J. Hum. Gen., 386-406 (1985).

Preferably the heterologous gene comprises a reporter gene whose product is used to assess regulation of transcription via a promoter and a regulatory element/oligonucleotide sequence of the present invention. The expression of this "reporter sequence" results in the formation of a reporter product protein) which is readily detectable. The reporter sequence will preferably be selected such that the reporter molecule will have a physical and chemical characteristics which will permit or facilitate its identification or detection by means well known in the art. In one i s~ll 11 3~

I

WO 95/28492 PCT/US95/04511 21 embodiment, the presence of the reporter molecule will be detected through the use of an antibody or an antibody fragment, capable of specific binding to the reporter molecule. In another embodiment, a reporter such as P-galactosidase or luciferase can be assayed enzymatically or immunologically.

A preferred reporter molecule is LUC, well known in the art. See, e., J. R. De-Wet et al., 7 Mol. Cell Bio., 725 (1987). Because this is an insect gene, it is absent from mammalian cells and the enzyme product can be directly assayed in a cell extract. The level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression. In addition, LUC mRNA may also be measured directly.

Typically, a plasmid containing the recombinant DNA molecule of the present invention, including the LUC gene, is introduced into mammalian cells, which are then grown to, at or near confluency. In this regard, any host cell capable of activating one or more transcriptional regulatory proteins in response to an appropriate signaling molecule or molecules can be transfected with the DNA constructs of the present invention. Preferably, such host cells comprise mammalian cells, such as HepG2 and U937 cells (available from ATCC, Rockville, Maryland).

The reporter cells are treated with a compound or sample suspected of containing a signaling molecule capable of inducing or activating a transcriptional regulatory protein, for example, a medium that has been in contact with cells suspected of producing a signaling molecule, such as a cytokine. The LUC-producing reporter cells are extracted, and the soluble extracts are supplemented with luciferin and ATP.

In the presence of these compounds the action of luciferase generates light, which is detected using a luminometer. The amount of light produced is directly related to the amount of luciferase present in the cellular extract.

With a suitable DNA construct transfected into a host cell, the present invention provides a method for the controlled expression of a gene of interest. Thus, application of a signaling molecule, such as cytokine, to transfected host cells can be used to drive the expression of a heterologous gene to yield defined quantities of a desired product, such as human growth hormone, by any of a variety of cell culture and fermentation techniques well known to those of skill in the art.

WO 95/28492 PCT/US95/04511 22 Alternatively, when the DNA construct conprises a reporter sequence, such as the gene for luciferase, transfection of the DNA construct into a host cell provides a convenient means for measuring the transcriptional activity of a reporter product in response to a signaling molecule, such as a cytokine or a medium that has been in contact with cells suspected of producing a signaling molecule, such as a cytokine.

Importantly, when transcription of LUC is activated by the transcriptional regulatory protein being assayed, LUC synthesis is increased relative to a control lacking the transcriptional regulatory protein. Thus, the amount of LUC enzyme produced is an indirect measure of transcription induced by the activated transcriptional regulatory protein binding to the regulatory elements/oligonucleotide sequences of the present invention, which is operably linked to the LUC gene.

When a preferred host cell, such as a HepG2 cell, is transfected with a reporter DNA construct according to the present invention, it can be utilized in assays to detect agonists and antagonists of signaling molecules that induce gene transcription via activated transcriptional regulatory proteins. As used herein, agonists or antagonists of gene transcription include compounds that intervene at any point within the signaling pathway from interaction between the signaling molecule and a cell surface receptor through activation of one or more transcriptional regulatory proteins and binding of the same to DNA regulatory elements, the end result of which is modulation of gene transcription. Further, as used herein, agonists and antagonists of gene transcription also include potentiators of known compounds with such agonist or antagonist properties. Agonists can be detected by contacting the transfected host cell with a compound or mix of compounds and, after a fixed period of time, determining the level of gene expression the level of luciferase produced) within the treated cells. This expression level can then be compared to the expression level of the reporter gene in the absence of the compound(s). The difference between the levels of gene expression, if any, indicated whether the compound(s) of interest agonize the activation of intracellular transcriptional regulatory proteins in an analogous fashion to a known agonist signaling molecule, such as a cytokine. Further, the magnitude of the level of reporter product expressed between the treated and untreated cells provides a relative II-- WO 95/28492 PCT/US95/04511 23 indication of the strength of that compound(s) as an agonist of gene transcription via a transcriptional regulatory protein pathway.

Alternatively, such a transfected host cell can be used to find antagonists of known agonists, cytokines such as JFNy, of gene transcription utilizing host cells transfected with the DNA constructs according to the present invention. In such an assay, the compound or compounds of interest are contacted with the host cell in conjunction with one or more known agonists cytokines) held at a fixed concentration. The extent to which the compound(s) depress the level of gene expression in the host cell below that available from the host cell in the absence of compounds, but presence of the known agonist, provides an indication and relative strength of the antagonist properties of such compound(s).

Thus, the present invention provides methods to assay for agonists and antagonists of gene transcription utilizing the regulatory elements/oligonucleotides of the present invention in appropriate DNA constructs and transfected host cells. In addition, the present invention provides a unique selective assay for agonists and antagonists of STAT heterodimer pathways, including the STAT1a/STAT3 heterodimer pathway. Further, the agonist and antagonist compounds discovered utilizing these methods can serve as pharmaceutical agents in the intervention of various cytokine-induced disease states and conditions, or to ameliorate disease states caused by cytokine deficiency, such as inflammation, infection, anemia, cytopenia and cancerous or precancerous conditions.

