CA2191189A1 - Methods and associated reagents for detecting modulators of cytokine action - Google Patents
Methods and associated reagents for detecting modulators of cytokine actionInfo
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- CA2191189A1 CA2191189A1 CA002191189A CA2191189A CA2191189A1 CA 2191189 A1 CA2191189 A1 CA 2191189A1 CA 002191189 A CA002191189 A CA 002191189A CA 2191189 A CA2191189 A CA 2191189A CA 2191189 A1 CA2191189 A1 CA 2191189A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
Abstract
The present invention provides DNA constructs that contain oligonucleotide sequences comprising DNA regulatory elements of the general sequence TTNXAA
that bind activated transcriptional regulatory proteins in response to signaling molecules, such as cytokines, an operably linked promoter and operably linked heterologous gene. The present invention also provides host cells transfected with such DNA constructs, as well as methods for measuring the ability of compounds to act as agonists and antagonists of gene transcription utilizing these DNA constructs and transfected host cells.
that bind activated transcriptional regulatory proteins in response to signaling molecules, such as cytokines, an operably linked promoter and operably linked heterologous gene. The present invention also provides host cells transfected with such DNA constructs, as well as methods for measuring the ability of compounds to act as agonists and antagonists of gene transcription utilizing these DNA constructs and transfected host cells.
Description
DOC~-: NO. 016-0030.WO
19118~
METHODS AND ASSOCIATED
REAGENTS FOR DETECTING
MODULATORS OF CYTOKlNE ACTION
Field of the Invention ~.
This invention relates to methods for detecting modulators of cytokine action, and to DNA constructs and transfected host cells useful in said assays.
Ba~ of thc Inventi~n ~'~ . In many cellular systems, . A l ",, . Ili ,1-. signaling molecules, such as poly~ JLidc Ggands, bind to receptors on the surface of the cells, thereby triggering an ",1, . . ll~ ,1- signaling pathway that ultimately regulates gene Ll~la~ JLio.l within the cells. For example, cytokines and growth factors, which comprise a large and dGverse 15 family of soluble p~ly~ id. ~ that control the growth, diLI'e~ Liull and function of m~mm~l ~ cells, bind to specific cell surface receptors, that in some way transduce signals that elicil a specific phenotypic response. A. ~/Iiyajama 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.Ulirich and J. Srhl~Ccin~ r~ 61 Cell, 203 (1990). Abundant 20 evidence shows that changes in the ~ I rate of specific genes are an important component of this response. This is thought to be a ~ e of alterations in the .. ...
~;- amount or the activity of specific DNA-binding proteins.
In some instances, progress has been made in defirling the pathway that leads from a receptor-Ggand 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 initiates a cascade of aclillw'Llu ~ ull~ pllOa~ ol ~lrL;u~a through MAP kinases leading to pllOa~ lrLiol~ of DNA binding proteins, thereby changing their tr~n~rriptir,r ~1IIIOdUIGLOIY 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., l ~ Mol. Cell. Rir,l 915 (1992).
DOCKE~ NO 016-0030.WO 2 ~ 9 1 1 8 9
19118~
METHODS AND ASSOCIATED
REAGENTS FOR DETECTING
MODULATORS OF CYTOKlNE ACTION
Field of the Invention ~.
This invention relates to methods for detecting modulators of cytokine action, and to DNA constructs and transfected host cells useful in said assays.
Ba~ of thc Inventi~n ~'~ . In many cellular systems, . A l ",, . Ili ,1-. signaling molecules, such as poly~ JLidc Ggands, bind to receptors on the surface of the cells, thereby triggering an ",1, . . ll~ ,1- signaling pathway that ultimately regulates gene Ll~la~ JLio.l within the cells. For example, cytokines and growth factors, which comprise a large and dGverse 15 family of soluble p~ly~ id. ~ that control the growth, diLI'e~ Liull and function of m~mm~l ~ cells, bind to specific cell surface receptors, that in some way transduce signals that elicil a specific phenotypic response. A. ~/Iiyajama 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.Ulirich and J. Srhl~Ccin~ r~ 61 Cell, 203 (1990). Abundant 20 evidence shows that changes in the ~ I rate of specific genes are an important component of this response. This is thought to be a ~ e of alterations in the .. ...
~;- amount or the activity of specific DNA-binding proteins.
In some instances, progress has been made in defirling the pathway that leads from a receptor-Ggand 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 initiates a cascade of aclillw'Llu ~ ull~ pllOa~ ol ~lrL;u~a through MAP kinases leading to pllOa~ lrLiol~ of DNA binding proteins, thereby changing their tr~n~rriptir,r ~1IIIOdUIGLOIY 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., l ~ Mol. Cell. Rir,l 915 (1992).
DOCKE~ NO 016-0030.WO 2 ~ 9 1 1 8 9
2 Despite these advances, the signal ~ pathways utilized by many growth factors and cytokines to alter gene expression remain unclear. Thus,although known second messengers have been implicated m signal l " ~ in response to some of these factors, their role in modulatmg gene expression remains speculative. Miyajama, 10 Annu. Rev. Tmm~lnf~l 295 and D.E. Lev,v 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 ~ 99s (1992) arld Levy, 2 New Biol.. 923.
- Progress in resolving these issues has been made recently m the interferor (IFN) system. L Ns and 13 (type 1) act as a primary non-specific defense against viral infections. S. Petska and J.A Langer, 56 Annu. Rev. Biochem.. 727 (Ig87). IFN~
(type II) has anti-viral properties but also plays a major role in regulation of the immune response. Id. Type I and type lI IFNs bmd to distinct cell surface receptors and cause rapid alterations in gene expression. Aguet, 55 Cell, 273; Uze, 60 Cell, 225; and G.C.
Sen and P. Lengyell 267 J. Biol. Chem.. 5017 (1992). Specific sequence elements have been identified in the promoters of genes that respond to IFNa, termed interferona stimulated response elements (ISREs), that are both necessary and sufficient forregulation by IFNa. Sen, 267 J. Biol. Chem.. 5017. Speci~ically, activation of the IFNa receptors stimulates t,vrosine ~ u~ u~ iu~ of a family of proteins that serve as DNA
- 20 binding proteins, and ac~,ù~ ly as Ll~ Liull }egulatory factors via the ISRE. C.
Schindler 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 ~
(STATs), assemble into a multimeric complex, translocate to the nucleus, and bind cis-25 acting enhancer elements in the appropriate regulatory regions. D.E. Levy et al., 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 30 Proc. Natl. Acad. Sci.. 8555 (1990). ISGF3 is a complex of 4 binding proteins, called DOCKE~NO.0160030.WO 21 9 1 i 8q ::
.. ~ .
p48, p84 (STAT1,~), p91 (STATI) Gnd pl 13 (STAT2). Recently, cDNAs encoding the proteins that constitute ISGF3 have been isolated and ~ , i. X-Y Fu et al., 89 P}oc. Natl. Acad. Sci.. 7840 (1992); C. Schindler et al.. 89 P}oc. NAtl Arqri Sci..
7836 (1992) and S.A Veals et al., 12 Mol. ('~ll Bif l 3315 (1992). p48 is the DNA
5 binding component of ISGF3 and has homolog,v to myb. Veals, 12 Mol. Cell. Ri.ll 3315. p84 and p91 are probably Gl~clllaLi~ spliced products of the sarne gene and are related to pl 13. X-Y Fu, 89 Proc. Natl. ~f~q~ Sci.. 7840 and Schindler, 89 Proc. NatL --Acad. Sci.. 7836. p84, p9~ and pl 13 Gre novel proteins that contain SH2 and SH3.f -. domains and are found in the c-ytoplasm of untreated cells. Schindler, 257 ~ç~, 809 and X.Y. Fu, 70 ~1~ 323-335 (1992). Thus, IFNa treatment of cells results m rapid tyrosine ~lloa~llùlylGliull of p84, p91 and pll3, causing them to associate and form a Il~.,c~ull..,.;c complex with p48 to fomm ISGF3, which then LIG lal~,G~ca to the nucleus and binds to ISREs, stimulating I~ Id.; Dale, 86 Proc. Natl. Acad. Sci..
1203 and Kessler, 4 Genes Dev. 1753.
Regulation in response to IFN^l is conferred by a distinct sequence from the ISRE, the gamma activated sequence (GAS). T. Decker et al., 10 EMBO J.. 927 :~
(1991); K.D. Khan et al., 90 Proc. NA~l Acad. Sci.. 6806 (1993) and D.J. Lew et al., 11 Mol.Cell.Biol.. 182(1991). TreatmentofcellswithlFNyresultsintyrosine pllûa~ u~ylGLicll of p91 (STATla), which then Lll.alo~,GLta to the nucleus and binds to - 20 the GAS. Decker, 10 EMBO J.. 927 and K. Shuai et al., 258 Science. 1808 (1992~.
Thus the specificity of binding of either IFN o} IFN~ to their receptors is translated into a specific ~llva~llulylGLiùll pattem within a related family of latent LlGIIa.,lil)Liu factors (i.e. DNA binding proteins). This pattem of IJllOa,ullulylG~iull dictates the association state of the proteins, which detemlines âpecificity of binding to either an 25 ISRE or a GAS, and tbe subsequent LlG l,...;~,Liul.GI response.
Yet another cytokine, Interleukin^6 (~-6) plays a major role in the induction of the acute phase response in l~ JGlU~.yLt~. The acute phase response is ~,I.G G~.Ltl i~ by the drarnatic ~ l upregulation of a distinct set of genes1 temmed acute phase response genes. P.C. Heinrich et al, 265 Biochem. J.. 621-6363 0 (1990). Studies of the promoter regions of these genes have identified specific DNA
DOCKE~ NO. 016-0030.WO 2 ~ 9 1 1 8 9 sequences that are required for induction of acute phase response genes by IL-6. See __ D.R Kunz et al., 17 Nuc. ~ Res.. 1121-1138 (1989); M. Hattori et al., 87 Proc.
Natl. ~!r~ Sci USA, 2364-2368 (1990); K,A Won and H. Bauman4 10 Mo~, CelL
~j~" 3965-3978 (1990) and D.R Wllson et al., 10 Mol. Cell. E~iol.. 6181-6191(1990).
5 These sequences are termed acute phase response elements (APREs). One type of APRE shows many similarities to the GA~S elements that confers induction by IFNr.
Yuan et al., 14 Mol. Cell. Biol.. 1657-1668 (1994). Proteins that bind to this class of APREs have been ~ ala~,Ltl~,l and purified. U. M. Wegenka et al., 13 Mol. Cell. p~in~
~-- 276-288 (1993); T. Ito et al., 17 Nuc. Ari(~ Res.. 9425-9435 (1989) and Hattori, 87 10 Proc. Natl. Acad. Sci. U,S~ 2364-2368. A cDNA clone encoding the IL-6 - induced APRE-binding protein has been isolated (Zhong, 264 ~iÇI~Ç~ 95 (1994); Akira et al, 77 ~, 63 (1994); Zhong et al. 91 Proc. Natd. ~ Sri 4806 (1994) and Raz et al., 269 J. Biol. (~hf~m 24391(1994)), and was found to encode a protein that shows ~ u~ r .-hlr homology to p91 (STATlc~). For this reason the protein is termed 15 STAT3. Like STATIc~, STAT3 is a latent L- all~ ,Lio-~ factor that is activated to bind DNA by rapid tyrosine pl~v~ v~JI~Livll.
Interleukin~ (IL-4) is a pleiotropic cytokine that elicits biological responses in a variety of both Iymphoid and non-lymphoid cell types. IL-4 is a ~IY~,V~JI uL~,.- of a,ul~ Wdilll~ 1 9 kD produced primarily by the Th2 subset of activated 20 T-ceas. IL-4 has since been shown to play an important role in B-cell ~u. uli{'cl dLiull, the regulation of ;.,.. ~gl. 1,~ . expression, in T-cell regulation and in the growth and dill~l.tlltiaLOl~ of h.,llla~v~vi~,Li-. precursor cells. IL-4 exerts its biological effects thrûugh a specif c high-af~inity receptor on the surface of h.,~llaLO~Ju;~,L~, as well as certain non h . . ~ .~ l up. . - I l~ cell lines. One chain of its receptor, the ~c chain, is sbared by the IL-2, IL-7, IL-9 and IL-13 receptors. M. Kondo et al., 262 Science 1874 (1993), M. Noguchi et al., 262 ~a~ 1877 (1993), S. Russell et al., 262 Sçience 1880 (1993), and M. Kondo et al., 263 ~Çi~ 1453 (19g4).
Binding of IL-4 to its receptor on the cell surface results in the activation of an ;..L- a~ ld- tyrosine kinase and the rapid ~ o~ u. ylaLivll of several proteins on 30 tyrosine. These initial events appear to be directly related to th~ immediate effects of , . , .. . .. . .. . .... .. _ . . . _ _ . . . ..
DOC~E~NO. 016-0030.WO 2 1 9 1 1 8 9 s L-4 on target gene ~ In particular, L-4 up-regulates in responsive cell lines the expression of several ceLI-surface antigens including class II MHC, the low afiinity Fc receptor for IgE (Fc~RlI, CD23), LFA-I and LFA-3, CD40 and surface IgM. B.
Aggarwal and J. U. Gutterman, Human Cy~nkin~ T~nflhogk fnr Basic Chernical = . _ .
S Researcb Blackwell Scientific PubL,~Lulla, Boston, MA (1992). Perhaps the mostprominent role of IL-4 is in B-cell ~ICI c~lLi~lLio4 where L-4 acts as a "switch factor"
promoting an Ig heavy chain class switch to IgE, the majo} mediator of Type I allergic reactions. W.E. Paul, 77 BlQod 1859 (1991). Evidence that L-4 operates through ar ', STAT signal ~ ladu-,Livli system is based upon the Ol/a.,- v~l~ion that L-4 rapidly lû activates in a variety of ceLI lines l~hGal~llu~yl uaillc-containing protein complexes that bind to a GAS-Like DNA sequence element. H. Kotanides and N. Reich 262 Sciençe 126j (1993) and C. Scbindler et al., 13 E~BO J. 1350 (L994~, P. Lamb et al., 83 Blood, 2û63 (1994) and I Kohler and E.P. Rieber, 23 Eur. J Tmmlln~l 3û66 (1993). A
STAT activated by L-4 in THP-I ceUs has béen cloned recently (called STAT-L-4 orSTAT6) and is likely a constituent of all of the reported L-4 induced complexes. J.
Hou et al., 265 ~n~, 17ûl (1994) and J.N. Ihle et al., 11 Trends in Genetics. 69(1995).
Interleukin 13 (IL- 13) is a pleiotropic cytokine that shares many of the biological activities of IL-4. G. Zurawski and J. E. de Vries, 15 Im.~munol. TQ~,V 19 _G 20 (1994). IL-13 has roughly 3û% sequence identit,v with IL4 and exhibits IL4-like activities on ~u~o~,~t.,J~ u~'1&27_~ and B-ceLls (A Minty et al., 362, ~h~ 248 (1993) and A N.J. McKenzie et al., 9û PrQc. Natl. ~ 1 Sci. U.~A 3735 (1993).
However, unlike ~-4, IL-13 has no effect on T-cells. The biological activity of IL-13 is mediated through binding to its specific highaffirlity cell surface receptors consisting of 25 an IL-13 binding subunit and one or more receptor ..~" ,l ~,. ,...l ~ that are shared with the L-4 receptor (the ~L4R' subunit and/or the yc subunit). G. Aversa et al., 178 J. Exp.
Med. 2213 (1993). Evidence that L-13, like L4, operates through a STAT signal system is based upon the observation that L-13 rapidly activates in a variety of cell lines phoa~.l,uLy-ua;"~-containing protein complexes very similar to those . . .
DOCKETNO. 016-00~0.WO - -2191 ~9 induced by IL-4 that bind to a GAS-like DNA sequence element . I. Kohler et al., 345 FEBS Lett. 187 (1994).
GM-CSF belongs to a group of growth factors termed colony stimulating factors which are involved m the survival, clonal expansion, and di~tlcllLialiul~ of 5 i~ Jr\~ proger~itor ceUs. J. Gasson, 77 ~Q~i 1131 (1991) and N.A, Nicola, 58 Annu Rev. Biûchem. 45 (1989). GM-CSF acts on a set of partiaUy committed progenitor ceUs and causes them to divide and dilI'c~cuLia~e in the gr mulocyte-. a~..ul,~ a~ pathways. GM-CSF can also activate mature granulocytes and llà~,l u~ . In addition to effects on ~ h,llloll~cytic lineages, GM-CSF can promote 10 the ~-ulircl aLiOIl of erythroid amd Ill~aLalyucy~(: progenitor cells. Gl~-CSF, an 18-22 kD ~;;y~.UIJlU~,;.l, is produced by a variety of cells, includmg T-ceUs, B-cells, macrophages, mast cells, endothelial cells and fibroblasts, in response to immune or ;.,nA..,.,. l..,y stimuii.
GM-CSF eYerts its effects by interacting with ceU surface receptors on specific target cells. The receptor is composed oftwo chains, GM-CSF-cL and GM-CSF-~. L.S. Park et al., 89 Proc. ~Atl Acad. Sci. 4~95 (199~). The G~-CSF~ is specific to GM-CSF, while the GM-CSF-,~ is identical to the f~ subunit of the IL-5 and IL-3 receptors. G. Goodall et al., 8 Growth Factor~ 87 (1993). Although neither GM-CSFo~ or GM-CSF,~ have intrinsic kinase activity, GM-CSF treatment of ceils results in ~: 20 rapid tyrosme pllo~l~olylaLiull of multiple proteins Evidence that GM-CSF operates t_rough a STAT signal Llaul~du~,Lioll system is based upon the obs~l va~iun that GM-CSF
rapidly activates in a variety of cell lines pllo~l,lloLy- o~ -containing protein complexes that bind to a GAS-like DNA sequence element A C Larner et al., 261 Scien.ce 1730 (1993) and P. Lamb et al, 83 Blood 2063 (1994) It has been reported that GM-CSF
activates STAT5, which is likely a constituent of aU of the reported Gl~-CSF activated complexes. ~le et al, 11 Trends in Gl~nptir~ 69 (1995).
Interleukin-3 (IL-3) is a pleiotropic cytokine produced primarily by activated T-cells Its effects include stimulating the proliferation amd L~clc~lLiaLioll of both pluripotent l~ r~r - I ;C precursor cells as well a wide variety of lineagecommitted cells Ihle, J.N. in Pectide (~rowth Factors ~n~ th~ir Rece~tors Springer-... ..... .... .. .. .. . . ., .. _ ., .. _ .. ... . . . .
DOCKET.NO. 016-0030.WO 2 1 9 1 1 8 9 Verlag, New York (1991). The mature protein has an apparent molecular weight of 28,000, and binds to a cell surface rçceptor (IL-3R) that consists of at least two poly~ idc chains, IL-3Rcc and L-3R~. The IL-3R~ chain is also a component of theIL-5 and Gl~-CSF receptors, whereas the IL-3c~ chain is unique to the IL-3R
5 Miyajama et al 82 Blood 1960, (1993). Binding of IL-3 to its receptor eauses the aetivation of the tyrosine kinase JAK2 and the rapid tyrosine pl~ ylaLiol~ of a set of ~,y~ llc proteins. O. Silvemnoinen et al., 90 Proc. ~atl. Aead Sci. 8429 (1993). A
GAS-binding eomplex that eontains a member of the STAT family ean be deteeted irl f: . extraets from eells trçated with IL-3. AC. Larner et al., 261 Sciencç 1730 (1993); J.N.