The invention will be further illustrated by reference to the following non-limiting Examples.

I c- WO 95/28492 PCT/US95/04511 24 EXAMPLE 1 In order to determine the relative affinities of the regulatory elements of the present invention for different cytokine-activated DNA-binding proteins, the following experiment was performed. Nuclear extracts from cells treated with either interferon-y (IFNy), IL-6, IL-4 or GM-CSF were incubated with various concentrations of unlabeled competitor DNA potential regulatory elements/ oligonucleotide sequences of the present invention: the "test" sequence) prior to the addition of radiolabeled DNA consisting of the Ly6E GAS element. Cytokine-induced GASbinding complexes were then resolved on non-denaturing polyacylamide gels, which were then subject to autoradiography. The ability of a "test" sequence to compete with the labeled Ly6E GAS element for the binding of a particular cytokine-induced complex was assessed by the reduction in the intensity of the band in the autoradiographs representing the cytokine-induced complex. See eg, FIG. 2. Details of these experiments are given below.

HepG2 cells (obtained from the American Type Culture Collection, Rockville MD) were grown in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) containing 2 mM glutamine, 10% fetal calf serum and 100 U/ml penicillin and streptomycin on 90 mm tissue culture dishes. Cells were treated with 9 ng/ml human interferon-y Darnell, Rockefeller University New York NY: commercially available from Genzyme, Cambridge, MA) or 20 ng/ml human IL-6 (R&D Systems, Minneapolis, MN) for 15 minutes when the cells had reached confluency. U937 cells Damell, Rockefeller University New York NY: commercially available from ATCC) were grown in RPMI medium (BioWhittaker) containing 2 mM glutamine, 10% fetal calf serum and 100 U/ml penicillin and streptomycin in 75 cm 2 tissue culture flasks. Cells were treated with 20 ng/ml human IL-4 or 10 ng/ml human GM-CSF (both from R&D Systems, Minneapolis, MN) for minutes when the cells were at a density of 2-5x105 cells/ml.

Nuclear extracts were prepared by NP40 lysis as described in H.B.

Sadowski and MZ. Gilman, 362 Nature, 79 (1993), the disclosure of which is herein incorporated by reference. Protein concentrations were measured using the Bradford dye binding assay (BioRad Laboratories, Hercules, CA). The GAS oligonucleotide 1 WO 95/28492 WO 9528492PCT/US95/04511 based on an lIFN7 responsive element in the Ly6E gene promoter Khan et al., Proc. Natl. Acad. Sci., 6806, (1993)), was formed by annealing oligonucleotides with the sequences: 5' 3,

GATCATATI'CCTGTAAGTG

TATAAGGACATTCACCTAG

SEQ ID NO. SEQ ID NO. 76 Further, the double-stranded probe oligonucleotides, incorporating the regulatory elements of the present invention, used in the Electrophoretic Mobility Shift Assays (EMSAs) were formed by annealing oligonucleotides with the sequences: 3' 5' 3' 3' T'1TECCTGTAAGTG

AAAAGGACATTCAC

TGTTCCTGTAAGTG

ACAAGGACArI'CAC

TCTI'CCTGTAA.GTG

AGAAGGACATTCAC

TAATCCTGTAAGTG

ATTAGGACAflCAC 3' 31 5' 3' 3' 3' 3'

GATCATAGTCCTGTAAGTG

TATCAGGACArTCACCTAG

GATCATACTCCTGTAAGTG

TATGAGGACATTCACCTAG

TATACCTGTAAGTG

ATATGGACATI'CAC

TATGCCTGTAAGTG

ATACGGACA ITCAC

TATCCCTGTAAGTG

ATAGGGACATTCAC

GATCATATTACTGTAAGTG

TATAATGACATI'CACCTAG

SEQ ID NO. SEQIB) NO. 16 SEQ ID NO. 17 SEQ ID NO. 18 SEQ ID NO. 19 SEQ ID NO. SEQ ID NO. 21 SEQ ID NO. 22 SEQ ID NO. 23 SEQ ID NO, 24 SEQ ID NO. SEQ ID NO. 26 SEQ ID NO. 27 SEQ ID NO. 28 SEQ ID NO. 29 SEQ ID NO. SEQ ID NO. 31 SEQ ID NO. 32 SEQ ID NO. 33 SEQ ID) NO. 34 SEQ ID NO. 51 TATW," AGTG WO 95/28492 WO 9528492PCT/US95/0451 I' 31 ATAAAGACAflCAC

TATTGCTGTAAGTG

ATAACGACATTCAC

TATTCATGTAAGTG

ATAAGTACAITCAC

TATTCTTGTAAGTG

ATAAGAACA TCAC

TATTCGTGTAAGTG

ATAAGCACATTCAC

3' 51 5' 3' 3' 3' 2 5 3' 3Y 5' 31 3' 51 3' 51 3' 31 5' 3'

TAITCCAGTAAGTG

ATAAGGTCATTCAC

TAITCCGGT-AAGTG

ATAAGGCCA TCAC GATCATATrCCCGTAAgTG

TATAAGGGCATTCACCTAG

GATCATATTCCTATAAGTG

TATAAGGATh LTCACCrAG

TATTCCTTAAGTG

ATAAGGAAATTCAC

GATCATATI'CCTCTAAGTG

TATAAGGAGATJTCACCrAG

TATI'CCTGAAAGTG

ATAAGGACTTCAC

TATTCCTGIzAAGTG

ATAAGGACCTI'CAC

TA~rCCTGCAAGTG ATAAGGACGTrCAC

TATTCCTGTGAGTG

ATAAGGACACTCAC

SEQ ID NO. 36 SEQ ID NO. 37 SEQ ID NO. 38 SEQ ID NO. 39 SEQ IID NO. SEQED NO. 41 SEQ ID NO. 42 SEQ ID NO. 43 SEQ ID NO. 44 SEQ ID NO. SEQ ID NO. 46 SEQ ID NO. 47 SEQ ID NO. 48 SEQ ID NO. 49 SEQ ID NO. SEQ ID NO. 51 SEQ ID NO. 52 SEQ ID NO. 53 SEQ ID) NO. 54 SEQ ID SEQ IOD NO. 56 SEQ, ID NO. 57 SEQ ID NO. 58 SEQ ID NO. 59 SEQ ID NO. SEQ ID NO. 61 SEQ ID NO. 62 SEQ ID NO. 63 SEQ ID NO. 64 SEQ ID NO. SEQ ID NO. 66 3 5' 3' 5' 31 5' 3' 5' 3' 5' 3' 5'

TATTCCTGTTAGTG

ATAAG'ACAAd'CAC WO 95/28492 PCT/US95/04511 27 TATTCCTGTCAGTG 3' SEQ ID NO. 67 3' ATAAGGACAGTCAC 5' SEQ ID NO. 68 5' TATTCCTGTAGGTG 3' SEQ IDNO. 69 3' ATAAGGACATCCAC 5' SEQ ID NO. TATTCCTGTATGTG 3' SEQ ID NO. 71 3' ATAAGGACATACAC 5' SEQ ID NO. 72 TATTCCTGTACGTG 3' SEQ ID NO. 73 3; ATAAGGACATGCAC 5' SEQ ID NO.74 where the nucleotide sequences shown in bold face type correspond to the nucleotide sequences, including their double stranded complement, tested for activity as regulatory elements according to the present invention.

The annealed oligonucleotides were labeled by filling in the overhanging ends with Klenow fragment (Boehringer Mannheim) in the presence of [a 32 dGTP or dATP (Amersham Corporation, Arlington Heights, IL). Oligonucleotides were S0 purchased from National Biosciences (Plymouth MN) or Integrated DNA Technologies (Coralville, IA). EMSAs were performed in 13 mM HEPES buffer (Sigma Chemical, St. Louis, MO) (pH 80 mM sodium chloride, 3 mM sodium fluoride, 3 mM sodium molybdate, 1 mM DTT, 0.15 mM EDTA, 0.15 mM EGTA and 8% glycerol (including contributions from the nuclear extract) and contained 75 lig/mi poly d(I-C) poly approximately 0.2 ng of radiolabeled Ly6E GAS element, either 0.5, 5.0 or 50.0 ng of competition sequence and 10-15 jig of protein. Reactions containing all components except the radiolabeled Ly6E GAS element were incubated on ice for minutes, followed by a further 20 minute incubation at room temperature following the addition of radiolabeled Ly6E GAS element. Reactions were then resolved on 3 0 polyacrylamide gels containing 0.25X TBE (IX TBE is 89 mM Tris borate, 1 mM EDTA [pH 8.0] and 5% glycerol. Gels were run at 4°C in 0.25X TBE at 20 V/cm, then dried and autoradiographed.

The ability of each "test" oligonucleotide (SEQ ID NOs. 15-74) to compete for the binding of the Ly6E GAS element to the DNA-transcriptional WO 95/28492 PCT/US95/04511 28 regulatory protein binding complexes induced by IFN, IL-6, IL-4 and GM-CSF were visually rated and scored according to the following scale: 0 No competition evident 1 Less than 50% reduction in intensity of the band corresponding to specific complex seen with 50 ng of competitor.

2 Greater than 50% reduction in intensity of the band corresponding to specific complex seen with 50 ng of competitor.

3 Less than 50% reduction in intensity of the band corresponding to specific complex seen with 5 ng of competitor.

4 Greater than 50% reduction in intensity of the band corresponding to specific complex seen with 5 ng of competitor.

Less than 50% reduction in intensity of the band corresponding to specific complex seen with 0.5 ng of competitor.

6 Greater than 50% reduction in intensity of the band corresponding to specific complex seen with 0.5 ng of competitor.

Thus higher numbers indicate a greater ability to compete for particular complexes that comprise or include transcriptional regulatory proteins. Numbers were assigned by visual inspection of the autoradiograms by two independent scientists, and differences in ratings resolved by consensus. If desired, the use of a phosphoimager or densitometer (commercially available from e.g. Bio-Rad Laboratories) could provide a means to assess the differences described here quantitatively. The specific visual ratings of binding affinities for the regulatory elements of oligonucleotide SEQ ID NOs.

15-74 are shown in FIG. 1. In this regard, when interpreting the results shown in FIG.