Ihle et al., 19 Trends Eliorhp~n Sci. 222 (1994). It has been reported that IL 3 activates STAT5, which is thus likely a constituent of the reported IL-3-activated complexes.
J.N. ~le et al., 11 Trends jn Genetics. 69 (1995) EryLll~ uk ~ul (Epo) is the major hormone responsible for the proliferation and maturation of red blood cell precursors. S.B. Krantz, 77 Blood 419 (1991). In vitro evidence indicates that it also plays a role in thrombocytopoiesis. An et al., 22 Exp. ~m~t 149 (1994). The protein, which has an apparent molecular wei~ht of 30,000, is produced mainly in the kidneys and is induced by conditions of tissue hypoxia.
It acts by binding to a cell sur~ace receptor (EpoR) that consists of a single poly~,t";de chain that is a member of the l , -l~J~ receptor family. ~ D'Andrça et al 57 ~; ~ 20 277 (1989). An early event following the binding of Epo to EpoR is the activation of the tyrosine kinase JAK2, which assoeiates non-covalerltly with the ~,y~ Jla;,llu~. domain of the receptor chain. B. Witthuhn et al, 74 Cell 227. Aetivation of JAK2 by Epo is correlated with induction of tyrosine ~ o~ o.~laii~..l of the EpoR and ~.y~o~Jla~llu~, proteins. Epo treatment of cells also results in the rapid induetion of a GAS-binding 25 activity that contains STAT proteins that are thought to contribute to Epo-induced changes in gene expression. P. Lamb et al., 83 Blood 2063 (1994); Fnbloom et al., 14 Mol. Cell Biol. 2113 (1994). It has been reported that Epo activates STAT5, which is thus likely a constituent of the reported Epo-activated complexes. J.N. Ihle et al., 11 Trends in Gençtiçs. 69 (1995).
DOCKE~Nb.016-0030.WO 21 91 1 8~
' 8 G-CSF is a pleiotropic cytokine best known for its specific effects on the ~ulircl~ioll,dirrcl~ .iio4andactivationofl- ~ yv :;~cellsoftherl~ lul,lul;~, ~ ulfJ~.y~c lineage. G-CSF has also been reported to have ~ h 1~ ;f activity for human ~lulo~y~cs and monocytes as well as for l.lc~el.~,l.y.,.al cells includingS fibroblasts, smooth muscle cells and ~u~. ubl~L~. These m vitro functions reflect the potential in vivo roles for G-CSF in the of steady state 1~
defense against infectio4 ;l l n - - .... ,,~ ;,,., and repair. When G-CSF was a lulul.~c. cd to various ani~nal models, an elevation of circulating neutrophils has been observed. G-- CSF is now used clinically in patients that have ~ lulùp~.. ua as a result of receiving 10 ~,Il.,.lluLIl~. ~y or receiving ' " - - "~ .. caa;Vc agents after organ ~
M AS. Moore, 9 Annu. Rev. Immunol. 159 (1991), N.A Nicola, 58 Annu. Rev.
Biochem. 45 (1989), and E. Pimentel, (1994) in Handbook of Growth Factors. Vol mE. Pimentel, ed., CRC Press, Boca Raton, p. 177.
G-CSF e.~erts its biological activity through binding to G-CSFr. The 15 receptor for G-CSF (G-CSFr) is a member of the type I cytokine receptor ~uu~,.rA~ILly that lacks a kinase domain ar.d appears to consist of a single ~oly~ id~ chain .D;.l.~ aLiu~l of two G-CSFr chains forms a high aftmity binding site for G-CSF.
Among the various 1. ,Alup~;l; receptor ~u~,.rAIllily members, G-CSFr is most closely relatedtogpl30,thesignal-llolla.luc;llgcomponentoftheL-6,oncostatinM,and ~-20 leukemiainhibitoryfactorreceptors. Recentstudieshave.l,,~lAlr,lthatinmyeloid leukemia cell lines, G-CSF treatment results in rapid tyrosine pl.o*,llolyldLoll of G-CSFr, JAK I and JAK2 tyrosine kinases and the members of the STAT family of factors. S.E. Nicholson et al. 91 Proc. Natl. Af~ad. Sci.USA 2985 (1994) and S.S. Tian et. al., 84 BlQQd 1760 (1994).
It has previously been reported that many cytokines, including IL-3, GM-CSF, Epo, G-CSF, L4 and IL-13, activate STAT or STAT-like complexes that bind toDNA sequence elements related to the GAS elements that were first characterized in the promoters of ~N^,~-responsive genes. However, to date there has been no reported.~... ,.)..~, Al ;.~l l that the DNA sequences reported to bind to the STAT or STAT-like 30 complexes activated by IL-3, GM-CSF, Epo, G-CSF, L4 and L-13 can mediate DOCKE~ NO. 016-0030.WO
2191 18~
. .
t induction in response to those cytol~nes. Accordingly, the ;~ . ,l ,l ;, ,,l ,....
of DNA sequence-elements capable of mediating 1~ activation in response to cytokines such as :L 1, GM-CSF, aCSF and Epo, for example, would be usefill tools that would allow the responses mediated by various cytokine-activated DNA-5 binding proteins to be ~U~ y assayed.
The disclosures of the above-cited references are hereby ;~ by reference in their entirety.
~ ..
DOCK~T N0. 016-0030.WO
~ ~ 2!91189 .
Summanf of the Invention The present invention is directed to methods for screening for modulators ~i.e., agonists and antagonists) of cytokine-mediated l l A 11~ , and to the DNAconstructs and cytokine-responsive host cell lines transfected with such DNA constructs 5 used in such screening methods. In a preferred f..,l,~.l"..~ .,l the present invention is directed to methods for screening for cytokine modulators mvolved in the STAT5 protein and/or STAT6 protem siOonaling pathway. In this regard, the DNA constructs of the present invention include F~ m 1- ~F~V~ ' sequences containing regulatory elements -- that selectively bind activated STAT5 and/or STAT6 proteins, and modulate10 1~ of the associated l~ loOuus gene, in response to ~IJl Upl i~ signaling molecules, such as the cytokines IL 3, IL4, L-13, Epo, G-CSF and GM-CSF~
Surprisingly, and contrary to the teaching in the art, only a limited subset of the regulatory elements that bind activated STAT5 and/or STAT6 proteins actually modulate ~ of the associated 'A,t.,. ulo~uus gene m the assays of the present 15 invention.
In particular, the present invention provides a DNA construct comprising (a) an ol;Ou~.uclFJliJ~ sequence comprising a reOulatory element of the nucleotide sequence TTNXAA, operably linked to (b) a promoter, operably linked to (c) a h.,Lc:luloOuu~ gene, wherein N is ," l~ y selected from A. T, C or G and x is 4, 5, r- ~ 20 6 or 7, and whereim the DNA construct is operably linked in such a manner that the ~l~l ulogùus gene is under the l ~ ;F~ ~AI control of the promoter and I F sequence when the r.~ . 1. ul ;, t~ sequence is bound by a STAT
protein activated in response to IL-2, ~-3, IL-4, IL-7, ~-9, IL-13, G-CSF, GM-CSF, Epo or Tpo Also provided is a cytokine-responsive host cell transfected with this DNA
25 construct.
The present invention also provides a DNA construct comprising (a) an .. 1. ul;~ sequence comprising a regulatory element ofthe nucleotide sequence ANTTCNNNNGAF~NA (SEQ ID NO. 3) operably linked to (b) a promoter, operably linked to (c) a Lu,LcluloOuu~ gene, wherein N is ~ ~f ~ y selected from A, T, C or 30 G, and wherein the DNA construct is operably linked in such a manner that the DOCKE~T NO. 016-0030.WO
h~ lOIO~,UU:i gene is under the ~ C. ' 1~ control ofthe promoter and Li.if sequence when the GL8U....LCIf~U~idf sequence is bound by a protein complex comprising a STAT6 protein activated in response to a cytokine Also provided is a cytokine-responsive host cell transfected with this DNA construct Further, the present invention provides methods for measuring the ability of a compound to act as an agonist of gene tlPnc~rtion comprising (a) contacting the compound with the transfected host cells described above under conditions in which the heterologous gene is capable of being pressed in response to the compound, and ~b) - , comparing the level of gene expression in step (a) with the level of gene eYpression from lû the host cells 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 ~ O.l comprising (a) contacting the compound with the transfected host cells described above in the presence of a ~ amount of a cytokine under conditions in which the l~ . ul~uus gene is capable of being expressed in response to the cytokine, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cells in the presence of the cytokine, but the absence of the compound. In both these methods, the l~ ulo~;uu~ gene may be any appropriatereporter gene such as the heterologous gene for luciferase, ~llo"....l.l.. : ..l acetyl transferase, green fiuorescent protein or ~-~a~ ei~i~cf.
These and various other advantages and features of novelty which . the invention are pointed out with p~ Li~ukuily in the claims annexed hereto and forming a part hereof However, for a better I ' ' ,, of the invention, its advantagec, and objects obtained by its use, reference should be had to the fl-,~,UIII~ llJill~, drawings and descriptive matter, in which there is illustrated and 25 described preferred f-l l ll ~O~ ` of the invention.
DOCKEI NO. 016-0030.WO
~ 2191 189 Definitions For the purposes of this invention:
~ ol~"",.. ;F~ " or "DNA" molecule or sequence refers to a molecule comprised of the dcv~y~ I; vl ;~ adenine (A), gL~anine (G), thymine (T) and/or 5 cytosine (C), in either smgle-stranded form or a double-stranded helix, and comprises or mcludes a "regulatory element" according to the present invention, as that term is defned herein. The exact size, ~ and orientation (i.e. 3' to 5', or 5' to 3') will depend upon many factors, which, in turn, depend upon the ultimate function and use of the~lig~ lFvl~ 0fthepresent3nvention. Thus,theterrn''i~l;s..~ lrv~ lor ,, 10 "DNA" includes double-stranded DNA found m linear DNA molecules or fragments,viruses, plasmids, vectors, ..~..u.~.o~v.l.~.~ or ~yllLL~,.i. ''y 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 dcv.~ylilJ- "~"~lrv~ sequence 15 comprising the whole, or a portion of, an ol ~ I/ vl ~ sequence to which an activated ~ AI regulatory protein, or a complex comprising one or more activated ~ Al regulatory proteins, binds so as to ~ Ally modulate the expression of an associated gene or genes, including, heterologous genes.
"Signalmg molecule" refers to an . . ~ ' pGIy~ ide ~
. . 20 or other rlon-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 i~ cell.ll~
pathway which includes the activation of one or more L~ "l regulator~v proteins that bind to a regulatory element, thereby L. A -`- ;~ Y modulating the expression of an associated gene or genes. As used herein, "sigmaling molecule" includes naturally 25 occurring molecules, such as cytokines, peptidyl and non-peptidyl hormones, antibodies, cell-surface antigens, or synthetic mimics of any of these signaling molecules, or synthetic molecules that mi~nic the action of any of these si~,maling molecules."Cytokines" refer to a diverse grouping of soluble poly~,~Lid~, including growth factors and hormones, that control the growth, li~tltlll;~tioll and function of 30 cells in such a manner as to ultimately elicit a phenotypic response in an organism.
, . _ _ _ ..... ..... . ... .........
DOCKE~NO. 016-0030.WO 2 l 91 1 89 Preferred cytokines useful with tbe regulatory elements and associated methods of tbe present invention include IL-3, IL-4, Il:~-13, GM-CSF, G-CSF, Epo and Tpo.
"TIA~ I regulatory protein" refers to ~Luyl~~ , or nuclear proteins that, when activated, bind the regulatory elements/,-l:~,. ..1. ,. 1~ vl ;~1 sequences of S the present invention either directly, or indirectly through a complex Of ~
regulatory proteins or other adapter proteins, to L A~ r ".~ modulate the activity of an associated gene or genes. Thus, 1~ 1 regulatory proteins can bind diréctly 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 10 bound to a DNA regulatory element of the present mvention. See e.A~.. S.A Veals et al., 13 Molec. Cell. Biol.. 196-206 (1993). As used berein, L~ regulatory proteins, mclude, but are not limited to, those proteins referred to in the art as STAT
proteins (Z. Zhong et al., 264 Science. 95) STF proteins (C. Schindler et al., 13 E~,IBO . =
J, 1350 (1994)), Mammary Gland-Specific Nuclear Factor (M. Schmidt-Ney et al., 6Mol. F~ - l."- ~f~l 1988 (1992) and H. Wakao et al., 267 J. Biol. Chem.. 16365 (1992)), APRF (Wegenka, 13 Mol, CelL~io.. 276), GHIF (Mayer, 269 J. Biol. Chem..4701), GHSF and EPOSF (Finbloom, 14 Mol. Ceil Bio,. 2113), as well as to all ~ul~:~L~I~;ul~ ov-.~ analogs and allelic variations thereo "T~ iyliolial!~ modulate the expression of an associated gene or ~-.20 genes" means to change the rate of L- r. L,.,I iy~iOIl of such gene or genes.
"STAT protein" refers to those 1. All~ IA1 regulatory proteins designated as "Signal Transducers amd Activators of TI . ,~ " (STAT) by Dr. J.E.Darnell of Rockefeller University. See Zhong, 264 Sciençç 95. As used herein, STAT
proteins include the p91 (STATI), p84 (STATI), pl 13 (STAT2) proteins and the STAT-associated p48 family of proteins. S.A. Veals et al., 12 Mol. Cf~ll Ri~-l 3315 (1992). Further, STAT proteins also include a binding protein designated as STAT3 (Zhong, 264 Science 95), and a binding protein designated as STAT4 (~.). In addition, MGF is now renamed STAT5 (Gouilleux et al., 13 EMBO J.~ 43614369 (1994)) and STAT-IL4 (or STAT6) has recently been cloned. Hou et al., 26i Science~ 730 (1994) .
DOCKE~NO.016-0030.WO 2~ 91 1 ~9 , ~ .
amd J.N. Ihle et al. I 1 Trends in Genetics. 69 (1995). Also included are ~ub~LdllL;~
r,lr.~ c analogs and allelic variations of all of the 2bove STAT proteins.
"Activate", "activated", "activation" or derivatives thereof, means that one or more ~ ,11 regulatory proteins within a cell are modified post-5 ; ' ".~/, or are cul~LiLui~ active, such that they can bind directly or indirectlyto DNA regulatory elementr/ ~ sequences of the present irlvention m a sequence-specific manner. This ".,,.1.~ ;".. will typically comprises pllu~llolylaLioll of the ~ c~ l regulatory proteins via a variety of ' , including, but not ~=. limitedtoactivatiorlbyvariousprotemlcinases. See,e.~" (Shuai,258~ciencç, 1808an P. Cohen, 17 T~S. 408 ~1992)).
"DNA construct'' refers to any genetic element, including, but not limited to, plasmids, vectors, ~ ulllOSul~ and viruses, that incorporate the ,~
sequences of the present invention. For example7 the DNA construct can be a vector comprising a promoter that is operably linked to an ,,1~ .."rl~ul"l~ sequence of the 15 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 bindmg directly or mdirectly to RNA polymerase in a cell and initiating Ll all~ iOII of a duwll lLl calll (3' direction) coding sequence. For purposes of the present invention, the promoter is ~: .. 20 bounded at its 3' terminus by the L~ r initiation site and extends upstream (5' direction) to mclude the minimum number of bases or elements necessary to initiate rl~l at levels detectable above ba~,h~uull~. Within the promoter will be found a l, i - ,~- ,1 .1 ;. ,.l initiation site (~c,--v ~Ih~ LIy def ned by mapping with S I nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of 25 RNA pc~ ,.aac. Eukaryotic promoters will often, but not always, contain "TATA"
boxes and "CCAT" boxes. P~uho~yu~iu 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 ;. ~ ., . required for the normal regulated production of a particular protein.
30 A "heterologous" region of a DNA construct (i.e. a heterologous gene) is an identifiable DOCKET NO. 016-0030.WO ~ ~ 2 1 9 1 l 8 9 . ~ .
segrnent of DNA within a larger DNA construct that is not found in association wLth the other genetic ~~ of the construct in nature. Thus, when the 1. .~ ulO~;uua geneencodes a r~m~ n 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 construct7 including an ~ f ol ;~1 sequence according to the present invention, is "operably linked" to a h~,.c. ulogu~ls gene when the presence of the promoter inrduences ~ n from the h~ lulc~;uua gene, including genes for reporter sequences such as luciferase, ~ acetyl - transferase, ~ and secreted placental alkaline r~ c. Operably linked I û 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 linhed if ~ "l l; " c ~" "~- ,-: ,~ in the promoter will produce an RNAtranscript of the reporter sequence. In order to be "operably linked" it is not necessary that two sequences be u~uil~,L~lL~ adjacent to one another.
A host cell has been "L- ular~:l,Lt;d ~ by exogenous or ~ olo~;uus DNA
(e g. a DNA construct) when such DNA has been introduced inside the cell. The l, .,"~rr, l",f~ DNA may or may not be integrated (covalently lirlked) into ~,luullluaul~
DNA mahing up the genome of the cell. In ~luhdlyuL~a, yeast, and m~mm~ n cells for example, the l l ~ rr- I, "o DNA may be maintained on an episomal element such as a .~ 20 plasmid. With respect to eukaryotic cells, a stablely transfected cell is one in which the 1 " r, I . ,j~ DNA has become integrated into a ~lu ulllOa~ c 50 that it is inherited by daughter cells through clu ullluaulliC repGcation. This stabiGty is d~ ~IIOIIall d~Cd by the ability of the eukaryotic cell to establish cell Gnes or clones comprised of a population of daughter cells contairling the Ll~lar~,~,Lul~ DNA
"Cytohine-responsive host cell" refers to a cell line that e7spresses, either nor~nally or after I I ,~ rr~ of the requisite cDNAs, the relevant cytokine receptor c~ , JAK proteins, STAT proteins, and accessory factors such that, upon cytohine binding to the cell surface, STAT-mediated gene Ll~u~s~ Liu~ is affected.
DOCN~T NO. 016-0030.WO
Brief DescriPtion of the Drawi The invention may be fulther illustrated by reference to the Z~ U~II~)dll,y;~l~
Drawing wherein:
FIG. I is a lc~ludu~.Liull ûfEl~,hu~l~u-cLi~. Mobility Shift Assay (EMSA) 5 A llu~ gl A -` that shûw the bindi~g patterns of h~ regulatory protein-DNA binding complexes activated by L-4 and IL-13. The EMSA's were performed as described in the Examples herein. The ~ tl, double-stranded U~
probes utilized in the EMSAs of FTGS. IA and lB were made by arnealing the of SEQ ID NOs. 14-23 (~IG. lA) and 24-35 (FIG. lB).
~ .
DOC~ET NO. 016-0030.W(~ ~
~ 1 9 1 ~ 89 Detailed Descrii~tion of of the Invention The present inventors have discovered that o~ly a select group of regulatory elements that bind activated ~ ''~,l regulatory proteins, such as 5 STAT proteins, actually modulate the ~ of an operably Gnked L~.~e~ uloguu~
gene in a cell-based screen. This unexpected result is in direct . .~" ,l, A~l;. .1 ;~ " ' to the teaching in the art that a regulatory element that binds such activated ~
regulatory proteins will also activate the l A ~ of an associated gene. Thus, the ~ . binding of a regulatory element to an activated L~ o~ regulatory protein is not correlated, and provides no l~l~dl~,l~;l;Ly, with respect to those regulatory elements that will activate ~ of an associated gene in response to this binding. It is only through the teaching of the present inventors that one will be able to select, without resorting to undue ~ , regulatory el.,.~ Lal~ vl ,,1 sequences that will both bind to, and cause Ll~laa~.Li\~a~ioll frvm, activated l~au~ io.~dl reg~latory proteins, such as STAT proteins.