1, the data should be analyzed for trends as opposed to specific numerical values. This is due to the inherent lack of fine sensitivity in the EMSA assay. This variability can arise, at least in part, from differences in the quality of nuclear extracts used, cell line differences, and variability in the protein concentration determinations.

An example of an autoradiograph of an EMSA used to generate the data in FIG. 1 is shown in FIG. 2. In particular, FIG. 2 shows the ability of oligonucleotide sequence 8T (SEQ ID NO. 53-54) to compete for specific cytokine-activated transcriptional regulatory protein complexes. In evaluating these FIGS., one should be ~d ar4 ~s W O 95/28492 PCT/US95/04511 29 aware that IL-6 induces 3 specific DNA-binding complexes in HepG2 cells. P. Lamb et al., 83 Blood 2063 (1994). Accordingly, for the column marked IL-6, the complex being rated is the slowest-migrating IL-6 induced complex whose position is indicated by the in FIG. 2, and which consists of a homodimer of STAT3 Zhong et al., 264 Science, 95 (1994); Raz, et al., 269 J. Biol. Chem. 24391-24391 (1994)), and thus is not reflective of complexes that contain STAT1cl (p91), which is rated in the IFN complex.

Analysis of the data in FIG. 1 shows that point mutations in various positions within the Ly6E GAS element profoundly effect the ability of the resulting sequences to bind to cytokine-activated transcriptional regulatory proteins. Some of the mutations eliminate or drastically weaken binding to the transcriptional regulatory proteins activated by IFN-7, IL-6, GM-CSF and IL-4. Other mutations result in sequences that bind more tightly to these proteins than to the native Ly6E GAS WT) sequence, for example sequence 9G, (SEQ ID NO. 59-60). Surprisingly, some mutations selectively alter the ability of the resulting sequences to bind to cytokineactivated transcriptional regulatory proteins. These sequences fall into 2 classes; those that bind to the IFN-y activated protein STATla, but not to the proteins activated by IL-6, GM-CSF or IL-4, and those that bind to the IFN- 7 activated proteins STATla and to the IL-6 activated protein STAT3, but not to the proteins activated by GM-CSF and IL-4. An example of the former class of sequences is sequence 8T (SEQ ID NO. 53- 54), and of the latter class is sequence 7C (SEQ ID NO. 49-50).

The remarkable selectivity of sequence 8T is demonstrated in FIG. 2.

This sequence competes for binding to the STATla complex induced by IFN-y (indicated by the in FIG. as evidenced by the reduction in the intensity of this band as increasing amounts of 8T are added to the reaction. However, the STAT3 complex induced by IL-6 (labeled in FIG. 2) and the complexes induced by GM- CSF and IL-4 are unaffected by the presence of this sequence. Note that IL-6 induces a small amount of STATla in HepG2 cells Lamb et al., 83 Blood 2063), and this complex is also reduced in intensity by the addition of sequence 8T.

i I as .L--~j-LP~P~I WO 95/28492 PCT/US95/04511 The selectivity demonstrated by the sequences that bind only to the IFNactivated proteins (STATla) or to both the IFN-y and IL-6 activated proteins (STATla and STAT3) will hold true for other signaling molecules that activate STATI and/or STAT3, such as growth hormone, G-CSF, LIF, Oncostatin M, CNTF, EGF, PDGF, IL- 11 EXAMPLE 2 In order to determine the ability of the Ly6E GAS element and regulatory elements of the present invention to mediate transcriptional induction in response to test cytokines, reporter plasmids were constructed by cloning the regulatory elements into a modified pZLUC plasmid that contains the HSV TK promoter from to +10 as described in Shuai et al., 261 Science, 1744-1746 (1993), the disclosure of which is herein incorporated by reference. HepG2 cells were transfected by calcium phosphate coprecipitation. Cells were seeded at 2X10 5 /ml the day before transfection.

Cells were exposed to a calcium phosphate precipitate containing 15 jg/ml of the above-described reporters and 5 gg/ml of the p-galactosidase expressing plasmid pCH110 for 5 hours. When included, STAT1a (Shuai et al., 1993), STAT3 (Zhong et al., 1994) or empty expression vectors were present at 10 tg/ml. The medium was then changed and the cells allowed to recover for 16 hours. Recombinant cytokines were then added directly to the medium (IFN-y, 5 ng/ml; IL-6, 10 ng/ml; LIP 10 ng/ml and OSM 10 ng/ml) and the cells harvested 5 hours later. Cells were lysed and luciferase and p-galactosidase activities determined using standard techniques. For each sample the normalized response was determined by dividing relative light units obtained from the luciferase assay with the P-galactosidase activity in the same lysate as determined using a chromogenic substrate. Each point represents the average normalized response from three experiments.

Reporter plasmids, constructed as described above, and containing a single copy of the Ly6E GAS element or the 4G (SEQ ID NOs. 29,30), 8T (SEQ ID NOs. 53,54), 6T (SEQ ID NOs. 41,42), 2C (SEQ ID NOs. 19,20), 9G (SEQ ID NOs.

I-~slplrl ~a~l~-pr~ WO 95/28492 PCT/US95/04511 31 59,60) and 7C (SEQ ID NOs. 49,50) regulatory element-containing oligonucleotide sequences of the present invention were introduced into HepG2 cells and tested for their ability to induce luciferase activity in response to either IFN-y, IL-6, LIF or OSM, A control plasmid, TK luc, that lacks a regulatory element, was als, tested. The results are shown in FIG. 3.

In all cases, IFN-y or LIF treatment of transfected cells gave no induction of luciferase activity. Surprisingly, both IL-6 and OSM treatment resulted in induction of luciferase activity over background for several of the reporter constructs. The degree of induction by IL-6 or OSM correlated with the ability of the regulatory elements of the selected oligonucleotides to bind to STAT proteins. The construct containing the 7C oligonucleotide, which binds to STATsla and 3 well, gave the largest inductions, whereas constructs containing regulatory elements that bind STATs less well, such as oligonucleotides 2C and 6T, gave lower inductions. We conclude from these experiments that IL-6 and OSM, but not IFN-y or LIF, can efficiently activate transcription from a single regulatory element of the present invention in these cells.

Thus reporter plasmids containing single regulatory elements according to the present invention exhibit surprising selectivity in their cytokine responsiveness.

We next tested the effect of multimerizing selected regulatory elements.

Reporter constructs containing 4 copies of the regulatory elements 4G, 7C, 8T and 8A, as well as 4 copies of the Ly6E GAS element were assayed for responsiveness to four test cytokines (FIG. All constructs except 4x4G and 4x8T were responsive to IL-6 and OSM, and gave much larger inductions than the constructs with the corresponding single elements. The degree of induction was not always related to the ability of the elements to bind to STATs in vitro, however. Thus the 4x7C reporter gives lower inductions than the wild type 4xLy6E reporter, despite binding STATsla and 3 better in vitro. This is the result of increases in the level of transcription in the absence of cytokine that are larger than the increases in the presence of cytokine, which results in a lowered fold induction. In contrast to the results with single elements, the 4xLy6E, 4x7C and 4x8A constructs also gave sizable inductions in response to IFN-y. We also observed differences between the 4x8A construct and the other constructs with respect WO 95/28492 PCT/US95/04511 32 to the ratio of IFN-y to IL-6 or OSM induction. The 4x8A construct gave reproducibly larger inductions in response to IFN-y than to IL-6 or OSM, in contrast to the other constructs tested. This probably reflects the preferential binding of the 8A mutant to STATIa versus STAT3 containing complexes, and demonstrates the importance of the precise sequence of the regulatory element in determining response to cytokines.

The constructs containing 4 copies of the 4G and 8T regulatory elementcontaining oligonucleotide sequences did not give responses to any cytokine tested.

Both of these oligonucleotides contain regulatory elements that bind preferentially to STATla in the in vitro binding assay, although they do so less avidly than the wild type Ly6E GAS element, or the 7C or 8A regulatory-element containing oligonucleotide sequences, scoring at a level of 3 as opposed to 4 or 5 (FIG. Taken together with the results with the 4x7C, 4x8A and 4xLy6E constructs, these data lead to the prediction that only regulatory elements having an in vitro binding affinity of 4 or more for STAT1xa on our scale, will mediate transcriptional induction in response to cytokines that activate STATI such as IFN-y (FIG. The differential ability of IFN-y, IL-6, OSM and LIF to activate transcription from regulatory element-containing promoters prompted us to characterize the STATs induced by these cytokines in HepG2 cells. Cells were treated with either IFN-y, IL-6, OSM or LIF, and lysates were immunoprecipitated with either anti-STAT1 or anti-STAT3 sera (available from Upstate Biotechnology, Inc., New York). The immunoprecipitates were fractionated on SDS-polyacrylamide gels, blotted and proteins detected using a phosphotyrosine antibody. As controls, aliquots of the same immunoprecipitations were blotted and detected with either anti-STAT1 or anti-STAT3 sera. The results are shown in FIG. 6.

IFN-y induces rapid tyrosine phosphorylation of STAT1a but no detectable phosphorylation of STAT3. In contrast, LIF induces tyrosine phosphorylation primarily of STAT3, with very low amounts of STATIa becoming phosphorylated. IL-6 and OSM induce tyrosine phosphorylation of both STATla and STAT3, OSM inducing slightly more phosphorylation of both STATs than IL-6. These patterns of STAT activation have been confirmed by gel retardation and antibody Lm~ 1"11~--~1 WO 95/28492 PCT/US95/04511 33 supershift experiments (data not shown). Consistent with previous data (Sadowski et al. (1993); Raz et al. (1994) and Zhong et al. (1994)), we interpret these results to indicated that IFN-y activates a homodimer of STAT 1 a, LIP induces primarily a homodimer of STAT3 and EL-6 and OSM induce homodimers of STATla and STAT3 as well as heterodimers of STATIa and STAT3. The differences in the ability of IFNy, IL-6, OSM and LIF to activate transcription from regulatory element containing reporters is therefore accompanied by differences in their ability to activate STATla, STAT 3, or both.

These data document the novel finding that reporter plasmids containing single regulatory elements, such as oligonucleotides 7C, 9G and 2C, are responsive to cytokines that activate two STATs, in this case STATla and STAT3, such that they form heterodimers. The heterodimer is able to activate transcription from single regulatory elements, whereas homodimers of STATla or STAT3 are not. Reporters containing single regulatory elements, including the elements of the present invention, are therefore useful for selectively detecting agonists that mimic the ability of cytokines such as IL-6 to activate both STAT1a and STAT3, allowing heterodimer formation. In addition, the DNA constructs are also useful for detecting antagonists of these cytokines.