The ~ v~ sequences, comprising DNA regulatory elements, and that are i l~vll~o-AL~d into the DNA constructs of the present invention are selected from the nucleotide sequence TTNXAA, wherein N is ;",~ l ly selected from A, T, C or G and x is 4, 5, 6 or 7. More preferably, the regulatory elements comprise ~i 20 ~ ,., --" l, vL;~ sequences that bind and ~l~la~ ~iv.L~t: in response to activated STAT5 and/or STAT6 proteins. These preferred ~ vl ,,~, 5 sequences are selected from the group consisting of TTCNNNGAA (SEQ ~ NO. 1), TTCNNNNGAA (SEQ lD
NO. 2) and ANTTCNNNNGAANA (SEQ. ID NO. 3), including their double stranded ...",,~,1..,....1~, where N is ;,"l~ l,.."l..~ ly selected from Al T, C or G. Especially 25 preferred ~.l;~.. " .. ., li ~,l ;~ sequences according to the present invention include:
ACTTCCCAAGAACA (SEQ ~ NO. 4), ACTTCCCCGGAACA (SEQ ID NO. 5), ACTTCCCCAGAACA (SEQ ID NO. 6), ACTTCCCAGGAACA (SEQ ID ~O. 7), ACTTCCTAAGAACA (SEQ ~ NO. 8), ACTTCTTAAGAACA (SEQ ID NO. 9), TTCCCGGAA (SEQ ID NO. 10), TTCCCCGAA (SEQ ~:) NO. 11), TTCTAAGAA
30 (SEQ ID NO. 12) and TTCTCAGAA (SEQ ~ NO. 13).
DOCKET NO 016-0030.WO
2~ 9 1 ~ 89 The ~ sequences of the DNA constructs of the present inYention can comprise the entire regulatory element alone, or can incl~de additional 'danking nudeotide sequences. In this regardl it is preferable that such ~.l 2 ~ ; vLid~, sequences comprise between 8 and 200 n--rl~oti~ , including the regulatory elements of S the present invention. However, sequences in excess of 200 ,A~AIrot~ that contain such regulatory elements, and that are capable of binding activated l ~
regulatory proteins, and of L~ modulating the expression of one or more genes thereby, are also considered to be within the scope of the present inYention ~-- The ~ sequence component of the DNA constructs of the present invention can also comprise multimers of t~vo or more "units" of the basic regulatory elements. In this regard, such multimer ~ ul ;~ 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, Lll.,o~Li~.dlly, there is no limit to the number of regulatory elements within such a multimer ~1 G~ Ulid~ sequence.
When used in the DNA construct, including a promoter and k.,.~uloguu~ gene, according to the present invention, a multimer of the regulatory elements can enkance the expression of the gene from the DNA construct in response to various c~Ytokines or other signaling molecules.
A variety of signaling molecules activate LIAI ~ AI regulatory proteins that bind directly or indirectly to the DNA constructs of the present mvention, and modulate l,, ~ . . of the operably linked heterologous gene. Nonlimiting examples of such signaling molecules include polrt,~,~,Lid~,~ such as cytokines and antibodies, and cell-surface antigens, ol;~ 7 typically found at or near the surface of cell, non-peptidyl molecules such as TlJBag4 OE Constant et al., 264 ~, 267 (1994)) and synthetic mirnics any of these molecules, in both their free and bound forms. Thus, the present invention includes cell to cell or cell to substrate ~
regulatory protein activation via signaling molecules bound to or near the surface of a cell or other substrate Preferably, the signaling molecules according to the present invention 30 comprise cytokines that activate ~ AI regulatory proteins, such as STAT
DOCKE~`NO. 016-0030.WO
19 2~91189 proteins, that bind to the regulatory el~.. ,.. L~ ;g~ o~ide sequences of the present invention. Exarnples of such cytokines include7 but are not limited to, L-l~- lu~i.~ 2, 3, 4, 5, 6, 7, 9, 10, I l, 12, 13 and 15 (IL-2, IL-3, IL-4, IL-5, IL-6, ~-7, L-9, IL-10, IL-11, IL-12, IL-13 and IL-15), ~llulul~uy~e-l~la~lu~ull~c~ colony stimulating factor (GM-S CSF), ~ululo~.LyLe colony stimulating factor (G-CSF), colony stimulating factor I
(CSF-I), interferons alpha, beta, arld garmna (IFNa, IFN~, IFNy), epidermal ~rowth factor (EGF), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF), Oncostatin M, nerve growth factor (NGF), ciliary ~uL u,ul~ic factor (CNTF), brain-derived rlc.~uL~u~l~ic factor (BDNF), ~yLluu~uk.,iul (Epo), Lluulll~ùlJoieLul (Tpo), growth hormone and prolactin. Particularly preferred cytokines according to the present invention include those that activate the STAT5 protein and/or STAT6 protein pathways, including, but are not lirnited to, ~-2, IL-3, IL4, IL-S, iL-7, IL-9, IL-13, IL-15, G-CSF, GM-CSF, Epo, Tpo and growth hormone.
The le-,ulll~ -lL DNA construct, such as a reporter plasrnid, according to the present invention, can be constructed using conventional molecular biology, lllhu ubioloy~y~ and c~,ulllbi~ lL DNA techniques well hnown to those of skiil in the art.
Such techniques are explained fully in the literature, including Maniatis, Fritsch &
Sambrooh, "Molecular Cloning: A I,aboratory Manual" (1982); "DNA Cloning: A
Practicai Approach," Volumes I and II (D.N. Glover ed. 1985); "O!;L~ r f~ ` 20 Synthesis"(M.J.Gaited. 1984);"NucleicAcidIIyb~ iu~ 3.D Hames&S.J.Higgins, eds (i984)]; "Animai Cell Culture" [RI. Freshney, ed. (1986)]; "T ' `'`Cells and Enymes" [IRL Press, (1986)] and B. Perbal, "A Practical Guide to Molecular Cloning" (1984), the disclosures of which are herein iU~,Vl ~.u. a~d by reference Promoter sequences useful in DNA constructs according to the present invention include all l,l uh~yu~iu, eukaryotic or viral promoters capable of driving 1"..,~ ;nnofah~Leluloguu~geneofinterestin~ witharegulatoryelement of the present invention, when transfected into an dlJ~I u~L i~e host cell. Suitable ~luh~uyu~ic promoters include, but are not limited to, promoters recognized by the T4, T3, Sp6, and T7 polyl.l~ r., the PR and PL promoters of b~l iu~llAg~" the 3û I~ nI~AI regulatory protein, recA, heat shock and lacZ promoters of ~ ~, the -DOCKET NO. 016-0030.WO
2 ~ 9 ~ 1 89 amylase and the -28-specific promoters of B. subtilis. the promoters of the ~ lr~ ofE~aciilus~sLlc~Lvlllyc~i~promoters1theintpromoterof~ r~
the bla promoter of the ~-lactamdse gene of pBR322 and the CAT promoter of the .f UI acetyl transferase gene of pPR325. ~, ç~, B.R Giick I J. Ind. .. = . .Microbiol.. 277-282 (1987); Y. Cenatiempo, 68 Biochimie. 505-516 (1986); J.D.Watson et ai., Il;: Molecular Biolo. Y Qf the Gerle. Fourth Edition, Benjamin Cummins, Meio Park CA (1987) and S. Gottesman, 18 Arln. Rev. GPnf~t 415-442 (1984), the disclosures of which are herein i~ Ul~UldLt~ by refererlce. Preferred eukaryotic, -~; promoters include the yeast cyc-l promoter, the promoter of the mouse "~ ' I
10 gene, the thymidine kinase promoter of the Herpes simplex vifus, the SV40 early promoter, and the yeast gal-4 gene promoter. See Guarante et ai., 78 Proc. Natl. Acad.
Sci. ~I~, 2199-2203 (1981), D. Hamer et ai., I J. Mol. Ap~ n 273-288 (1982), S. McKnight, 31 S~L 355-365 (1982), C. Benoist et ai., 290 ~ ~London), 304-310 (1981), S.A Johnston et ai., 79 Proc Natd. Acad. Sci. (USA), 6971-6915 (1982) and P.A Silver et ai., 81 Proc. Nati. Acad. Sf,i. (USA), 5951-5955 (1984), the disclosures of which are herein il.cuil)o-dLtd by reference herein. Preferably, a DNA constructaccording to the prent invention utilizes the thymidine i~inase gene promoter of the Herpes simplex virus.
The third component of the I c~,fj~-lY~A~ll DNA or construct molecuies of ( ~- 20 the present invention is a "heterologous gene" which may be composed of any set of nucleotides regardless of sequence. Nu~ ~ .IIL...~ examples of such h~.t~,. vl~ùu~ genes include the structurai genes for luciferase, ,~-Vd~ VIA~ acetyl transferase, secreted placental aikaiine l ' - .~ human growth hormone, tPA green fiuorescent protein and interferon. For a more extensive list of ~l~,t~l ul~uus genes 25 usable m the constructs and methods of the present invention, see Beaudet, 37 Hum. Gen.. 386-406 (1985).
Preferably the ~.,lcluloguu~ gene comprises a reporter gene whose product is used to assess reguiation of ~ via a promoter and a regulatory eleme~tlnl..~ l v~ sequence of the present invention. The expression of this 30 "reporter sequence" results in the formation of a reporter product ~e.g., protein) which is DOCKE~ NO. 016-0030.WO
21 2~9ll89 readily detectable. The reporter sequence will preferably be selected such that the reporter molecule wiU have a physical arld ehemical ~ r~ which wiU pemlit or faeilitate its i~ or detection by means weU known in the art. In one f~mhofiimf~nt~ the presence of the reporter molecule will be deteeted through the use of 5 an arltibody or an antibody fragment, capable of specifie binding to the reporter moleeule. In another ~ o~ , a reporter such as ~ t5 ~ or luciferase can be assayed C.~yll.,...;."LUy or immlmf~lf ~
A preferred reporter molecule is LUC, well known in the a}t. See, e.g..
J. R De-Wet et al., 7 Mol. Cell Bio.. 725 (1987). Because this is an insect gene, it is 10 abserlt from mammalian cells and the enzyrne product can be directly assayed in a ceU
extraet. The level of enzyme activity cul~e~ull~s to the amount of enzyme that was made, which in tum reveals the level of expression. In addition, LUC mRNA may also be measured directly.
Typically, a plasmid containing the . ~, ., . "1 " " - . l l DNA molecule of the15 present invention, including the LUC gene, is introduced irlto cytokine-responsive mammalian ceUs, which are then grown to, at or near confiuency. In this regard, arly cvtokine-responsive host cell capahle of activating one or more ~ ,I;fl..,.l regulatory proteins in response to an appropriate signaling moleeule or molecules can be transfeeted with the DNA construets of the present invention Preferably, such . . 20 eytokine-responsive host ceUs comprise m~rnm~ n eells, sueh as HepG2, U937, M~-180, TF-1 and NFS-60 eeUs.
The reporter eeUs are treated with a eompound or sample suspected of eontaining a signaling moleeule eapable of indueing or aetivating a l, ~ . ,~. . ;l.l ;~.fl~l regulatory protein, for example, an extraet of other eytokine-treated cells. The LUC-25 producing reporter ceUs are extraeted, and the soluble extracts are ~u~ cd withluciferin and ATP. In the presence of these efJllllJOIlllll:~ the aetion of luciferase generates light, which is detected using a 1..".,,...,... ~.. The amount of iight produced is direetly related to the amount of luciferase present irl the cellular extraet.
Wlth a suitable DNA construct of the present invention transfected into a 30 cytokine-responsive host cell, the present invention provides a convenient means for KET N . 016-0030.WO - ~
measuring the 1~ AAI activity of a reporter product in response to a signaling molecule, such as a cytokine or extract of cytokine-treated cells.
vl Li~ily, when I ~ I ;n~ of LUC is activated by the ~
regulatory protein being assayed, LUC synthesis is increased relative to a control lacking 5 the 1, A~ AI regulatory protein. Thus, the amount of LUC enzyme produced is an indirect measure of L Iis~ uu-l induced by the activated ~ Al regulatory protem binding to the regulatory el.,.. liJ~1 5. - "rl ~ sequences of the present invention which is operably linked to the LUC gene.
~~. When a preferred cytokine-responsive host cell, such as a HepG2 cell, is 10 transfected with a reporter DNA construct according to the present inventionl it can be utilized in assays to detect agonists and antagonists of sigmaling molecules that induce gene 1 l A-~ via activated 1, .... ;1,l; ~ IAI regulatory proteins. As used herein, agonists or antagonists of gene 1. A l C- I ;I~;nI~ include ~ , I,v~ that intervene at any point within the sigmaling pathway from interaction between the signaling molecule and 15 a cell surface receptor through activation of one or more ~ a~ iOl~dl regulatory proteins and binding of the same to DNA regulatory elements, the end result of which is mn~ lAtion of gene ~ 11 Further, as used herein, agonists and antagonists of gene transcription also include p-,~ of known Cvl~ vu~S with such agonist or antagonist properties. Agonists can be detected by contacting the transfected host cell 20 with a compound or mix of compounds and, after a fixed period of time, ~i t 1~ Ig the level of gene expression (e.g., the level of luciferase produced) within the treated cells.
This expression level can then be compared to the expression level of the reporter gene im the absence of the compound(s). The difference between the levels of gene expression, if any, mdicated whether the compound(s) of interest agonize the activation 25 of intracellular I . A ~ V~ ~AI 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 bet~veen the treated and untreated cells provides a relative indication of the strength of that compound(s) as an agonist of gene 1, A I ~ ' ;1'1;')~1 via a LIA~ I regulatory protein pathway.
DOCKET NO. 016-00~0.WO
2~ 91 1 8~
Altematively, such a transfected host cell can be used to find antagonists of known agonists, e.g., cytokines such as L4, of gene LI A I I~ utilizing host cells transfected with the DNA constructs according to the present inventiorl. In such ar assay, the compound or rl~mro~1n~C of interest are contacted with the host cell in 5 , , with one or more known agonists (e.g., cytokines) held at a fixed C~ 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 c~mrol-n-i~, 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 antagorlists of gene transcription utilizing the regulatory elements/ ~l:g.~ of the DNA constructs and transfected host cells of the present invention. Further, the agonist and antagonist ~mrol~n~C discovered utili~ing these methods can serve asAl agents irl the illLt~ ,.lLiU-- of various cytolcine-induced disease states and 15 conditions, or to arneliorate disease states caused by cytokine deficiency, such as ;llnAl.. AI IIJ.. , infection, ane-m--ia~ cytopenia and cancerous or yl~ uu~ conditions.
The invention will be &rther illustrated by reference to the following non-limiting Examples.
( ~- 20 EXAMPLE I
Rea~ents Ol.~.. ,. l~ul,.1. were obtained from Operon T~ c~ (Alameda, CA). R Pnr~mhinAnt human GM-CSF, L-3, IL4 and IL-6 were obtained firom R&D
Systems (Minn~rl~lic, MN). ~ omhinAnt human IL-13 was obtained from Biosource 25 (Cam~rillo, CA). l?.cc~mhin~-At human Epo and G-CSF were from Arngen, ~nc.
(Thousand Oaks, CA). Protease inhibitors and poly d(I-C) poly d(l-C) were from Boehringer Manmheim (T~
DOC~NO. 016-00,0.WO - ~
O 21911~q CeDs and cell culture U937 cells were obtained from Dr. J. E. Darnell (cù~ , available from ATCC) and grown in RPMI-1640 (13ioWhiKaker) ~ rd with fetal bovine serum (10% vlv), glutamine (Z mM) and gentamicin sulfate (50 ,uglmL). MEI-I 80 cells 5 were obtained from the ATCC and grown in McCoy's 5A (GibcolBRL, G._;h.,l abu MD) ~ r1 with fetal bovine serum (10% v/v), glutarnine (2 mM) and gentamicin sulfate (50 ,uglrriL). TF-1 cells were obtained from the ATCC and grown in RPMI-1640 (BioWhittaker) ~ rd with fe~al bovine serum (10% vlv), glutarnine ~- ~. (2 mM), gentamicin sulfate (50 ,uglrrlL), and GM-CSF (5 nglrnL). L-3-dependent 10 NFS-60 cells were obtained from Dr. J. N. Ihle (St. Jude Children's Research Hospital, Memphis, TN) and were maintained in RPMI-1640 5~ll.plf .~l. .llr~1 with fetal bovine serum (10% vlv), glutamine (2 rr~l), gentamicin sulfate (50 ,ug/rrlL) and 10% WEHI-3B-~. ,.,.1,1 lf .,..~11 medium (to provide IL-3). Factor-.l~lf l~f .~,i. .,l NFS-60 cells were selected by Will~dld~i~lg WEHI-3B-c- .,..~;l,. .."~l medium from the culture medium. In 15 about two weeks, the cells adjusted to the new growth conditions and l~u~ d~d as well as the parental NFS-60 cells. ME-18û cells were treated with cytokines at 50-75%
conrduency, U937, TF-I and NFS-60 cells at a density of 2 X 105 - 1 X 1061ml.
Cytokines were used at the following rnn~Pntr~tinn~ ~-6, lû ng/rnL, IL4, 10-30 nglml, GM-CSF, 5 nglml, Epo, 4-6 UlraL, L-3, 15-20 nglraL, IL-13, 60 nglrnL, and G-CSF, 20 nglmL.
P~ of nuclear e~tracts and E1f~ t;~ Mobilitv Shift Assa~s Nuclear extracts were prepared by NP40 Iysis as described in H. B.
Sadowski and M. Z. Gilman, 362 Nature. 79 (1993), the disclosure of which is herein ihl~,ùl~uldL~d by reference. Protein ~ were measured using Bradford dye binding assay (Bio-Rad T ~hnr~tnri-o~, Hercules, CA). Nuclear extracts were prepared either from untreated U937 cells, U937 cells treated for 30 min with GM-CSF, U937 cells treated for 30 min with IL-4, TF-1 cells starved of GM-CSF for 18 h and then either left untreated or treated for 30 rnin with Epo, L-3 or GM-CSF; ME-180 cells either left untreated or treated for 30 min with IL4 or ~-13; or NFS-60 cells starved of DOCKET NO. 016-0030.WO
25 2191l89 L-3 for 16-18 h then either left untreated or treated for 10 rnin with G-CSF, L-3 or IL-6. The double-stranded probe ~ c used in the El~ u~llu~ , Mobility Shi~ Assays (E~MSAs) were forrned by annealing ~ F ` with the sequences:
5 5'-GATCCACTTCCCAAGAACAGA -3' (SEQIDNO. 14)
- Progress in resolving these issues has been made recently m the interferor (IFN) system. L Ns and 13 (type 1) act as a primary non-specific defense against viral infections. S. Petska and J.A Langer, 56 Annu. Rev. Biochem.. 727 (Ig87). IFN~
(type II) has anti-viral properties but also plays a major role in regulation of the immune response. Id. Type I and type lI IFNs bmd to distinct cell surface receptors and cause rapid alterations in gene expression. Aguet, 55 Cell, 273; Uze, 60 Cell, 225; and G.C.
Sen and P. Lengyell 267 J. Biol. Chem.. 5017 (1992). Specific sequence elements have been identified in the promoters of genes that respond to IFNa, termed interferona stimulated response elements (ISREs), that are both necessary and sufficient forregulation by IFNa. Sen, 267 J. Biol. Chem.. 5017. Speci~ically, activation of the IFNa receptors stimulates t,vrosine ~ u~ u~ iu~ of a family of proteins that serve as DNA
- 20 binding proteins, and ac~,ù~ ly as Ll~ Liull }egulatory factors via the ISRE. C.
Schindler 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 ~
(STATs), assemble into a multimeric complex, translocate to the nucleus, and bind cis-25 acting enhancer elements in the appropriate regulatory regions. D.E. Levy et al., 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 30 Proc. Natl. Acad. Sci.. 8555 (1990). ISGF3 is a complex of 4 binding proteins, called DOCKE~NO.0160030.WO 21 9 1 i 8q ::
.. ~ .
p48, p84 (STAT1,~), p91 (STATI) Gnd pl 13 (STAT2). Recently, cDNAs encoding the proteins that constitute ISGF3 have been isolated and ~ , i. X-Y Fu et al., 89 P}oc. Natl. Acad. Sci.. 7840 (1992); C. Schindler et al.. 89 P}oc. NAtl Arqri Sci..
7836 (1992) and S.A Veals et al., 12 Mol. ('~ll Bif l 3315 (1992). p48 is the DNA
5 binding component of ISGF3 and has homolog,v to myb. Veals, 12 Mol. Cell. Ri.ll 3315. p84 and p91 are probably Gl~clllaLi~ spliced products of the sarne gene and are related to pl 13. X-Y Fu, 89 Proc. Natl. ~f~q~ Sci.. 7840 and Schindler, 89 Proc. NatL --Acad. Sci.. 7836. p84, p9~ and pl 13 Gre novel proteins that contain SH2 and SH3.f -. domains and are found in the c-ytoplasm of untreated cells. Schindler, 257 ~ç~, 809 and X.Y. Fu, 70 ~1~ 323-335 (1992). Thus, IFNa treatment of cells results m rapid tyrosine ~lloa~llùlylGliull of p84, p91 and pll3, causing them to associate and form a Il~.,c~ull..,.;c complex with p48 to fomm ISGF3, which then LIG lal~,G~ca to the nucleus and binds to ISREs, stimulating I~ Id.; Dale, 86 Proc. Natl. Acad. Sci..
1203 and Kessler, 4 Genes Dev. 1753.
Regulation in response to IFN^l is conferred by a distinct sequence from the ISRE, the gamma activated sequence (GAS). T. Decker et al., 10 EMBO J.. 927 :~
(1991); K.D. Khan et al., 90 Proc. NA~l Acad. Sci.. 6806 (1993) and D.J. Lew et al., 11 Mol.Cell.Biol.. 182(1991). TreatmentofcellswithlFNyresultsintyrosine pllûa~ u~ylGLicll of p91 (STATla), which then Lll.alo~,GLta to the nucleus and binds to - 20 the GAS. Decker, 10 EMBO J.. 927 and K. Shuai et al., 258 Science. 1808 (1992~.
Thus the specificity of binding of either IFN o} IFN~ to their receptors is translated into a specific ~llva~llulylGLiùll pattem within a related family of latent LlGIIa.,lil)Liu factors (i.e. DNA binding proteins). This pattem of IJllOa,ullulylG~iull dictates the association state of the proteins, which detemlines âpecificity of binding to either an 25 ISRE or a GAS, and tbe subsequent LlG l,...;~,Liul.GI response.
Yet another cytokine, Interleukin^6 (~-6) plays a major role in the induction of the acute phase response in l~ JGlU~.yLt~. The acute phase response is ~,I.G G~.Ltl i~ by the drarnatic ~ l upregulation of a distinct set of genes1 temmed acute phase response genes. P.C. Heinrich et al, 265 Biochem. J.. 621-6363 0 (1990). Studies of the promoter regions of these genes have identified specific DNA
DOCKE~ NO. 016-0030.WO 2 ~ 9 1 1 8 9 sequences that are required for induction of acute phase response genes by IL-6. See __ D.R Kunz et al., 17 Nuc. ~ Res.. 1121-1138 (1989); M. Hattori et al., 87 Proc.
Natl. ~!r~ Sci USA, 2364-2368 (1990); K,A Won and H. Bauman4 10 Mo~, CelL
~j~" 3965-3978 (1990) and D.R Wllson et al., 10 Mol. Cell. E~iol.. 6181-6191(1990).
5 These sequences are termed acute phase response elements (APREs). One type of APRE shows many similarities to the GA~S elements that confers induction by IFNr.
Yuan et al., 14 Mol. Cell. Biol.. 1657-1668 (1994). Proteins that bind to this class of APREs have been ~ ala~,Ltl~,l and purified. U. M. Wegenka et al., 13 Mol. Cell. p~in~
~-- 276-288 (1993); T. Ito et al., 17 Nuc. Ari(~ Res.. 9425-9435 (1989) and Hattori, 87 10 Proc. Natl. Acad. Sci. U,S~ 2364-2368. A cDNA clone encoding the IL-6 - induced APRE-binding protein has been isolated (Zhong, 264 ~iÇI~Ç~ 95 (1994); Akira et al, 77 ~, 63 (1994); Zhong et al. 91 Proc. Natd. ~ Sri 4806 (1994) and Raz et al., 269 J. Biol. (~hf~m 24391(1994)), and was found to encode a protein that shows ~ u~ r .-hlr homology to p91 (STATlc~). For this reason the protein is termed 15 STAT3. Like STATIc~, STAT3 is a latent L- all~ ,Lio-~ factor that is activated to bind DNA by rapid tyrosine pl~v~ v~JI~Livll.
Interleukin~ (IL-4) is a pleiotropic cytokine that elicits biological responses in a variety of both Iymphoid and non-lymphoid cell types. IL-4 is a ~IY~,V~JI uL~,.- of a,ul~ Wdilll~ 1 9 kD produced primarily by the Th2 subset of activated 20 T-ceas. IL-4 has since been shown to play an important role in B-cell ~u. uli{'cl dLiull, the regulation of ;.,.. ~gl. 1,~ . expression, in T-cell regulation and in the growth and dill~l.tlltiaLOl~ of h.,llla~v~vi~,Li-. precursor cells. IL-4 exerts its biological effects thrûugh a specif c high-af~inity receptor on the surface of h.,~llaLO~Ju;~,L~, as well as certain non h . . ~ .~ l up. . - I l~ cell lines. One chain of its receptor, the ~c chain, is sbared by the IL-2, IL-7, IL-9 and IL-13 receptors. M. Kondo et al., 262 Science 1874 (1993), M. Noguchi et al., 262 ~a~ 1877 (1993), S. Russell et al., 262 Sçience 1880 (1993), and M. Kondo et al., 263 ~Çi~ 1453 (19g4).
Binding of IL-4 to its receptor on the cell surface results in the activation of an ;..L- a~ ld- tyrosine kinase and the rapid ~ o~ u. ylaLivll of several proteins on 30 tyrosine. These initial events appear to be directly related to th~ immediate effects of , . , .. . .. . .. . .... .. _ . . . _ _ . . . ..
DOC~E~NO. 016-0030.WO 2 1 9 1 1 8 9 s L-4 on target gene ~ In particular, L-4 up-regulates in responsive cell lines the expression of several ceLI-surface antigens including class II MHC, the low afiinity Fc receptor for IgE (Fc~RlI, CD23), LFA-I and LFA-3, CD40 and surface IgM. B.
Aggarwal and J. U. Gutterman, Human Cy~nkin~ T~nflhogk fnr Basic Chernical = . _ .
S Researcb Blackwell Scientific PubL,~Lulla, Boston, MA (1992). Perhaps the mostprominent role of IL-4 is in B-cell ~ICI c~lLi~lLio4 where L-4 acts as a "switch factor"
promoting an Ig heavy chain class switch to IgE, the majo} mediator of Type I allergic reactions. W.E. Paul, 77 BlQod 1859 (1991). Evidence that L-4 operates through ar ', STAT signal ~ ladu-,Livli system is based upon the Ol/a.,- v~l~ion that L-4 rapidly lû activates in a variety of ceLI lines l~hGal~llu~yl uaillc-containing protein complexes that bind to a GAS-Like DNA sequence element. H. Kotanides and N. Reich 262 Sciençe 126j (1993) and C. Scbindler et al., 13 E~BO J. 1350 (L994~, P. Lamb et al., 83 Blood, 2û63 (1994) and I Kohler and E.P. Rieber, 23 Eur. J Tmmlln~l 3û66 (1993). A
STAT activated by L-4 in THP-I ceUs has béen cloned recently (called STAT-L-4 orSTAT6) and is likely a constituent of all of the reported L-4 induced complexes. J.
Hou et al., 265 ~n~, 17ûl (1994) and J.N. Ihle et al., 11 Trends in Genetics. 69(1995).
Interleukin 13 (IL- 13) is a pleiotropic cytokine that shares many of the biological activities of IL-4. G. Zurawski and J. E. de Vries, 15 Im.~munol. TQ~,V 19 _G 20 (1994). IL-13 has roughly 3û% sequence identit,v with IL4 and exhibits IL4-like activities on ~u~o~,~t.,J~ u~'1&27_~ and B-ceLls (A Minty et al., 362, ~h~ 248 (1993) and A N.J. McKenzie et al., 9û PrQc. Natl. ~ 1 Sci. U.~A 3735 (1993).
However, unlike ~-4, IL-13 has no effect on T-cells. The biological activity of IL-13 is mediated through binding to its specific highaffirlity cell surface receptors consisting of 25 an IL-13 binding subunit and one or more receptor ..~" ,l ~,. ,...l ~ that are shared with the L-4 receptor (the ~L4R' subunit and/or the yc subunit). G. Aversa et al., 178 J. Exp.
Med. 2213 (1993). Evidence that L-13, like L4, operates through a STAT signal system is based upon the observation that L-13 rapidly activates in a variety of cell lines phoa~.l,uLy-ua;"~-containing protein complexes very similar to those . . .
DOCKETNO. 016-00~0.WO - -2191 ~9 induced by IL-4 that bind to a GAS-like DNA sequence element . I. Kohler et al., 345 FEBS Lett. 187 (1994).
GM-CSF belongs to a group of growth factors termed colony stimulating factors which are involved m the survival, clonal expansion, and di~tlcllLialiul~ of 5 i~ Jr\~ proger~itor ceUs. J. Gasson, 77 ~Q~i 1131 (1991) and N.A, Nicola, 58 Annu Rev. Biûchem. 45 (1989). GM-CSF acts on a set of partiaUy committed progenitor ceUs and causes them to divide and dilI'c~cuLia~e in the gr mulocyte-. a~..ul,~ a~ pathways. GM-CSF can also activate mature granulocytes and llà~,l u~ . In addition to effects on ~ h,llloll~cytic lineages, GM-CSF can promote 10 the ~-ulircl aLiOIl of erythroid amd Ill~aLalyucy~(: progenitor cells. Gl~-CSF, an 18-22 kD ~;;y~.UIJlU~,;.l, is produced by a variety of cells, includmg T-ceUs, B-cells, macrophages, mast cells, endothelial cells and fibroblasts, in response to immune or ;.,nA..,.,. l..,y stimuii.
GM-CSF eYerts its effects by interacting with ceU surface receptors on specific target cells. The receptor is composed oftwo chains, GM-CSF-cL and GM-CSF-~. L.S. Park et al., 89 Proc. ~Atl Acad. Sci. 4~95 (199~). The G~-CSF~ is specific to GM-CSF, while the GM-CSF-,~ is identical to the f~ subunit of the IL-5 and IL-3 receptors. G. Goodall et al., 8 Growth Factor~ 87 (1993). Although neither GM-CSFo~ or GM-CSF,~ have intrinsic kinase activity, GM-CSF treatment of ceils results in ~: 20 rapid tyrosme pllo~l~olylaLiull of multiple proteins Evidence that GM-CSF operates t_rough a STAT signal Llaul~du~,Lioll system is based upon the obs~l va~iun that GM-CSF
rapidly activates in a variety of cell lines pllo~l,lloLy- o~ -containing protein complexes that bind to a GAS-like DNA sequence element A C Larner et al., 261 Scien.ce 1730 (1993) and P. Lamb et al, 83 Blood 2063 (1994) It has been reported that GM-CSF
activates STAT5, which is likely a constituent of aU of the reported Gl~-CSF activated complexes. ~le et al, 11 Trends in Gl~nptir~ 69 (1995).
Interleukin-3 (IL-3) is a pleiotropic cytokine produced primarily by activated T-cells Its effects include stimulating the proliferation amd L~clc~lLiaLioll of both pluripotent l~ r~r - I ;C precursor cells as well a wide variety of lineagecommitted cells Ihle, J.N. in Pectide (~rowth Factors ~n~ th~ir Rece~tors Springer-... ..... .... .. .. .. . . ., .. _ ., .. _ .. ... . . . .
DOCKET.NO. 016-0030.WO 2 1 9 1 1 8 9 Verlag, New York (1991). The mature protein has an apparent molecular weight of 28,000, and binds to a cell surface rçceptor (IL-3R) that consists of at least two poly~ idc chains, IL-3Rcc and L-3R~. The IL-3R~ chain is also a component of theIL-5 and Gl~-CSF receptors, whereas the IL-3c~ chain is unique to the IL-3R
5 Miyajama et al 82 Blood 1960, (1993). Binding of IL-3 to its receptor eauses the aetivation of the tyrosine kinase JAK2 and the rapid tyrosine pl~ ylaLiol~ of a set of ~,y~ llc proteins. O. Silvemnoinen et al., 90 Proc. ~atl. Aead Sci. 8429 (1993). A
GAS-binding eomplex that eontains a member of the STAT family ean be deteeted irl f: . extraets from eells trçated with IL-3. AC. Larner et al., 261 Sciencç 1730 (1993); J.N.
Ihle et al., 19 Trends Eliorhp~n Sci. 222 (1994). It has been reported that IL 3 activates STAT5, which is thus likely a constituent of the reported IL-3-activated complexes.
J.N. ~le et al., 11 Trends jn Genetics. 69 (1995) EryLll~ uk ~ul (Epo) is the major hormone responsible for the proliferation and maturation of red blood cell precursors. S.B. Krantz, 77 Blood 419 (1991). In vitro evidence indicates that it also plays a role in thrombocytopoiesis. An et al., 22 Exp. ~m~t 149 (1994). The protein, which has an apparent molecular wei~ht of 30,000, is produced mainly in the kidneys and is induced by conditions of tissue hypoxia.
It acts by binding to a cell sur~ace receptor (EpoR) that consists of a single poly~,t";de chain that is a member of the l , -l~J~ receptor family. ~ D'Andrça et al 57 ~; ~ 20 277 (1989). An early event following the binding of Epo to EpoR is the activation of the tyrosine kinase JAK2, which assoeiates non-covalerltly with the ~,y~ Jla;,llu~. domain of the receptor chain. B. Witthuhn et al, 74 Cell 227. Aetivation of JAK2 by Epo is correlated with induction of tyrosine ~ o~ o.~laii~..l of the EpoR and ~.y~o~Jla~llu~, proteins. Epo treatment of cells also results in the rapid induetion of a GAS-binding 25 activity that contains STAT proteins that are thought to contribute to Epo-induced changes in gene expression. P. Lamb et al., 83 Blood 2063 (1994); Fnbloom et al., 14 Mol. Cell Biol. 2113 (1994). It has been reported that Epo activates STAT5, which is thus likely a constituent of the reported Epo-activated complexes. J.N. Ihle et al., 11 Trends in Gençtiçs. 69 (1995).
DOCKE~Nb.016-0030.WO 21 91 1 8~
' 8 G-CSF is a pleiotropic cytokine best known for its specific effects on the ~ulircl~ioll,dirrcl~ .iio4andactivationofl- ~ yv :;~cellsoftherl~ lul,lul;~, ~ ulfJ~.y~c lineage. G-CSF has also been reported to have ~ h 1~ ;f activity for human ~lulo~y~cs and monocytes as well as for l.lc~el.~,l.y.,.al cells includingS fibroblasts, smooth muscle cells and ~u~. ubl~L~. These m vitro functions reflect the potential in vivo roles for G-CSF in the of steady state 1~
defense against infectio4 ;l l n - - .... ,,~ ;,,., and repair. When G-CSF was a lulul.~c. cd to various ani~nal models, an elevation of circulating neutrophils has been observed. G-- CSF is now used clinically in patients that have ~ lulùp~.. ua as a result of receiving 10 ~,Il.,.lluLIl~. ~y or receiving ' " - - "~ .. caa;Vc agents after organ ~
M AS. Moore, 9 Annu. Rev. Immunol. 159 (1991), N.A Nicola, 58 Annu. Rev.
Biochem. 45 (1989), and E. Pimentel, (1994) in Handbook of Growth Factors. Vol mE. Pimentel, ed., CRC Press, Boca Raton, p. 177.
G-CSF e.~erts its biological activity through binding to G-CSFr. The 15 receptor for G-CSF (G-CSFr) is a member of the type I cytokine receptor ~uu~,.rA~ILly that lacks a kinase domain ar.d appears to consist of a single ~oly~ id~ chain .D;.l.~ aLiu~l of two G-CSFr chains forms a high aftmity binding site for G-CSF.
Among the various 1. ,Alup~;l; receptor ~u~,.rAIllily members, G-CSFr is most closely relatedtogpl30,thesignal-llolla.luc;llgcomponentoftheL-6,oncostatinM,and ~-20 leukemiainhibitoryfactorreceptors. Recentstudieshave.l,,~lAlr,lthatinmyeloid leukemia cell lines, G-CSF treatment results in rapid tyrosine pl.o*,llolyldLoll of G-CSFr, JAK I and JAK2 tyrosine kinases and the members of the STAT family of factors. S.E. Nicholson et al. 91 Proc. Natl. Af~ad. Sci.USA 2985 (1994) and S.S. Tian et. al., 84 BlQQd 1760 (1994).
It has previously been reported that many cytokines, including IL-3, GM-CSF, Epo, G-CSF, L4 and IL-13, activate STAT or STAT-like complexes that bind toDNA sequence elements related to the GAS elements that were first characterized in the promoters of ~N^,~-responsive genes. However, to date there has been no reported.~... ,.)..~, Al ;.~l l that the DNA sequences reported to bind to the STAT or STAT-like 30 complexes activated by IL-3, GM-CSF, Epo, G-CSF, L4 and L-13 can mediate DOCKE~ NO. 016-0030.WO
2191 18~
. .
t induction in response to those cytol~nes. Accordingly, the ;~ . ,l ,l ;, ,,l ,....
of DNA sequence-elements capable of mediating 1~ activation in response to cytokines such as :L 1, GM-CSF, aCSF and Epo, for example, would be usefill tools that would allow the responses mediated by various cytokine-activated DNA-5 binding proteins to be ~U~ y assayed.
The disclosures of the above-cited references are hereby ;~ by reference in their entirety.
~ ..
DOCK~T N0. 016-0030.WO
~ ~ 2!91189 .