The data also document the novel finding that DNA reporter constructs according to the present invention containing multimers of regulatory elements, such as 7C and 8A, are responsive to cytokines that activate STATla homodimers or STAT1a/STAT3 heterodimers, but not STAT3 homodimers. These DNA reporter constructs are therefore useful for selectively detecting agonists that mimic the ability of cytokines, such as IFNy or IL-6, to activate STATla homodimers or STAT1a/STAT3 heterodimers. In addition, the DNA constructs are also useful for detecting antagonists of these cytokines.

While in accordance with the patent statutes, description of the preferred weight fractions, and processing conditions have been provided, the scope of the invention is not to be limited thereto or thereby. Various modifications and alterations maine WO 95/28492 PCT/US95/04511 of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.

Consequently, for an understanding of the scope of the present invention, reference is made to the following claims.

c- WO 95/28492 PCT/US95/04511 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: LIGAND PHARMACEUTICALS INCORPORATED STREET: 9393 TOWNE CENTRE DRIVE CITY: SAN DIEGO STATE: CA COUNTRY: US POSTAL CODE (ZIP): 92121 TELEPHONE: (619) 550-7675 TELEFAX: (619) 535-3906 (ii) TITLE OF INVENTION: DNA REGULATORY ELEMENTS RESPONSIVE TO CYTOKINES AND METHODS FOR THEIR USE (iii) NUMBER OF SEQUENCES: 76 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/228,934 FILING DATE: 14-APR-1994 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TATTCCTGGA AGT- WO 95/28492 PCT/US95/04511 36 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TATTCCGGTA AGT 13 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCTTCCTGTA AGT 13 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" ~C9 IR IPI~- WO 95/28492 PCT/US95/04511 37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TATTCTTGTA AGT 13 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATTCCTGTT AGT 13 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TATTCCCGTA AGT 13 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -aL -~C~rL WO 95/28492 PCT/US95/04511 38 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TATTCCTATA AGT 13 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TATTCCTGTC AGT 13 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TATACCTGTA AGT -4 TIC WO 95/28492 PCT/US95/04511 39 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATGCCTGTA AGT 13 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TATTCCTTTA AGT 13 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" ~a bl WO 95/28492 PCT/US95/04511 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TATTCCTCTA AGT 13 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TATTCCTGCA AGT 13 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION; SEQ ID NO: 14: TATTCCTGTA CGT 13 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic id STRANDEDNESS: single TOPOLOGY linear ~--~IYsa r~c~sl srrrr-~-- B~PRPIPI~PSPDIB3 WO 95/28492 PCT/US95/04511 41 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTTTCCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CAATTACAGG AAAA 14 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TGTTCCTGTA AGTG -I u I WO 95/28492 PCT/US95/04511 42 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CACTTACAGG AACA 14 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCTTCCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs (13) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA"

~A

WO 95/28492 PCT/US95/04511 43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CACTTACAGG AAGA 14 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TAATCCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CACTTACAGG ATTA 14 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 1 WO 95/28492 PCT/US95/04511 44 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GATCATAGTC CTGTAAGTG 19 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GATCCACTTA CAGGACTAT 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCATACTC CTGTAAGTG s~ _Ip- -911C~ep WO 95/28492 PCTUS95/04511 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GATCCACTTA CAGGAGTAT 19 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: TATACCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" Lu 1 LL Y WO 95/28492 PCT/US95/04511 46 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CACTTACAGG TATA 14 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TATGCCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /dese "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CACTTACAGG CATA 14 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 95/28492 PCT/US95/04511 47 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: TATCCCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CACTTACAGG GATA 14 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GATCATATTA CTGTAAGTG la~ WO 95/28492 PCT/US95/04511 48 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: GATCCACTTA CAGTAATAT 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATTTCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" WO 95/28492 PCT/US95/04511 49 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CACTTACAGA AATA 14 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: TATTGCTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: CACTTACAGC AATA 14 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 95/28492 PCT/US95/04511 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: TATTCATGTA AGTG 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CACTTACATG AATA 14 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: TATTCTTGTA AGTG

I_

WO 95/28492 PCT/US95/04511 51 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: CACTTACAAG AATA 14 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: TATTCGTGTA AGTG 14 INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear C -e i! WO 95/28492 PCT/US95/04511 52 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: CACTTACACG AATA 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATTCCAGTA AGTG 14 INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: CACTTACTGG AATA ~s)ly CI 9~r~lCC11 lsl 9- 1~ WO 95/28492 PCT/US95/04511 53 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: TATTCCGGTA AGTG 14 INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: CACTTACCGGAATA 14 INFORMATION FOR SEQ ID NO: 49: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" PIIII~' ~1 l~elk ~ararPB rlllP~BSi WO 95/28492 PCT/US95/04511 54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: GATCATATTC CCGTAAGTG 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDED! SS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCCACTTA CGGGAATAT 19 INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE YPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: GATCATATTC CTATAAGTG 19 INFORMATION FOR SEQ ID NO: 52: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear l-?ar~.~he~lOlsl WO 95/28492 PCT/US95/04511 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: GATCCACTTA TAGGAATAT 19 INFORMATION FOR SEQ ID NO: 53: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: TATTCCTITA AGTG 14 INFORMATION FOR SEQ ID NO: 54: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: CACTTAAAGG AATA M -0 WO 95/28492 PCT/US95/04511 56 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCATATTC CTCTAAGTG 19 INFORMATION FOR SEQ ID NO: 56: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: GATCCACTTA GAGGAATAT 19 INFORMATION FOR SEQ ID NO: 57: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /dese "OTHER NUCLEIC ACID, SYNTHETIC DNA" WO 95/28492 PCT/US95/04511 57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: TATTCCTGAA AGTG 14 INFORMATION FOR SEQ ID NO: 58: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: CACTTTCAGG AATA 14 INFORMATION FOR SEQ ID NO: 59: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: TATTCCTGGA AGTG 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