Summanf of the Invention The present invention is directed to methods for screening for modulators ~i.e., agonists and antagonists) of cytokine-mediated l l A 11~ , and to the DNAconstructs and cytokine-responsive host cell lines transfected with such DNA constructs 5 used in such screening methods. In a preferred f..,l,~.l"..~ .,l the present invention is directed to methods for screening for cytokine modulators mvolved in the STAT5 protein and/or STAT6 protem siOonaling pathway. In this regard, the DNA constructs of the present invention include F~ m 1- ~F~V~ ' sequences containing regulatory elements -- that selectively bind activated STAT5 and/or STAT6 proteins, and modulate10 1~ of the associated l~ loOuus gene, in response to ~IJl Upl i~ signaling molecules, such as the cytokines IL 3, IL4, L-13, Epo, G-CSF and GM-CSF~
Surprisingly, and contrary to the teaching in the art, only a limited subset of the regulatory elements that bind activated STAT5 and/or STAT6 proteins actually modulate ~ of the associated 'A,t.,. ulo~uus gene m the assays of the present 15 invention.
In particular, the present invention provides a DNA construct comprising (a) an ol;Ou~.uclFJliJ~ sequence comprising a reOulatory element of the nucleotide sequence TTNXAA, operably linked to (b) a promoter, operably linked to (c) a h.,Lc:luloOuu~ gene, wherein N is ," l~ y selected from A. T, C or G and x is 4, 5, r- ~ 20 6 or 7, and whereim the DNA construct is operably linked in such a manner that the ~l~l ulogùus gene is under the l ~ ;F~ ~AI control of the promoter and I F sequence when the r.~ . 1. ul ;, t~ sequence is bound by a STAT
protein activated in response to IL-2, ~-3, IL-4, IL-7, ~-9, IL-13, G-CSF, GM-CSF, Epo or Tpo Also provided is a cytokine-responsive host cell transfected with this DNA
25 construct.
The present invention also provides a DNA construct comprising (a) an .. 1. ul;~ sequence comprising a regulatory element ofthe nucleotide sequence ANTTCNNNNGAF~NA (SEQ ID NO. 3) operably linked to (b) a promoter, operably linked to (c) a Lu,LcluloOuu~ gene, wherein N is ~ ~f ~ y selected from A, T, C or 30 G, and wherein the DNA construct is operably linked in such a manner that the DOCKE~T NO. 016-0030.WO
h~ lOIO~,UU:i gene is under the ~ C. ' 1~ control ofthe promoter and Li.if sequence when the GL8U....LCIf~U~idf sequence is bound by a protein complex comprising a STAT6 protein activated in response to a cytokine Also provided is a cytokine-responsive host cell transfected with this DNA construct Further, the present invention provides methods for measuring the ability of a compound to act as an agonist of gene tlPnc~rtion comprising (a) contacting the compound with the transfected host cells described above under conditions in which the heterologous gene is capable of being pressed in response to the compound, and ~b) - , comparing the level of gene expression in step (a) with the level of gene eYpression from lû the host cells 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 ~ O.l comprising (a) contacting the compound with the transfected host cells described above in the presence of a ~ amount of a cytokine under conditions in which the l~ . ul~uus gene is capable of being expressed in response to the cytokine, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cells in the presence of the cytokine, but the absence of the compound. In both these methods, the l~ ulo~;uu~ gene may be any appropriatereporter gene such as the heterologous gene for luciferase, ~llo"....l.l.. : ..l acetyl transferase, green fiuorescent protein or ~-~a~ ei~i~cf.
These and various other advantages and features of novelty which . the invention are pointed out with p~ Li~ukuily in the claims annexed hereto and forming a part hereof However, for a better I ' ' ,, of the invention, its advantagec, and objects obtained by its use, reference should be had to the fl-,~,UIII~ llJill~, drawings and descriptive matter, in which there is illustrated and 25 described preferred f-l l ll ~O~ ` of the invention.
DOCKEI NO. 016-0030.WO
~ 2191 189 Definitions For the purposes of this invention:
~ ol~"",.. ;F~ " or "DNA" molecule or sequence refers to a molecule comprised of the dcv~y~ I; vl ;~ adenine (A), gL~anine (G), thymine (T) and/or 5 cytosine (C), in either smgle-stranded form or a double-stranded helix, and comprises or mcludes a "regulatory element" according to the present invention, as that term is defned herein. The exact size, ~ and orientation (i.e. 3' to 5', or 5' to 3') will depend upon many factors, which, in turn, depend upon the ultimate function and use of the~lig~ lFvl~ 0fthepresent3nvention. Thus,theterrn''i~l;s..~ lrv~ lor ,, 10 "DNA" includes double-stranded DNA found m linear DNA molecules or fragments,viruses, plasmids, vectors, ..~..u.~.o~v.l.~.~ or ~yllLL~,.i. ''y 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 dcv.~ylilJ- "~"~lrv~ sequence 15 comprising the whole, or a portion of, an ol ~ I/ vl ~ sequence to which an activated ~ AI regulatory protein, or a complex comprising one or more activated ~ Al regulatory proteins, binds so as to ~ Ally modulate the expression of an associated gene or genes, including, heterologous genes.
"Signalmg molecule" refers to an . . ~ ' pGIy~ ide ~
. . 20 or other rlon-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 i~ cell.ll~
pathway which includes the activation of one or more L~ "l regulator~v proteins that bind to a regulatory element, thereby L. A -`- ;~ Y modulating the expression of an associated gene or genes. As used herein, "sigmaling molecule" includes naturally 25 occurring molecules, such as cytokines, peptidyl and non-peptidyl hormones, antibodies, cell-surface antigens, or synthetic mimics of any of these signaling molecules, or synthetic molecules that mi~nic the action of any of these si~,maling molecules."Cytokines" refer to a diverse grouping of soluble poly~,~Lid~, including growth factors and hormones, that control the growth, li~tltlll;~tioll and function of 30 cells in such a manner as to ultimately elicit a phenotypic response in an organism.
, . _ _ _ ..... ..... . ... .........
DOCKE~NO. 016-0030.WO 2 l 91 1 89 Preferred cytokines useful with tbe regulatory elements and associated methods of tbe present invention include IL-3, IL-4, Il:~-13, GM-CSF, G-CSF, Epo and Tpo.
"TIA~ I regulatory protein" refers to ~Luyl~~ , or nuclear proteins that, when activated, bind the regulatory elements/,-l:~,. ..1. ,. 1~ vl ;~1 sequences of S the present invention either directly, or indirectly through a complex Of ~
regulatory proteins or other adapter proteins, to L A~ r ".~ modulate the activity of an associated gene or genes. Thus, 1~ 1 regulatory proteins can bind diréctly 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 10 bound to a DNA regulatory element of the present mvention. See e.A~.. S.A Veals et al., 13 Molec. Cell. Biol.. 196-206 (1993). As used berein, L~ regulatory proteins, mclude, but are not limited to, those proteins referred to in the art as STAT
proteins (Z. Zhong et al., 264 Science. 95) STF proteins (C. Schindler et al., 13 E~,IBO . =
J, 1350 (1994)), Mammary Gland-Specific Nuclear Factor (M. Schmidt-Ney et al., 6Mol. F~ - l."- ~f~l 1988 (1992) and H. Wakao et al., 267 J. Biol. Chem.. 16365 (1992)), APRF (Wegenka, 13 Mol, CelL~io.. 276), GHIF (Mayer, 269 J. Biol. Chem..4701), GHSF and EPOSF (Finbloom, 14 Mol. Ceil Bio,. 2113), as well as to all ~ul~:~L~I~;ul~ ov-.~ analogs and allelic variations thereo "T~ iyliolial!~ modulate the expression of an associated gene or ~-.20 genes" means to change the rate of L- r. L,.,I iy~iOIl of such gene or genes.
"STAT protein" refers to those 1. All~ IA1 regulatory proteins designated as "Signal Transducers amd Activators of TI . ,~ " (STAT) by Dr. J.E.Darnell of Rockefeller University. See Zhong, 264 Sciençç 95. As used herein, STAT
proteins include the p91 (STATI), p84 (STATI), pl 13 (STAT2) proteins and the STAT-associated p48 family of proteins. S.A. Veals et al., 12 Mol. Cf~ll Ri~-l 3315 (1992). Further, STAT proteins also include a binding protein designated as STAT3 (Zhong, 264 Science 95), and a binding protein designated as STAT4 (~.). In addition, MGF is now renamed STAT5 (Gouilleux et al., 13 EMBO J.~ 43614369 (1994)) and STAT-IL4 (or STAT6) has recently been cloned. Hou et al., 26i Science~ 730 (1994) .
DOCKE~NO.016-0030.WO 2~ 91 1 ~9 , ~ .
amd J.N. Ihle et al. I 1 Trends in Genetics. 69 (1995). Also included are ~ub~LdllL;~
r,lr.~ c analogs and allelic variations of all of the 2bove STAT proteins.
"Activate", "activated", "activation" or derivatives thereof, means that one or more ~ ,11 regulatory proteins within a cell are modified post-5 ; ' ".~/, or are cul~LiLui~ active, such that they can bind directly or indirectlyto DNA regulatory elementr/ ~ sequences of the present irlvention m a sequence-specific manner. This ".,,.1.~ ;".. will typically comprises pllu~llolylaLioll of the ~ c~ l regulatory proteins via a variety of ' , including, but not ~=. limitedtoactivatiorlbyvariousprotemlcinases. See,e.~" (Shuai,258~ciencç, 1808an P. Cohen, 17 T~S. 408 ~1992)).
"DNA construct'' refers to any genetic element, including, but not limited to, plasmids, vectors, ~ ulllOSul~ and viruses, that incorporate the ,~
sequences of the present invention. For example7 the DNA construct can be a vector comprising a promoter that is operably linked to an ,,1~ .."rl~ul"l~ sequence of the 15 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 bindmg directly or mdirectly to RNA polymerase in a cell and initiating Ll all~ iOII of a duwll lLl calll (3' direction) coding sequence. For purposes of the present invention, the promoter is ~: .. 20 bounded at its 3' terminus by the L~ r initiation site and extends upstream (5' direction) to mclude the minimum number of bases or elements necessary to initiate rl~l at levels detectable above ba~,h~uull~. Within the promoter will be found a l, i - ,~- ,1 .1 ;. ,.l initiation site (~c,--v ~Ih~ LIy def ned by mapping with S I nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of 25 RNA pc~ ,.aac. Eukaryotic promoters will often, but not always, contain "TATA"
boxes and "CCAT" boxes. P~uho~yu~iu 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 ;. ~ ., . required for the normal regulated production of a particular protein.
30 A "heterologous" region of a DNA construct (i.e. a heterologous gene) is an identifiable DOCKET NO. 016-0030.WO ~ ~ 2 1 9 1 l 8 9 . ~ .
segrnent of DNA within a larger DNA construct that is not found in association wLth the other genetic ~~ of the construct in nature. Thus, when the 1. .~ ulO~;uua geneencodes a r~m~ n 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 construct7 including an ~ f ol ;~1 sequence according to the present invention, is "operably linked" to a h~,.c. ulogu~ls gene when the presence of the promoter inrduences ~ n from the h~ lulc~;uua gene, including genes for reporter sequences such as luciferase, ~ acetyl - transferase, ~ and secreted placental alkaline r~ c. Operably linked I û 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 linhed if ~ "l l; " c ~" "~- ,-: ,~ in the promoter will produce an RNAtranscript of the reporter sequence. In order to be "operably linked" it is not necessary that two sequences be u~uil~,L~lL~ adjacent to one another.
A host cell has been "L- ular~:l,Lt;d ~ by exogenous or ~ olo~;uus DNA
(e g. a DNA construct) when such DNA has been introduced inside the cell. The l, .,"~rr, l",f~ DNA may or may not be integrated (covalently lirlked) into ~,luullluaul~
DNA mahing up the genome of the cell. In ~luhdlyuL~a, yeast, and m~mm~ n cells for example, the l l ~ rr- I, "o DNA may be maintained on an episomal element such as a .~ 20 plasmid. With respect to eukaryotic cells, a stablely transfected cell is one in which the 1 " r, I . ,j~ DNA has become integrated into a ~lu ulllOa~ c 50 that it is inherited by daughter cells through clu ullluaulliC repGcation. This stabiGty is d~ ~IIOIIall d~Cd by the ability of the eukaryotic cell to establish cell Gnes or clones comprised of a population of daughter cells contairling the Ll~lar~,~,Lul~ DNA
"Cytohine-responsive host cell" refers to a cell line that e7spresses, either nor~nally or after I I ,~ rr~ of the requisite cDNAs, the relevant cytokine receptor c~ , JAK proteins, STAT proteins, and accessory factors such that, upon cytohine binding to the cell surface, STAT-mediated gene Ll~u~s~ Liu~ is affected.
DOCN~T NO. 016-0030.WO
Brief DescriPtion of the Drawi The invention may be fulther illustrated by reference to the Z~ U~II~)dll,y;~l~
Drawing wherein:
FIG. I is a lc~ludu~.Liull ûfEl~,hu~l~u-cLi~. Mobility Shift Assay (EMSA) 5 A llu~ gl A -` that shûw the bindi~g patterns of h~ regulatory protein-DNA binding complexes activated by L-4 and IL-13. The EMSA's were performed as described in the Examples herein. The ~ tl, double-stranded U~
probes utilized in the EMSAs of FTGS. IA and lB were made by arnealing the of SEQ ID NOs. 14-23 (~IG. lA) and 24-35 (FIG. lB).
~ .
DOC~ET NO. 016-0030.W(~ ~
~ 1 9 1 ~ 89 Detailed Descrii~tion of of the Invention The present inventors have discovered that o~ly a select group of regulatory elements that bind activated ~ ''~,l regulatory proteins, such as 5 STAT proteins, actually modulate the ~ of an operably Gnked L~.~e~ uloguu~
gene in a cell-based screen. This unexpected result is in direct . .~" ,l, A~l;. .1 ;~ " ' to the teaching in the art that a regulatory element that binds such activated ~
regulatory proteins will also activate the l A ~ of an associated gene. Thus, the ~ . binding of a regulatory element to an activated L~ o~ regulatory protein is not correlated, and provides no l~l~dl~,l~;l;Ly, with respect to those regulatory elements that will activate ~ of an associated gene in response to this binding. It is only through the teaching of the present inventors that one will be able to select, without resorting to undue ~ , regulatory el.,.~ Lal~ vl ,,1 sequences that will both bind to, and cause Ll~laa~.Li\~a~ioll frvm, activated l~au~ io.~dl reg~latory proteins, such as STAT proteins.
The ~ v~ sequences, comprising DNA regulatory elements, and that are i l~vll~o-AL~d into the DNA constructs of the present invention are selected from the nucleotide sequence TTNXAA, wherein N is ;",~ l ly selected from A, T, C or G and x is 4, 5, 6 or 7. More preferably, the regulatory elements comprise ~i 20 ~ ,., --" l, vL;~ sequences that bind and ~l~la~ ~iv.L~t: in response to activated STAT5 and/or STAT6 proteins. These preferred ~ vl ,,~, 5 sequences are selected from the group consisting of TTCNNNGAA (SEQ ~ NO. 1), TTCNNNNGAA (SEQ lD
NO. 2) and ANTTCNNNNGAANA (SEQ. ID NO. 3), including their double stranded ...",,~,1..,....1~, where N is ;,"l~ l,.."l..~ ly selected from Al T, C or G. Especially 25 preferred ~.l;~.. " .. ., li ~,l ;~ sequences according to the present invention include:
ACTTCCCAAGAACA (SEQ ~ NO. 4), ACTTCCCCGGAACA (SEQ ID NO. 5), ACTTCCCCAGAACA (SEQ ID NO. 6), ACTTCCCAGGAACA (SEQ ID ~O. 7), ACTTCCTAAGAACA (SEQ ~ NO. 8), ACTTCTTAAGAACA (SEQ ID NO. 9), TTCCCGGAA (SEQ ID NO. 10), TTCCCCGAA (SEQ ~:) NO. 11), TTCTAAGAA
30 (SEQ ID NO. 12) and TTCTCAGAA (SEQ ~ NO. 13).
DOCKET NO 016-0030.WO
2~ 9 1 ~ 89 The ~ sequences of the DNA constructs of the present inYention can comprise the entire regulatory element alone, or can incl~de additional 'danking nudeotide sequences. In this regardl it is preferable that such ~.l 2 ~ ; vLid~, sequences comprise between 8 and 200 n--rl~oti~ , including the regulatory elements of S the present invention. However, sequences in excess of 200 ,A~AIrot~ that contain such regulatory elements, and that are capable of binding activated l ~
regulatory proteins, and of L~ modulating the expression of one or more genes thereby, are also considered to be within the scope of the present inYention ~-- The ~ sequence component of the DNA constructs of the present invention can also comprise multimers of t~vo or more "units" of the basic regulatory elements. In this regard, such multimer ~ ul ;~ 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, Lll.,o~Li~.dlly, there is no limit to the number of regulatory elements within such a multimer ~1 G~ Ulid~ sequence.
When used in the DNA construct, including a promoter and k.,.~uloguu~ gene, according to the present invention, a multimer of the regulatory elements can enkance the expression of the gene from the DNA construct in response to various c~Ytokines or other signaling molecules.
A variety of signaling molecules activate LIAI ~ AI regulatory proteins that bind directly or indirectly to the DNA constructs of the present mvention, and modulate l,, ~ . . of the operably linked heterologous gene. Nonlimiting examples of such signaling molecules include polrt,~,~,Lid~,~ such as cytokines and antibodies, and cell-surface antigens, ol;~ 7 typically found at or near the surface of cell, non-peptidyl molecules such as TlJBag4 OE Constant et al., 264 ~, 267 (1994)) and synthetic mirnics any of these molecules, in both their free and bound forms. Thus, the present invention includes cell to cell or cell to substrate ~
regulatory protein activation via signaling molecules bound to or near the surface of a cell or other substrate Preferably, the signaling molecules according to the present invention 30 comprise cytokines that activate ~ AI regulatory proteins, such as STAT
DOCKE~`NO. 016-0030.WO
19 2~91189 proteins, that bind to the regulatory el~.. ,.. L~ ;g~ o~ide sequences of the present invention. Exarnples of such cytokines include7 but are not limited to, L-l~- lu~i.~ 2, 3, 4, 5, 6, 7, 9, 10, I l, 12, 13 and 15 (IL-2, IL-3, IL-4, IL-5, IL-6, ~-7, L-9, IL-10, IL-11, IL-12, IL-13 and IL-15), ~llulul~uy~e-l~la~lu~ull~c~ colony stimulating factor (GM-S CSF), ~ululo~.LyLe colony stimulating factor (G-CSF), colony stimulating factor I
(CSF-I), interferons alpha, beta, arld garmna (IFNa, IFN~, IFNy), epidermal ~rowth factor (EGF), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF), Oncostatin M, nerve growth factor (NGF), ciliary ~uL u,ul~ic factor (CNTF), brain-derived rlc.~uL~u~l~ic factor (BDNF), ~yLluu~uk.,iul (Epo), Lluulll~ùlJoieLul (Tpo), growth hormone and prolactin. Particularly preferred cytokines according to the present invention include those that activate the STAT5 protein and/or STAT6 protein pathways, including, but are not lirnited to, ~-2, IL-3, IL4, IL-S, iL-7, IL-9, IL-13, IL-15, G-CSF, GM-CSF, Epo, Tpo and growth hormone.
The le-,ulll~ -lL DNA construct, such as a reporter plasrnid, according to the present invention, can be constructed using conventional molecular biology, lllhu ubioloy~y~ and c~,ulllbi~ lL DNA techniques well hnown to those of skiil in the art.
Such techniques are explained fully in the literature, including Maniatis, Fritsch &
Sambrooh, "Molecular Cloning: A I,aboratory Manual" (1982); "DNA Cloning: A
Practicai Approach," Volumes I and II (D.N. Glover ed. 1985); "O!;L~ r f~ ` 20 Synthesis"(M.J.Gaited. 1984);"NucleicAcidIIyb~ iu~ 3.D Hames&S.J.Higgins, eds (i984)]; "Animai Cell Culture" [RI. Freshney, ed. (1986)]; "T ' `'`Cells and Enymes" [IRL Press, (1986)] and B. Perbal, "A Practical Guide to Molecular Cloning" (1984), the disclosures of which are herein iU~,Vl ~.u. a~d by reference Promoter sequences useful in DNA constructs according to the present invention include all l,l uh~yu~iu, eukaryotic or viral promoters capable of driving 1"..,~ ;nnofah~Leluloguu~geneofinterestin~ witharegulatoryelement of the present invention, when transfected into an dlJ~I u~L i~e host cell. Suitable ~luh~uyu~ic promoters include, but are not limited to, promoters recognized by the T4, T3, Sp6, and T7 polyl.l~ r., the PR and PL promoters of b~l iu~llAg~" the 3û I~ nI~AI regulatory protein, recA, heat shock and lacZ promoters of ~ ~, the -DOCKET NO. 016-0030.WO
2 ~ 9 ~ 1 89 amylase and the -28-specific promoters of B. subtilis. the promoters of the ~ lr~ ofE~aciilus~sLlc~Lvlllyc~i~promoters1theintpromoterof~ r~
the bla promoter of the ~-lactamdse gene of pBR322 and the CAT promoter of the .f UI acetyl transferase gene of pPR325. ~, ç~, B.R Giick I J. Ind. .. = . .Microbiol.. 277-282 (1987); Y. Cenatiempo, 68 Biochimie. 505-516 (1986); J.D.Watson et ai., Il;: Molecular Biolo. Y Qf the Gerle. Fourth Edition, Benjamin Cummins, Meio Park CA (1987) and S. Gottesman, 18 Arln. Rev. GPnf~t 415-442 (1984), the disclosures of which are herein i~ Ul~UldLt~ by refererlce. Preferred eukaryotic, -~; promoters include the yeast cyc-l promoter, the promoter of the mouse "~ ' I
10 gene, the thymidine kinase promoter of the Herpes simplex vifus, the SV40 early promoter, and the yeast gal-4 gene promoter. See Guarante et ai., 78 Proc. Natl. Acad.
Sci. ~I~, 2199-2203 (1981), D. Hamer et ai., I J. Mol. Ap~ n 273-288 (1982), S. McKnight, 31 S~L 355-365 (1982), C. Benoist et ai., 290 ~ ~London), 304-310 (1981), S.A Johnston et ai., 79 Proc Natd. Acad. Sci. (USA), 6971-6915 (1982) and P.A Silver et ai., 81 Proc. Nati. Acad. Sf,i. (USA), 5951-5955 (1984), the disclosures of which are herein il.cuil)o-dLtd by reference herein. Preferably, a DNA constructaccording to the prent invention utilizes the thymidine i~inase gene promoter of the Herpes simplex virus.
The third component of the I c~,fj~-lY~A~ll DNA or construct molecuies of ( ~- 20 the present invention is a "heterologous gene" which may be composed of any set of nucleotides regardless of sequence. Nu~ ~ .IIL...~ examples of such h~.t~,. vl~ùu~ genes include the structurai genes for luciferase, ,~-Vd~ VIA~ acetyl transferase, secreted placental aikaiine l ' - .~ human growth hormone, tPA green fiuorescent protein and interferon. For a more extensive list of ~l~,t~l ul~uus genes 25 usable m the constructs and methods of the present invention, see Beaudet, 37 Hum. Gen.. 386-406 (1985).
Preferably the ~.,lcluloguu~ gene comprises a reporter gene whose product is used to assess reguiation of ~ via a promoter and a regulatory eleme~tlnl..~ l v~ sequence of the present invention. The expression of this 30 "reporter sequence" results in the formation of a reporter product ~e.g., protein) which is DOCKE~ NO. 016-0030.WO
21 2~9ll89 readily detectable. The reporter sequence will preferably be selected such that the reporter molecule wiU have a physical arld ehemical ~ r~ which wiU pemlit or faeilitate its i~ or detection by means weU known in the art. In one f~mhofiimf~nt~ the presence of the reporter molecule will be deteeted through the use of 5 an arltibody or an antibody fragment, capable of specifie binding to the reporter moleeule. In another ~ o~ , a reporter such as ~ t5 ~ or luciferase can be assayed C.~yll.,...;."LUy or immlmf~lf ~
A preferred reporter molecule is LUC, well known in the a}t. See, e.g..
J. R De-Wet et al., 7 Mol. Cell Bio.. 725 (1987). Because this is an insect gene, it is 10 abserlt from mammalian cells and the enzyrne product can be directly assayed in a ceU
extraet. The level of enzyme activity cul~e~ull~s to the amount of enzyme that was made, which in tum reveals the level of expression. In addition, LUC mRNA may also be measured directly.
Typically, a plasmid containing the . ~, ., . "1 " " - . l l DNA molecule of the15 present invention, including the LUC gene, is introduced irlto cytokine-responsive mammalian ceUs, which are then grown to, at or near confiuency. In this regard, arly cvtokine-responsive host cell capahle of activating one or more ~ ,I;fl..,.l regulatory proteins in response to an appropriate signaling moleeule or molecules can be transfeeted with the DNA construets of the present invention Preferably, such . . 20 eytokine-responsive host ceUs comprise m~rnm~ n eells, sueh as HepG2, U937, M~-180, TF-1 and NFS-60 eeUs.
The reporter eeUs are treated with a eompound or sample suspected of eontaining a signaling moleeule eapable of indueing or aetivating a l, ~ . ,~. . ;l.l ;~.fl~l regulatory protein, for example, an extraet of other eytokine-treated cells. The LUC-25 producing reporter ceUs are extraeted, and the soluble extracts are ~u~ cd withluciferin and ATP. In the presence of these efJllllJOIlllll:~ the aetion of luciferase generates light, which is detected using a 1..".,,...,... ~.. The amount of iight produced is direetly related to the amount of luciferase present irl the cellular extraet.
Wlth a suitable DNA construct of the present invention transfected into a 30 cytokine-responsive host cell, the present invention provides a convenient means for KET N . 016-0030.WO - ~
measuring the 1~ AAI activity of a reporter product in response to a signaling molecule, such as a cytokine or extract of cytokine-treated cells.
vl Li~ily, when I ~ I ;n~ of LUC is activated by the ~
regulatory protein being assayed, LUC synthesis is increased relative to a control lacking 5 the 1, A~ AI regulatory protein. Thus, the amount of LUC enzyme produced is an indirect measure of L Iis~ uu-l induced by the activated ~ Al regulatory protem binding to the regulatory el.,.. liJ~1 5. - "rl ~ sequences of the present invention which is operably linked to the LUC gene.
~~. When a preferred cytokine-responsive host cell, such as a HepG2 cell, is 10 transfected with a reporter DNA construct according to the present inventionl it can be utilized in assays to detect agonists and antagonists of sigmaling molecules that induce gene 1 l A-~ via activated 1, .... ;1,l; ~ IAI regulatory proteins. As used herein, agonists or antagonists of gene 1. A l C- I ;I~;nI~ include ~ , I,v~ that intervene at any point within the sigmaling pathway from interaction between the signaling molecule and 15 a cell surface receptor through activation of one or more ~ a~ iOl~dl regulatory proteins and binding of the same to DNA regulatory elements, the end result of which is mn~ lAtion of gene ~ 11 Further, as used herein, agonists and antagonists of gene transcription also include p-,~ of known Cvl~ vu~S with such agonist or antagonist properties. Agonists can be detected by contacting the transfected host cell 20 with a compound or mix of compounds and, after a fixed period of time, ~i t 1~ Ig the level of gene expression (e.g., the level of luciferase produced) within the treated cells.
This expression level can then be compared to the expression level of the reporter gene im the absence of the compound(s). The difference between the levels of gene expression, if any, mdicated whether the compound(s) of interest agonize the activation 25 of intracellular I . A ~ V~ ~AI 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 bet~veen the treated and untreated cells provides a relative indication of the strength of that compound(s) as an agonist of gene 1, A I ~ ' ;1'1;')~1 via a LIA~ I regulatory protein pathway.
DOCKET NO. 016-00~0.WO
2~ 91 1 8~
Altematively, such a transfected host cell can be used to find antagonists of known agonists, e.g., cytokines such as L4, of gene LI A I I~ utilizing host cells transfected with the DNA constructs according to the present inventiorl. In such ar assay, the compound or rl~mro~1n~C of interest are contacted with the host cell in 5 , , with one or more known agonists (e.g., cytokines) held at a fixed C~ 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 c~mrol-n-i~, 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 antagorlists of gene transcription utilizing the regulatory elements/ ~l:g.~ of the DNA constructs and transfected host cells of the present invention. Further, the agonist and antagonist ~mrol~n~C discovered utili~ing these methods can serve asAl agents irl the illLt~ ,.lLiU-- of various cytolcine-induced disease states and 15 conditions, or to arneliorate disease states caused by cytokine deficiency, such as ;llnAl.. AI IIJ.. , infection, ane-m--ia~ cytopenia and cancerous or yl~ uu~ conditions.
The invention will be &rther illustrated by reference to the following non-limiting Examples.
( ~- 20 EXAMPLE I
Rea~ents Ol.~.. ,. l~ul,.1. were obtained from Operon T~ c~ (Alameda, CA). R Pnr~mhinAnt human GM-CSF, L-3, IL4 and IL-6 were obtained firom R&D
Systems (Minn~rl~lic, MN). ~ omhinAnt human IL-13 was obtained from Biosource 25 (Cam~rillo, CA). l?.cc~mhin~-At human Epo and G-CSF were from Arngen, ~nc.
(Thousand Oaks, CA). Protease inhibitors and poly d(I-C) poly d(l-C) were from Boehringer Manmheim (T~
DOC~NO. 016-00,0.WO - ~
O 21911~q CeDs and cell culture U937 cells were obtained from Dr. J. E. Darnell (cù~ , available from ATCC) and grown in RPMI-1640 (13ioWhiKaker) ~ rd with fetal bovine serum (10% vlv), glutamine (Z mM) and gentamicin sulfate (50 ,uglmL). MEI-I 80 cells 5 were obtained from the ATCC and grown in McCoy's 5A (GibcolBRL, G._;h.,l abu MD) ~ r1 with fetal bovine serum (10% v/v), glutarnine (2 mM) and gentamicin sulfate (50 ,uglrriL). TF-1 cells were obtained from the ATCC and grown in RPMI-1640 (BioWhittaker) ~ rd with fe~al bovine serum (10% vlv), glutarnine ~- ~. (2 mM), gentamicin sulfate (50 ,uglrrlL), and GM-CSF (5 nglrnL). L-3-dependent 10 NFS-60 cells were obtained from Dr. J. N. Ihle (St. Jude Children's Research Hospital, Memphis, TN) and were maintained in RPMI-1640 5~ll.plf .~l. .llr~1 with fetal bovine serum (10% vlv), glutamine (2 rr~l), gentamicin sulfate (50 ,ug/rrlL) and 10% WEHI-3B-~. ,.,.1,1 lf .,..~11 medium (to provide IL-3). Factor-.l~lf l~f .~,i. .,l NFS-60 cells were selected by Will~dld~i~lg WEHI-3B-c- .,..~;l,. .."~l medium from the culture medium. In 15 about two weeks, the cells adjusted to the new growth conditions and l~u~ d~d as well as the parental NFS-60 cells. ME-18û cells were treated with cytokines at 50-75%
conrduency, U937, TF-I and NFS-60 cells at a density of 2 X 105 - 1 X 1061ml.
Cytokines were used at the following rnn~Pntr~tinn~ ~-6, lû ng/rnL, IL4, 10-30 nglml, GM-CSF, 5 nglml, Epo, 4-6 UlraL, L-3, 15-20 nglraL, IL-13, 60 nglrnL, and G-CSF, 20 nglmL.
P~ of nuclear e~tracts and E1f~ t;~ Mobilitv Shift Assa~s Nuclear extracts were prepared by NP40 Iysis as described in H. B.
Sadowski and M. Z. Gilman, 362 Nature. 79 (1993), the disclosure of which is herein ihl~,ùl~uldL~d by reference. Protein ~ were measured using Bradford dye binding assay (Bio-Rad T ~hnr~tnri-o~, Hercules, CA). Nuclear extracts were prepared either from untreated U937 cells, U937 cells treated for 30 min with GM-CSF, U937 cells treated for 30 min with IL-4, TF-1 cells starved of GM-CSF for 18 h and then either left untreated or treated for 30 rnin with Epo, L-3 or GM-CSF; ME-180 cells either left untreated or treated for 30 min with IL4 or ~-13; or NFS-60 cells starved of DOCKET NO. 016-0030.WO
25 2191l89 L-3 for 16-18 h then either left untreated or treated for 10 rnin with G-CSF, L-3 or IL-6. The double-stranded probe ~ c used in the El~ u~llu~ , Mobility Shi~ Assays (E~MSAs) were forrned by annealing ~ F ` with the sequences:
5 5'-GATCCACTTCCCAAGAACAGA -3' (SEQIDNO. 14)
3'- GTGAAGG(J~ ( :lAG -5' (SEQ D:) NO. 15) 5'-GATCTGCTTCCCCGGAACGT -3' (SEQ :O NO. 16) 3'- ACGAAGGGGCCTTGCACTAG -5' (SEQ IDNO. 17) .- ~ 5'-GATCTGCTTCCCCAGAACGT -3' (SEQ ID NO. 18) 3'- ACGAAGGGGTCTTGCACTAG -5' (SEQ IDNO. 19) 5'-GATCTGCTTCCCAAGAACGT -3' (SEQ ID NO. 20) 3'- ACGAAGGGTTCTTGCACTAG -5' (SEQ ~ NO. 21) 5'-GATCCACTTCCCCGGAACAGA -3' (SEQ ID NO. 2~) 3'- GTGAAGGGGCCTTGTCTCTAG -5' (SEQ ID NO. 23) 5'-GATCCACTTCCCCAGAACAGA -3' (SEQ ID NO. 24) 3'- GTGAAGGGGTCTTGTCTCTAG -5' (SEQ ~:) NO. 25) 5'-GATCCACTTCCCAGGAACAGA -3' (SEQ ID NO. 26) 3'- GTGAAGGGTCCTTGTCTCTAG -5' (SEQ IDNO. 27) 5'-GATCTACTTCCCAAGAACATA -3' (SEQ ID NO. 28) . :. 3'- ATGAAGGGTTCTTGTATCTAG -5' (SEQ ID NO. 29) 5'-GATCCGCTTCCCAAGAACGGA -3' (SEQ ~ NO. 30) 3'- GCGAAGGGTTCTTGCCTCTAG -5' (SEQ~NO. 31) 5'-GATCCACTTCTTAAGAACAGA -3' (SEQ n~ NO. 32) 3'- GTGAAGAAl l~ 1AG -5' (SEQ ID NO. 33) 5'-GATCCACTTTCCAAGAACAGA -3' (SEQ ID NO. 34) 3'- GTGAAAGGTTCTTGTCTCTAG -5' (SEQ ~ NO. 35) 5'-GATCTGCTTCCCGGAACGT -3' (SEQ ID NO. 36) 3'- ACGAAGGGCCTTGCACTAG -5' (SEQ ID NO. 37) 5'-GATCGATTTCCCCGAAATG -3' (SEQ ID NO. 38) 3'- CTAAAGG&GCTTTACCTAG -5' (SEQ ID NO. 39) 5'-GATCATATTCCTGTAAGTG -3' (SEQ ID NO. 40) DOCKE~ NO. 016-0030.WO - ~ ~ ~
3'- TATAAGGACATTCACCTAG -5' (SEQ ID NO. 41) 5'-GATCATATTCCCGTAAGTG -3' (SEQ ID NO. 42) 3'- TATAAGGGCATTCACCTAG -5' (SEQ ID NO. 43) 5'-GATCCATTTCTGGAAATG -3' (SEO~ ID NO. 44) 3'- GTAAAGACCTTTACCTAG -5' (SEQ ID NO. 45) 5'-GATCCATTTCCCGTAAATC -3' (SEQ ID NO. 46) 10 3'- GTAAAGGGCATTTAGGATC -5' (SEQ ID NO. 47) 5'-GATCATATTACCAGAAATG -3' (SEQ ID NO. 48) 3'- TATAATGGTCTTTACCTAG . -5' (SEQ ID NO. 49) 15 5'-GATCATTTTCCAGTAACAG -3' (SEQ IDNO. 50) 3'- TAAAAGGTCATTGTCCTAG -5' (SEQ ID NO. 51) 5'-GATCCAATTTCTAAGAAAGGA -3' (SEQ ID NO. 52) 3'- GTTAAAGATTCTTTCCTCTAG -5' (SEQ ID NO. 53) 5'-GATCTGCTTCCCGAACGT -3' (SEQ ID NO. 54) 3'- ACGAAGGGCTTGCACTAG -5' (SEQ ID NO. 55) 5'-GATCTGCTTCTCAGAACGT -3' (SEQ ID NO. 56) 25 3'- ACGAAGAGTCTTGCACTAG -5' (SEQ IDNO. 57) 5'-GATCTGCTTCCCCGAACGT -3' (SEQ ID NO. 58) 3'- ACGAAGGGGCTTGCACTAG -5' (SEQ ID NO. 59) 30 where the nucleotide sequences shown in bold type fa~e correspond to nucleotide sequences, including their double-stranded c...,.l,l..,...,l, tested for activity as regulatory elements according to the present invention.
The annealed ~-~l s ~ l;u~ were labeled by filling in the O~ a,.~llg ends with Klenow fragment (Boehringer Mannheim) in the presence of [a-32P]-dGTP
35 and/or [a-32P]-dATP (Amersham Corporation, Arlington Heights, IL). El._~,tlu~llu.~;c mobility shift assays (EMSA's) were performed in HEPES buffer (13 mM, pH 7.6, Sigma Chemical, St. Louis, MO), containing sodium chloride (80 rnM), sodium fiuoride (3mM), sodium molybdate (3mM), DTT (ImM), EDTA (0.15mM), EGTA (0.15rnM), glycerol (8% v/v, including c~ from the nuclear extract), poly d(I-C) poly d(I-40 C) (75,ug/mL)",..l,"l l.. IP.J probe (~lu~ill~tly 0.2ng) and nuclear extract containing DOCKETNO. 016-0030.WO
21 9 1 1 8~
5-10~Lg of total protein. Reactions were incubated at room Lt.ll~,ld~W~ for 20 minutes then resolved on 5% pOlya~ ldl~ldt gels containing 0.25X TBE [IX TBE is Tris borate (89 mM), pH 8.0 containing EDTA (I mM)] and glycerol (5% v/v). Gels were run at 4C in 0.25X TBE at 20V/cm, then dried and auLu-ddi~_. ' 1.