I

WO 95/28492 PCT/US95/04511 58 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CACTTCCAGG AATA 14 INFORMATION FOR SEQ ID NO: 61: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: TATTCCTGCA AGTG 14 INFORMATION FOR SEQ ID NO: 62: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: CACTTGCAGG AATA

IT~

WO 95/28492 PCT/US95/04511 59 INFORMATION FOR SEQ ID NO: 63: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: TATTCCTGTG AGTG 14 INFORMATION FOR SEQ ID NO: 64: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: CACTCACAGGAATA 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc '"OTHER NUCLEIC ACID, SYNTHETIC DNA" -L i I_-IC WO 95/28492 PCT/US95/04511 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATTCCTGTT AGTG 14 INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: CACTAACAGG AATA 14 INFORMATION FOR SEQ ID NO: 67: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: TATTCCTGTC AGTG 14 INFORMATION FOR SEQ ID NO: 68: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 95/28492 PCTIUS95/04511 61 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: CACTGACAGG AATA 14 INFORMATION FOR SEQ ID NO: 69: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: TATTCCTGTA GGTG 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CACCTACAGG AATA I ~s WO 95/28492 PCT/US95/04511 62 INFORMATION FOR SEQ ID NO: 71: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: TATTCCTGTA TGTG 14 INFORMATION FOR SEQ ID NO: 72: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: CACATACAGG AATA 14 INFORMATION FOR SEQ ID NO: 73: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" -h Re ~Ll~s WO 95/28492 PCTIUS95/04511 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: TATTCCTGTA CGTG 14 INFORMATION FOR SEQ ID NO: 74: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: CACGTACAGG AATA 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATCATATTC CTGTAAGTG 19 INFORMATION FOR SEQ ID NO: 76: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear I_ I~B-d~ d BeC lbI-U WO 95/28492 64 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "OTHER NUCLEIC ACID, SYNTHETIC DNA" PCTUS95/0451 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: GATCCACTTA CAGGiAATAT

Claims (26)

1. An oligonucleotide sequence comprising a regulatory element that binds to an activated transcriptional regulatory protein in the presence of a signaling molecule, said regulatory element having a nucleotide sequence of TATTCCTGGAAGT, TATTCCGGTAAGT, TCTTCCTGTAAGT, TATTCTTGTAAGT, TATTCCTGTTAGT, TATTCCCGTAAGT, TATTCCTATAAGT, TATTCCTGTCAGT, TATACCTGTAAGT, TATGCCTGTAAGT, TATTCCITTAAGT, TATTCCTCTAAGT, TATTCCTGCAAGT, or TATTCCTGTACGT.

2. An oligonucleotide sequence according to claim 1, wherein the signaling molecule is selected from the group consisting of a polypeptide, an oligosaccaride, a non-peptidyl molecule, and synthetic mimics thereof.

3. An oligonucleotide sequence according to claim 2, wherein the polypeptide is selected from the group consisting of a cytokine, a hormone, an antibody, a cell-surface antigen, and synthetic mimics thereof. 2 C 4. An oligonucleotide sequence according to claim 1, wherein the transcriptional regulatory protein comprises a STAT protein. An oligonucleotide sequence according to claim 4, wherein the STAT protein is selected from the group consisting of STATIa protein, STATI3 protein, STAT2 protein, STAT3 protein, STAT4 protein, STATS protein and STAT6.

6. An oligonucleotide sequence according to claim 1, wherein the regulatory element selectively binds to an activated transcriptional regulatory protein comprising STATla protein in response to a cytokine that activates the STATla protein. i Ir WO 95/28492 PCT/US95/04511 66

7. An oligonucleotide sequence according to claim 6 having a nucleotide sequence of TATACCTGTAAGT, TATGCCTGTAAGT, TATTCCTTTAAGT, TATTCCTCTAAGT, TATTCCTGCAAGT, or TATTCCTGTACGT.

8. An oligonucleotide sequence according to claim 1, wherein the regulatory element selectively binds to an activated transcriptional regulatory protein comprising STAT1a protein and/or STAT3 protein in response to a cytokine that activates the STAT1a protein and/or the STAT3 protein.