Relative binding affinities, as determined from the EMSA results for J.J...,. I. u~ r SEQ lD NOs 14-59, were visually rated and assigned according to the following scale:
(-) band ~.~)ll C:~JUlldill~8 to specific complex on the EMSA d' ~ r ~ I dlU (See e.g., FIG. lA, lane 7) barely discernible or not discernible.
(+) band Cullt*lù~dlllg to specific complex on the ElMSA dULOlddio~.a.~. (See e.g., FIG. lA~ lanes 8 and 9) easily discernible but of weak intensity.
(++) band ~,~?llt:~lJulld;llg to specific complex on the EMSA ~ " d~ 8, dlll (See e.g., FIG. lA, lanes S and 6) easily discernible and of moderate intensity.
15 (+~+) band CG-It~lJOlldlllg to specific compleY on the EMSA q~-t~r~qs~ gram (See e.g., FIG. IA, lanes 7 and 3) easily discernible and of strong intensity This visual rating system is sufficient to analyze ~ v~
differences and trends in the EMSA bmding data as opposed to specific numerical ~- - 20 values. If desired~ the use of a phosphor imager or d ,~ (colll~ / aVailable from e.g., Bio-Rad T qborqt~ri~) cûuld provide â means to assess the differencesdescribed here ~ua~LiLdLi~'J. Specific visual ratings of binding affinities for the regulatory elements of ~ . . ,., l ul ;~ir, SEQ ID NOs 14-41, 44-53 and 56-59 are shown in Table I below. Specific visual ratings of binding aftinities for the regulatory elements of oli~ul,u~ ùLid~ SEQ n~ NOs 36-57 are shown in Table 2 below.
DOCN~TNO. 016-0030.WO
. ~ 21911~9 Table 1: Relative EMSA binding affinities for a series of regulatory elements of double stranded ~ differing in fiar~ng and spacing sequences to 1 ~ r- ~ regulatory proteins activated in responSe to the cytokines L4 and IL-13 i~ U-937 or ME-180 cells.
SEQ Core 1~
ID Element IL,4 IL-13 14 CACTTCCCAAGAACAGA +++ +++
16 TGCTTCCCCGGAACGT ++ ++
18 TGCTTCCCCAGAACGT + +
~~20 TGCTTCCCAAGAACGT ++ ++
22, CACTTCCCCGGAACAGA ++ ++
24 CACTTCCCCAGAACAGA ++ ++
26 CACTTCCCAGGAACAGA ++ ++
28 TACTTCCCAAGAACATA ++ ++
30 CGCTTCCCAAGAACGGA ++ ++
32 CACTTCTTAAGAACAGA ++ ++
34 CACTTTCCAAGAACAGA ++ ++
36 TGCTTCCCGGAACGT ++ ++
38 GATTTCCCCGAAATG ++ ++
ATATTCCTGTAAGTG + n.d.
44 CATTTCTGGAAATG ++ n.d.
46 CATTTCCCGTAAATC ++ n.d.
48 ATATTACCAGAAATG + n.d.
ATTTTCCAGTAACAG + n.d.
52 CAATTTCTAAGAAAGGA ++ n.d.
56 TGCTTCTCAGAACGT ++ ++
58 TGCTTCCCCGAACGT ++ n.d.
n.d.= not d~t.,.
DOCKET NO. 016-0030.WO
29 2191 ~9 Table 2: Relative EMSA binding affinities for a series of regulatory elements ofdouble stranded ~ L~ula~iOllj differing in fianking and spacing sequences to ~ regulatory proteins activated in response to the cytokines IL-3, GM-CSF, G-CSF.
GM-Epo ~3 G-CSF G-CSF
SEQ ID No. CSF (comple~ 1) (comple~ 2) 36 ++ +
38 +++ +++
+ + ++ n.d. n.d.
~--42 + n.d. + n.d. rl.d.
44 ++ n.d. + n.d. n.d.
46 ++ + ++ ++ ++
48 + + ++ n.d. n.d.
+ n.d. + n.d. n.d.
52 ++ +++ +++ n.d. n.d.
54 - n.d. - +++
56 ++ + ++ ++
n.d.= not determined ~- - The data im Table I show that the IL-4- and IL-13-activated STAT
complexes cam bimd to all of the Gsted sequences of general structure Tl~T6AA with ~0 similar aff nity (with the exception of SEQ ID NO. 18, which was slightly lower in affinity). The ~4 and IL-13-activated STAT complexes can also bind to all ofthe hsted sequences of general structure TTNsAA with varying affinities.
A specific example of the data ~"" ,., IAI; ~ -'i in Table I can be seen with respect to the EMSA autoradiograms of FIGS. IA and IB In panel IA" A ii~
15 double-stranded ~ probes made by annealing the ~ c of SEQ
ID NOs. 14-23 were used. Lanes marked '~N7 represent c~ using extracts from untreated cells. Other lanes are marked according to the inducin=, cytokineActivated complexes can be identified by their absence in untreated eA~racts and their DOCKET NO. 016-OOiO.WO
2~ql ~89 presence m extracts treated by cytokine. The STAT complexes activated by IL4 andlL-13 bound to all of the ~ P p}obes with similar affinities (except SEQ ~
NO. 18, which bound with a slightly lower affnity), as can be seen m Lanes 2, 3, 5, 6, 8, 9, Il, 12, 14 and 15 of panel IA In parlel IB",~ A, double-st}anded ~ .rlr~ lc probes made my annealing ~ ,.. IrUl ;~lf; of SEQ ID NOs. 24-35 were used. Lanes marked 'UN' represent ~ usmg extracts from untreated cells. Other larles are marked according to the irlducing cytokine. Activated complexes carl be identified by their absence in untreated extracts and their presence in extracts - treated by cytokine. The STAT complexes activated by lL-4 and IL-13 bound to all of 10 the r l;~. ,.. ,~1.. ~1 ;~r probes with similar afr~nities, as can be seen in Lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17 and 18 of panel IB.
The data in Table 2 show that the STAT complexes activated by GM-CSF, Epo, IL-~ and G-CSF can bind to a variety of DNA sequences of geQeral structure TTNsAA with varying aftinities. rn NFS-60 cells, GCSF activated two STAT
comple.Yes that were, ~ . .;cl .~ by their differing mobilities in arl EMSA. Theslower-migrating complex (Complex 1) cornigrated with the STAT3 homodimer stimulated by LL-6 and was shown to contain STAT3 by antibody supershift ~A,u~
using a specific STAT3 antiserum (available from Upstate l~ j~tPrhnr lr oy Ll~,u~ul ~td, New York), and would selectively bind to a DNA sequence with a TTN4AA structure ` ~ 2û ~e.g., SEQ ID NO. 54). The faster-migratirlg complex (Complex 2) contained an .;.1 .l;l~ ;1 STAT complex that migrated like the STAT complexes activated by IL-3 and GM-CSF. The two G-CSF-activated complexes had markedly different affinities for some ofthe regulatûry elements (e.g. SEQID 54 vs SEQID 56).
Transient Trnnsfection Assal~s The reporter plasmids SEQID14x4-TK-LUC, SEQID 16x4-TK-LUC, SE~ID18x4-TK-LUC, SEQLD2ûx4-TK-LUC, SEQID22x4-lX-LUC, SEQL~24x4-TK-LUC, SEQL~26x4-TK-LUC, SEQlD28x4-TK-LUC, SEQlD3ûx4-lK-LUC, SEQlD32x4-TK-LUC, SEQ~34x4-TK-LUC, SEQID36x4-TK-LUC, SEQlD38x4-TK-LUC, SEQID40x4-TK-LUC, SEQ~42x4-TK-LUC, SEQlI)44x4-TK-LUC, DOCKE~NO. 016-0030.WO
2191 ~9 SEQID46x4-TK-LUC, SEQID48x4-TK-LUC, SEQID50x4-TK-LUC, SEQ~52x4-TK-LUC, SEQID52x6-TK-LUC. SEQID54x4-T~-LUC, SEQID56x4-TK-LUC, and SEQID58x4-TK-LUC contain four copies (or six copies for SEQID52x6-TK-LUC) of the same SEQ ID NOs 14-58 as those used m the EMSA's, linked to the promoter of the 5 Herpes Simplex virus thymidine kinase gene at position -35 with respect to the cap site.
See FIG. 1. The reference reporter, l~-LUC (P. Lamb et al., 83 Blood 2063 (1994)), the disclosure of which is herein Ul~.Ul l)UI dL~:d by reference, is the parent vector that contains no response element. These chimeric promoters drive the expression of the ,~ structural gene for firerdy luciferase.
ME-I 80 cells were transfected with the reporter plasmids of above by calcium phosphate Cu~ d~iOIl. Cells were seeded at 1-4X105/ml the day before .r~ 11 Cells were exposed to a calcium phosphate precipitate containing the above reporter plasmids (10-20 llglml) and the ~ -C~ aill~ plasmid pCHl 10 (5 llg/ml, Cul~ .; lI,y available from Pharmacia Biotech, Piscataway, NJ) for 15 IZ h. The medium was then changed and the cells allowed to recover for 16-18 h.
~ -nmhinAnt cytokines were then added prediluted in growth medium and the cellsharvested after 6 h. Cells were Iysed and luciferase and l3~ AI~ IrISj~ - activities determined using standard techniques. See, e.~. J. R De Wet et al., 7 Mol. CeU. Biol.
725 (1987) and S_mbrook et al., Molecular ('.llmir~g A Laboratorv Manual 2nd ed., ~ 20 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). For each sample the normalized response was determined by dividing light units obtamed from the luciferase assay with the ~ SAt~ if tA`r- activity in the same Iysate as detern~ined using a ~L. UlllO~ substrate. The results of these l l A 11~ are shown below in Table 3 .
Numbers given are the mean fold inductions ~A-fold induction' is defined as the normalized 25 response in a cytokine-treated sample divided by the normalized response in an untreated sample). The value in parentheses is the number of i" t l, ,rt, ,l c.~ a included to calculate the mean.
TF-l cells were transfected with the reporter plasmids of above by the DEAE-dextran method as described (J. Suzow and A.D. Friedman, 13 Mol. Cell. ~iol.
2141 (1993)) with the following ".. ~ test reporter constructs we~e added to a DOCKE-l NO. 016-0030.WO
of 3 .~glmL during the trAn~rtinn~ pMsvcAT yector was not added to the L~ art~.Liùll rnixtures, the growth medium used was as described above for TF-1 cells, and cytokine inductions were carried out for 4-5 h. Cells were Iysed and luciferase activity deterrrined using standard techniques. T~ r~ were performed in a batch,S and identical numbers of transfected cells were then separately induced with cytokine for the 4-5 h induction period. The results of these L~ rr~ are shown below in Table4. Numbers given are the mean fold i~ductions ~Afold induction' for TF-1 l, O~r~- l;ol~ is defined as the luciferase response in a cytokine-treated sample divided by the lucrferase 5 ~ response in an untreated sample). The value in ~ .ILIle~ is the number of 10 ;"~ I ` included to calculate the mean.
NFS-60 cells were transfected with the reporter plasmids described above by the DEAE-dextran method as described in the preceding para~raph with the following .~ test reporter constructs were added to a ~,UllCcllLl4Liull of 6 ,ug/mL during the Ll~art~_iiull, and cytokine inductions were carried out for 2.5h. The 15 results of these l . A~ ~rr~ are shown below in Table 5.
Stable Transfection of NFS-60 Cells Stable Ll Allarr,~,Liull of NFS-60 cells was A- ~ U' ~ If ~l as described by H. Pahl et al., 19 Exp. Hematol.. 1038-1041 (1991). In brief, 1.4 x 107 factor~ ..J~ 'FS-60cells were washed twice with RPMI-1640 and then ~calla~ ded in RPMI-1640 (0.5 ~~ 20 mL); linearized reporter DNA (16 llg) and EcoRI-digestcd pSV2NEO plasmid (4 llg, Cu~ ly available from ATCC) were then added. Cells and plasmids were incubated for 5 min at RT in a 0.4 cm ~ 1IU~UIdLiUI~ cuvette (Bio-Rad), then subjected to a 330V, 960 ~F pulse, using a Bio-Rad Gene Pulser. Cells were il~ ,diALtly incubated on ice for 15 min, then placed in normal growth medium (10 mL). Two days later, G418 (300 ~Lg/mL, Boehringer-Mannheim, Illdi~di)olia, IN) was added to the culture. Stablely transfected cells were cloned by limiting dilution. Al.yluAilllALely 400 clones were screened for G-CSF-inducible luciferase actiYit,Y, and 16 positive clones were identified and .,Il,_~.Lt~ further. The results for four of the positiYe clones are . d in Table 6. The number of; ~ . ..l CA~)t:lilll~,.l~ included to calculate0 the mearl is indicated iri y~ c~ ,scs.
DOCKET~NO. 016-0030.WO
~ ~ 219~189 Table 3: T, ~ iU~ induction in ME-I80 cells of reporter constructs ,UI~)UI clLlllg multiple copies of test STAT regulatory elements. The values given are mean fold inductions in response to the irldicated cytokine. The value in the p~LlCll~ .S~S is the number of S included to calculate the mean.
Reporter CoreElement IL 4 IL-13 TK-LUC none û.9 (3) I.û (3) SEQ~14x4TK-LUC CACTTCCCAAGAACAG 22 (3) 9.7 (3) SEQD~16x4TK-LUC TGCTTCCCCGGAACGT 1.3 (3) I.û (3) SEQn~18x4TK-LUC TGCTTCCCCAGAACGT 1.2 (3) 1.1 (3) SEQrD20x4TK-LUC TGCTTCCCAAGAACGT 1.5 (3) 1.2 (3) SEQII)22~s4TK-LUC CACTTCCCCGGAACAG 2.7 (3) 1.6 (3) SEQrD24x4TK-LUC CACTICCCCAGAACAG 8.0 (3) 3.0 (3) SEQrD26x4TK-LUC CACTTCCCAGGAACAG lû (3) 6.4 (3) SEQrD28x4TK-LUC TACTTCCCAAGAACAT 3.0 (3) 1.5 (3) SEQID30x4TK-LUC CGCTTCCCAAGAACGG 1.5 (3) 1.3 (3) SEQrD32x4TK-LUC CACTTCTTAAGAACAG 7.3 (3) 3.3 (3) SEQlD34x4TK-LUC CACTTTCCA.~GAACAG 1.7 (3) 1. I (3) SEQrD36x4TK-LUC TGCTTCCCGGAACGT I . I (3) n.d.
SEQrD38x4TK-LUC GATTTCCCCGAAATG 0.8 (3) n.d.
SEQn~40x4TK-LUC ATATTCCTGTAAGTG 1.2 (3) n.d.
SEQrD44x4TK-LUC CATTTCTG&AAATG 1.1 (3) n.d.
SEQrD46x4TK-LUC CATTTCCCGTAAATC 1.0 (3) n.d.
SEQD~48x4TK-LUC ATATTACCAGAAATG 12 (3) n.d.
SEQrD50x4TK-LUC ATTTTCCAGTAACAG 1.0 (3) n.d.
SEQrD52x4TK-LUC CAATTTCTAAGAAAGGA 0.8 (3) n.d.
SEQrD56x4TK-LUC TGCTTCTCAGAACGT 1.7 (3) n.d.
SEQlD58x4TK-LUC TGCTTCCCCGAACGT 1.2 (3) n.d.
n.d.= not d~t~
21911~9 Table 4 T ~ 1 induction in TF-I cells of reporter constructs ~ICul yOI ~Lilg mu~tiple copies of test STAT regulatory elements The values given are mean fold i~ductions in response to the indicated cytokine The value in the ~-,~1l.,~ is the number of l ,~1 ; ,t~
included to calculate the mean Reporter 11,4 GM-CSF Epo ~3 TK-LUC 0 7 (2) 0 8 (2) 0 8 a) û 7 a) SEQID36x4TK-LUC n.d. 3 4 (2) 18 (2) 2 8 (2) SEQID38x4TK-LUC 14 (2) 9 6 (4) 3 1 (4) 6 8 (3) - SEQID40x4TK-LUC n.d. n.d. 12 (2) 13 (2 SEQID42x4TK-LUC n.d. n.d. 10 (2) 12 (2 SEQID44x4TK-LUC n.d. n.d. 18 (3) 0 9 SEQID46x4TK-LUC n.d. n.d. 0 8 (2) 0 9 (2 SEQID48x4TK-LUC n.d. n.d. , 12 (2) 1 I (2) SEQID50x4TK-LUC n.d. n.d. 15 (2) 3 1 (2) SEQlD52x6TK-LUC n.d. 7 6 (2) 3 5 (2) 7 3 (2 SEQlD14x4TK-LUC 3 3 (3) 13 (2) 0 8 (2) 13 (2 n.d.= not d~t~A ' ,-?:._ 10 Table 5 T, . ~ 1 induction in NFS-60 cells of reporter constructs UI LJUI dLillg multiple copies of test STAT regulator,v elerllents The values given are mean fold inductions in response to the indicated cytol~ne The value in the pcu t~lth~ .s is the number of ~ 1, ,", , included to calculate the mean Reporter Core Element G-CSF ~3 IL,6 SEQID54x4TK-LUC TTCCCGAA 14 (2) 10 (V 41 (2) SEQID36x4TK-LUC TTCCCGGAA 24 (2) 3 5 (2) 4 8 (2) SEQID56x4TK-LUC TTCTCAGAA 3 1 (2) 14 (2) 17 (2) SEQlD38x4TK-LUC TTCCCCGAA 24 (2) 61 (2) 6 9 (2) DOCKET NO. 016-0030.WO
~ ~ 2191189 Table 6: TIA~IC~ AI induction in NFS-60 cells stablely transfected with the SEQID38x4TK-LUC reporter plasmid. The values given are mean fold inductions in response to the indicated cytokine. The value in the ~,~cl~L~ ses is the number of ~ included to calcuiate the 5 mean.
Clone G-CSF ~3 I~6 r l ~.
IEII 17.2 (3) 16.5 (3) 3.4 (3) 6G8 17.3 (3) 20.6 (3) 3.6 (3) IB10 16.8 (3) 27.6 (3) 2.7 (3) 4C2 12.8(3) 20.1 (3) 2.3 (3) The data ~Ill,,.r.A. ,~d in Table 3 when compared to the in vitro binding 10 data described above clearly d~ ollOlldLe that in vitro binding is not predictive of Ll f..~c.il,~ivl.dl activity. Thus, all of the DNA elements that were il.~ Ltd as multimers into the reporter vectors bound the STAT complexes activated by lL4 and IL-13 with a similar affinity; however, oUI1.1lioill~l~ not all could mediate ~
induction in response to lL-4 and lL-13 (defined as greater than a 2-fold induction).
- ~ 15 Although it has been reported that a sequence element found in the promoter of the FcRIIb gene (SEQID52) is necessary for the IL4 It~/O~ o:l ofthis gene and can bind the STAT complex activated by IL4 in vitro (I. Kohler et al., 345 FEBS Lett. 187 (1994)), it is clear from the data in Table 3 (SEQID52x4TK-LUC entry) that this element is not sufticient on its own to mediate IL-4 I~ e~ further "...~. . 5f ~20 the .l ~ .,... lf l ;...l between in vitro binding data and functional, l . A ,~ u~Al activity The data 5111~ I in Table 4 when compared to the in vitro binding data described above again clearly d~ that in vitro binding cannot be relied on to be predictive of ~ AI activity. Thus, many of the DNA elements that were ih~c~ oldL~d as multimers into the reporter vectors bound the STAT complexes 25 activated by IL4, GM-CSF, Epo and ~-3 with v~ing affnities (Table 2); however, DOCKET NO. 016-00~0.WO
2~ 8~
most could not mediate a ~ induction in response to these c-ytokines (defined as greater than a 2-fold induction).