9. An oligonucleotide sequence according to claim 8 having a nucleotide sequence of TATTCCCGTAAGT, TATTCCTATAAGT, TATTCCTGTCAGT, TATACCTGTAAGT, TATGCCTGTAAGT, TATTCCTTTAAGT, TATTCCTCTAAGT, TATTCCTGCAAGT, or TATTCCTGTACGT. An oligonucleotide sequence according to claim 1, wherein the regulatory element binds to activated transcriptional regulatory proteins comprising STATla protein, STAT3 protein, STATS protein and STAT6 protein in response to a cytokine that activates the STATla protein, STAT3 protein, STAT5 protein and/or STAT6 protein.

11. An oligonucleotide sequence according to claim 10, having a nucleotide sequence of TATTCCTGGAAGT, TATTCCGGTAAGT, TCTTCCTGTAAGT, TATTCCTGTTAGT, or TATTCTTGTAAGT.

12. An oligonucleotide sequence according to claim 3, wherein the cytokine is selected from the group consisting of IFNy, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 GM-CSF, Oncostatin M, growth hormone, G-CSF, Epo, Tpo, EGF, PDGF, CNTF and LIF. L WO 95/28492 PCTIUS95/04511 67

13. An oligonucleotide sequence according to claim 1 which is double stranded.

14. A DNA construct comprising the oligonucleotide sequence of claim 1 operably linked to a promoter, which promoter is operably linked to a heterologous gene, wherein the DNA construct is linked in such a manner that the gene is under the transcriptional control of the oligonucleotide sequence and promoter.

15. A DNA construct according to claim 14, wherein the promoter is selected from the group consisting of the gene promoter of the Herpes simplex virus thymidine kinase, adneovirus Elb and yeast alcohol dehydogenase.

16. A DNA construct according to claim 14, wherein the heterologous gene comprises the structural gene for luciferase, chloramphenicol acetyl transferase, 3- galactosidase, secreted placental alkaline phosphatase, human growth hormone, t-PA, green fluorescent protein or interferon.

17. A DNA construct according to claim 14, wherein the oligonucleotide sequence comprises a multimer of at least one of the regulatory elements of claim 1.

18. A DNA construct according to claim 14, wherein the promoter comprises the gene promoter of the Herpes simplex virus thymidine kinase and the heterologous gene comprises the gene for luciferase.

19. A host cell transfected with the DNA construct of claim 14. A host cell according to claim 19, wherein the cell comprises a HepG2 cell or a U937 cell. I~P~IIY I~I1I~II~ e~ a rrsr(81 WO 95/28492 PCT/US95/04511 68

21. A method for the controlled expression of a heterologous gene of interest comprising culturing the cells of claim 19 in the presence of a signaling molecule and a tanscriptional regulatory protein.

22. A method for controlled expression according to claim 21, wherein the signaling molecule comprises a cytokine and the transcriptional regulatory protein comprises a STAT prftein.

23. A method for detecting the presence of a transcriptional regulatory protein in a sample comprising contacting the sample with an oligonucleotide sequence according to claim 1 under conditions where the transcriptional regulatory protein is activated and binds with the oligonucleotide sequence to form a complex, and detecting the presence of the complex in the sample.

24. A method according to claim 23, further comprising, separating the complex from the sample and the oligonucleotide sequence from the complex to yield the transcriptional regulatory protein. A method for measuring the ability of a compound to act s an' agonist of gene transcription comprising: contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed in response to the compound; and comparing the level of gene expression in step with the level of gene expression from the host cell in the absence of the compound.

26. A method according to claim 25, wherein the heterologous gene comprises the structural gene for luciferase, chloramphenicol acetyl transferase, 3- galactosidase, green fluorescent protein or secreted placental alkaline phosphatase. -I WO 95/28492 PcTIUS95451 69

27. A method for measuring the ability of a compound to act as an antagonist of gene transcription comprising: contacting the compound with a host cell according to claim 19 in the presence of a predetermined amount of a signaling molecule under conditions in which the heterologous gene is expressed in response to the signaling molecule; and comparing the level of gene expression in step with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound.

28. A method according to claim 27, wherein the heterologous gene comprises the structural gene for luciferase, chloramphenicol acetyl transferase, p- galactosidase, green fluorescent protein or secreted placental alkaline phosphatase.

29. An oligonucleotide sequence comprising a regulatory element having a nucleotide sequence of TATTCCTGGAAGT, TATTCCGGTAAGT, TCTTCCTGTAAGT, TATTCTTGTAAGT, TATTCCTGTTAGT, TATTCCCAGAAGT, TATTCCTATAAGT, TATTCCTGTCAGT, TATACCTGTAAGT, TATGCCTAGAAGT, TATTCCTTAAGT, TATTCCTCTAAGT, TATTCCTGCAAGT, TATTCCTGTACGT, or their complements. An oligonucleotide sequence according to claim 29, wherein the oligonucleotide sequence comprises a multimer of at least one of the regulatory elements of claim 29. WO 95/284192 PCT/US95/04511

31. A method for selectively measuring the ability of a compound to agonize or antagonize the induction of STAT heterodimers comprising: contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed in response to the compound, wherein the host cell is transfected with a DNA construct comprising a single copy of an oligonucleotide sequence comprising a regulatory element that that is capable of selectively binding to an activated STAT heterodimer; and comparing the level of gene expression in step with the level of gene expression from the host cell in the absence of the compound.

32. A method according to claim 31, wherein the STAT heterodimer comprises a STAT1a/STAT3 heterodimer. ,Y