The data ~"".., - ;,. .l in Table S again show that in vitro binding data do notreliablycorrelatewiththeabilityoftheelementstomediateall""~. .;1,1;.~.~,.1 5 induction. As described above, G-CSF activates two complexes in NFS-60 cells, one containing STAT3 and one containing an ~ STAT protein resembling the complex activated by IL-3 in NFS-60 cells. Response elements that could bind to both G-CSF-activated complexes, such as SEQID38 and SEQID36, mediated a strong . . ,~ l induction in response to G-CSF, and the response element that bound ~ ~ 10 strongly only to the STAT3-containing, G-CSF-activated complex (SEQ~54) was activated by G-CSF (though not quite as strongly as were the sequences that bound both complexes). However, ~ul~Jliaiul~ly, the response element that bound only the IL-3-activated complex (SEQID56) was not activated in response to IL-3 (def~ned as less than a 2-fold induction) and was only weaklyr activated by G-CSF.
The data ~.. ,,~1~, ;,. ~1 in Table 6 show that the NFS-60 clones stablely transfected with SEQID38x4TK-LUC respond well to both G-CSF, IL-3 and IL-6 (though the response to IL-6 was slightly lower than what was obtained in the transient Also, compared to the transiently transfected NFS-60 cells, the stable NFS-60 clones appear to respond more robustly to IL-3. N~ , in general, the 20 transient ~. ~. ,~ data are a good indicator of what to expect when the reporter is stablely tr_nsfected into the NFS-60 cells.
It has previously been reported that many cytokines, including IL-3, GM-CSF, Epo, aCSF, IL~ and IL-13 activate STAT or STAT-like complexes that bind to DNA sequence elements related to the GAS element that was first .1"., ". I rl 1 ~ in the 25 promoters of IFNy-responsive genes. The data in Example I .,~ ,lu ,;~ ~,Iy show that, ~u~ , in vitro binding is not predictive of L.~.~ ,L;ol~cl activation for the cytokines IL-3, IL4, IL-13, GM-CSF, G-CSF and Epo. One can certainly not therefore assume that the in vitro STAT complex binding observed in previously published work is directly ~. c.~lcL~l~ into a functional reporter assay. To date there has been no 30 reported d~ ollaLI CLi.,.l that the DNA sequences reported to bind to the STAT or . ~
DOCKE~T NO 016-OOiO WO . . - -2 ~ 9 STAT-lilce compleYes activated by IL-3, IL ~1, L-13, GM-CSF, G-CSF or Epo can mediate ~ 1 induction in response to those cytokines~ Because it is not possible to eYtrapolate from in vitro binding data that a g,iven sequence will be functional, the d "" ~ of functional activity such as that shown in the eYample 5 above is absolutely critical.
While in accordance with the patent statutes, description of the preferred weight fractions, and processing conditions have been provided, the scope of theinvention is not to be limited thereto or thereby. Various ~ and alterations of the present invention will be apparent to those skilled in the art without departing ` ~ 10 from the scope and spirit of the present invention.
C~ y, for an u~ , of the scope of the present invention, reference is made to the following claims Pa~. Docket No. 016-0030.W0 ~ 2~91 1~9 SEQUE~CE LI-SI~G
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(D) STATE: CA
(E) COUNl RY: US
(Ii) POSTAL CODE (ZrP): 92121 (G) TELEPHONE: (619) 550-7675 (H) TELEFAX: (619) 535-3906 (ii) TITLE OF INV~TION: METHODS AND ASSOCIATED REAGENTS FOR
DETECTING MODULATORS OF CYTOKINE ACTION
~iu) NUMBER OF SEQUENCES: 59 v) COMPUTER READAr'LE FOR~
(A) MEDIUM TYPE: Floppy disk (B) COl~PUTER: IBM PC cornpatible ~C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentrn Release "1.0, Version ~1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
30 (i) SEQUENCE CHARACTERISTIC
. ~ (A) LENGTH: 9 base pairs S.
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
40 (xi) SEQUENCE DESCR~PTION: SEQ ID NO: 1:
TTCNN~.GAA 9 Pat` Docket No. 016-0030.WO
2/23 2 1 9 ~ 1 89 (2) INFORMATION FOR SEQ ID NO: 2:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A)DESCRIPTION: Idesc="OTHERNUCLEICAClD, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
--~ 15 TTCNNNNGAA 10 (2) INFORMATION FOR SEQ ID NO: 3:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE mE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTE~TIC DNA"
30 (~i) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
1._ . .
(2) INFORMATION FOR SEQ ID NO: 4:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNl~ETIC DNA"
, . . .. . . . .
Pat` Docket No. 016-0~30.WO
3/23 2~ 91 1 ~9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~i) MOLECULE TYPE: other nucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICACID
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) rNFOR~rATION FOR SEQ ID NO: 6:
25 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~) SYNTHETIC DNA"
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
40 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid 45 (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pa~. Docket No. 016-0030.WO
2191 ~89 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
5 (xi) SEQUI~NCE DESCRIPTION: SEQ ID NO: 7:
(2) INFORMATION FOR SEQ lD NO: 8:
(i) SEQUENCE CHARACTERISTICS:
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(2) ~FOR~IATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Pat.~Docket No. 016-0030.WO
5/23 2191 ~89 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs S (B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE mE: other ~ucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICACID
SYNTHETIC DNA"
(xi) SEQUENOE DESCR~TION: SEQ ID NO: 10:
(2) INFORMATION FOR SEQ ID NO: 11:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinole 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRrPTION: Idesc = ~OTHER NUCLEIC ACID, SYNTHETIC DNA"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
35 (2) ~IFORMATION FOR SEQ ID NO: 12:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs (B) TYPE: nucleic acid 40 (C) STRANDEDNESS: sin_le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~
SYNTHETIC DNA"
Pat Docket No. 01~-0030.WO
6/23 21 9~ 1 ~9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
TTCTAAGAA g (2) INFORMATION FOR SEQ ID NO: 13:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: other nucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICAClD
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
(2) INFORMATION FOR SEQ ID NO: 14:
25 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pai~s (B) TYPE: nucleic acid (C) STRAN.DEDNESS: single (D) TOPOLOGY: linear t. ~ ~Li) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~, SYNTHETIC DNA"
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
(2) ~FORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Par. Docket No. 01-6-0030.~0 7l23 2 7 9 ~
i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
5 (~) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
10 (2) INFORMATION FOR SEQ ID NO: 16:
Cl) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~: 15 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /de; c = "OTHER NUCLEIC ACID, SY~THETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
(2) lNFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~li) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Pat~ Doclcet No. 016-0030~WO
. ~ .
8l23 2 ~ 9 ~
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 20 base pairs 5 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
~.
(2) INFORMATION FOR SEQ ID NO: 19:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE. other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AClD
SYNTHETIC DNA"
30 (Xl~ SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GATCACGTTC TG&GGAAGCA 20 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
PaC. DocketNo. 016-0030.WO
9/23 2~91 189 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
(2) LNFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear _ (ii) MOLECULE TYPE: other nucleic acid .. 15 (A)DESCRIPTION: /desc="OTHERNUCLEICACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
r ('') INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE C~IARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear ~,. (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~D, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat. DocketNo. 016-0030.WO
-- 10/23 21qll89 (u) MOLECULE TYPE: other nucleic acid (A) DESCRrPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRlPTION: SEQ ID NO: 23:
10 (2) ~FORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs ~a (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTE~R NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
= 30 (A) LENGTH: 21 base pairs ~_- (B) TYPE. nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ lD NO: 25:
Pat. Docket No. 016-0030.WO
11/23 2~91~89 ~2) lNFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) MOLECULE TYPE: other nucleic acid 10 (A) DESCRIPTION: /desc = ~OTHERNUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENOE DESCRIPTION: SEQ ID NO: 26:
~3' ~_'.T 15 GATCCACTTC CCAGGAACAG A 21 (2) INFORMATION FOR SEQ ID NO: 27:
20 (i) SEQUENCE CHARACTER~STICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
,, 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
35 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid 40 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc= "OTHER~IJCLEIC ACID
S~IIC DNA"
Pat: Docket No. 016-0030.WO
2191 ~8~
(xi) SEQUENOE DESCRIPTION: SEQ ID NO: 28:
(2) lNFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 21 base pairs ~) mE: nucleic acid (C) STRANDEDNESS: single . (D) TOPOLOGY: linear , r (ii) MOLECULE mE: other nucleic acid ~: - 15 (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
(2) INFORMATION FOR SEQ ID NO: 30:
25 (i) SEQUENCE CHARACTERTSTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~-~~ (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
(2) INFORMATION FOR SEQ ~ NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat`DocketNo. 016-0030.WO
(ii) MOLECULE TYPE: other nucleic acid (A) DESCF~IPTION: Idesc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ lD NO: 31:
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs _ (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Gnear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = nOTHER NUCLEIC ACID, SYl~HETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEO ID NO: 32:
(Z) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTE~TIC DNA"
Pat`DocketNo. 016-0030.WO
2 1 9 1 1 8q (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
(2) INFORMATION FOR SEQ lD NO: 34:
(~) SEQUENCE CHARACTERISTICS:
(A) LENGlX: 21 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ; (A) DESCRIPTION: Idesc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
(2) INFORMATION FOR SEQ ~ NO: 35:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRA.~DEDNESS: sin~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid f -' (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTEI: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat. Docket No. 016-0030.WO
i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
10 (V ~FORMATION FOR SEQ ID NO: 37:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid 15 (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SY~THETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
(2) INFORMATION FOR SEQ ~) NO: 38:
(i) SEQUENCE CHARACTERISTICS:
30 (A) LENGTH: 19 base pairs - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SE ID NO: 38:
Q
Pat. Docket No. 016-0030.WO
21 9~ 1 89 (2) ~FORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRAMDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 10 (A) DESCRIPTION: /desc = "OTHERNUCIEIC ACID, SYNTHETIC DNA"
(xi) SEQllENCE DESCRIPTION: SEQ ID NO: 3g:
'~- ~ 15 GATCCATTTC GGGGAAATC 19 (2) ~FORMATION FOR SEQ ID NO: 40:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLE~IC ACID, SYNTHETIC DN~"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
35 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lg base pairs (13) TYPE: nucleic acid 40 (C~ STRANDEDNESS: sin~le (D) TOPOLOGY: linear ~ui) MOLECULE TYPE: other rlucleic acid (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID, SYNTHETIC DNA"
Pat'Docket No. 016-0030.WO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
(2) INFORMATION FOR SEQ lD NO: 42:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) MOLECULE mE: other nucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICAClD, SYNTHETIC DNA"
(xi) SEQUENCE DESCRrPTION: SEQ ID NO: 42:
(2) INFOR~IATION FOR SEQ lD NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) T~PE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .~ (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
(2) lNFORMATION FOR SEQ ID ~O: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairS
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat.~DocketNo. 016-00~0.WO
2' 91 ~ ~9 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTE~R NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ~ NO: 44:
(2) INFORMATION FOR SEQ ID NO: 45:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs , tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTEIETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
(2) INFORMATION FOR SEQ ID NO: 46:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH 19 base pairs (B) TYPE: nudeic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other ~ucleic acid (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYN~HETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Pat. Docket No. 016-0030.WO - -l9/23 2 1 9 1 1 8 9 (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHERNUCLEIC ACID
SYNTHETIC DNAN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
(2) INFORMATION FOR SEQ ID NO: 48:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs ~B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GATCATATTA CCAGAAATG l9 (2) ~FORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGl~I: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
Pat` Docket No. 016-0030.WO
2~91 '~9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ~: 15 (A)DESCRrPTION: /desc="OTHERNUCLEICACID, SYNTEIETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
(2) INFORMATION FOR SEQ lD NO: 51:
25 (i) SEQUENCE CIIARACTERlSTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
35 (xi) SEQUENCEDESCRIPTION: SEQIDNO: 51:
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 21 base pairs (~3) TYPE: nuc~eic acid (C) STRANDEDNESS: single (D) TOPOLOGY: lin~r Pa~. Docket No. 016-0030.WO
21/23 219~189 (ii) MOLEC~LE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
10 (2) INFORMATION FOR SEQ ID NO: 53:
) SEQUENCE CEIARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid t,_" 1s (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
20 . SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ lD NO: 53:
(2) INFORMATION FOR SEQ ID NO: 54:
~I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~li) MOLECU~E TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Pat. Oocket No. 01~-003~.WO
2V23 2~91 18q (2) ~FORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A)DESCr~lPTION: /desc="OTHERNUCr,EICACrD
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
t~ 15 GATCACGTTC GGGAAGCA 18 (2) rNFORMATION FOR SEQ ~ NO: 56:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OT~R NUCLEIC ACID
SYNTHETIC DNA"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
(2) INFORMATION FOR SEQ ID NO: 57:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~
SYNTHETIC DNA"
, . . . . . . .
. Pat. Docket No. 016-00~0.WO
. ~ .
23/23 2 ~ 9 1 ~ ~9 (xi) SEQllENCE DESCRIPTION: SEQ ID NO: 57:
(2) ~IFORMATION FOR SEQ ID ~O: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear IOLECULE TYPE: other nucleic acid ~- .. 15 (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
(~) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
(2? INFORMATION FOR SEQ ID NO: 59:
25 (i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~- (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
3'- TATAAGGACATTCACCTAG -5' (SEQ ID NO. 41) 5'-GATCATATTCCCGTAAGTG -3' (SEQ ID NO. 42) 3'- TATAAGGGCATTCACCTAG -5' (SEQ ID NO. 43) 5'-GATCCATTTCTGGAAATG -3' (SEO~ ID NO. 44) 3'- GTAAAGACCTTTACCTAG -5' (SEQ ID NO. 45) 5'-GATCCATTTCCCGTAAATC -3' (SEQ ID NO. 46) 10 3'- GTAAAGGGCATTTAGGATC -5' (SEQ ID NO. 47) 5'-GATCATATTACCAGAAATG -3' (SEQ ID NO. 48) 3'- TATAATGGTCTTTACCTAG . -5' (SEQ ID NO. 49) 15 5'-GATCATTTTCCAGTAACAG -3' (SEQ IDNO. 50) 3'- TAAAAGGTCATTGTCCTAG -5' (SEQ ID NO. 51) 5'-GATCCAATTTCTAAGAAAGGA -3' (SEQ ID NO. 52) 3'- GTTAAAGATTCTTTCCTCTAG -5' (SEQ ID NO. 53) 5'-GATCTGCTTCCCGAACGT -3' (SEQ ID NO. 54) 3'- ACGAAGGGCTTGCACTAG -5' (SEQ ID NO. 55) 5'-GATCTGCTTCTCAGAACGT -3' (SEQ ID NO. 56) 25 3'- ACGAAGAGTCTTGCACTAG -5' (SEQ IDNO. 57) 5'-GATCTGCTTCCCCGAACGT -3' (SEQ ID NO. 58) 3'- ACGAAGGGGCTTGCACTAG -5' (SEQ ID NO. 59) 30 where the nucleotide sequences shown in bold type fa~e correspond to nucleotide sequences, including their double-stranded c...,.l,l..,...,l, tested for activity as regulatory elements according to the present invention.
The annealed ~-~l s ~ l;u~ were labeled by filling in the O~ a,.~llg ends with Klenow fragment (Boehringer Mannheim) in the presence of [a-32P]-dGTP
35 and/or [a-32P]-dATP (Amersham Corporation, Arlington Heights, IL). El._~,tlu~llu.~;c mobility shift assays (EMSA's) were performed in HEPES buffer (13 mM, pH 7.6, Sigma Chemical, St. Louis, MO), containing sodium chloride (80 rnM), sodium fiuoride (3mM), sodium molybdate (3mM), DTT (ImM), EDTA (0.15mM), EGTA (0.15rnM), glycerol (8% v/v, including c~ from the nuclear extract), poly d(I-C) poly d(I-40 C) (75,ug/mL)",..l,"l l.. IP.J probe (~lu~ill~tly 0.2ng) and nuclear extract containing DOCKETNO. 016-0030.WO
21 9 1 1 8~
5-10~Lg of total protein. Reactions were incubated at room Lt.ll~,ld~W~ for 20 minutes then resolved on 5% pOlya~ ldl~ldt gels containing 0.25X TBE [IX TBE is Tris borate (89 mM), pH 8.0 containing EDTA (I mM)] and glycerol (5% v/v). Gels were run at 4C in 0.25X TBE at 20V/cm, then dried and auLu-ddi~_. ' 1.
Relative binding affinities, as determined from the EMSA results for J.J...,. I. u~ r SEQ lD NOs 14-59, were visually rated and assigned according to the following scale:
(-) band ~.~)ll C:~JUlldill~8 to specific complex on the EMSA d' ~ r ~ I dlU (See e.g., FIG. lA, lane 7) barely discernible or not discernible.
(+) band Cullt*lù~dlllg to specific complex on the ElMSA dULOlddio~.a.~. (See e.g., FIG. lA~ lanes 8 and 9) easily discernible but of weak intensity.
(++) band ~,~?llt:~lJulld;llg to specific complex on the EMSA ~ " d~ 8, dlll (See e.g., FIG. lA, lanes S and 6) easily discernible and of moderate intensity.
15 (+~+) band CG-It~lJOlldlllg to specific compleY on the EMSA q~-t~r~qs~ gram (See e.g., FIG. IA, lanes 7 and 3) easily discernible and of strong intensity This visual rating system is sufficient to analyze ~ v~
differences and trends in the EMSA bmding data as opposed to specific numerical ~- - 20 values. If desired~ the use of a phosphor imager or d ,~ (colll~ / aVailable from e.g., Bio-Rad T qborqt~ri~) cûuld provide â means to assess the differencesdescribed here ~ua~LiLdLi~'J. Specific visual ratings of binding affinities for the regulatory elements of ~ . . ,., l ul ;~ir, SEQ ID NOs 14-41, 44-53 and 56-59 are shown in Table I below. Specific visual ratings of binding aftinities for the regulatory elements of oli~ul,u~ ùLid~ SEQ n~ NOs 36-57 are shown in Table 2 below.
DOCN~TNO. 016-0030.WO
. ~ 21911~9 Table 1: Relative EMSA binding affinities for a series of regulatory elements of double stranded ~ differing in fiar~ng and spacing sequences to 1 ~ r- ~ regulatory proteins activated in responSe to the cytokines L4 and IL-13 i~ U-937 or ME-180 cells.
SEQ Core 1~
ID Element IL,4 IL-13 14 CACTTCCCAAGAACAGA +++ +++
16 TGCTTCCCCGGAACGT ++ ++
18 TGCTTCCCCAGAACGT + +
~~20 TGCTTCCCAAGAACGT ++ ++
22, CACTTCCCCGGAACAGA ++ ++
24 CACTTCCCCAGAACAGA ++ ++
26 CACTTCCCAGGAACAGA ++ ++
28 TACTTCCCAAGAACATA ++ ++
30 CGCTTCCCAAGAACGGA ++ ++
32 CACTTCTTAAGAACAGA ++ ++
34 CACTTTCCAAGAACAGA ++ ++
36 TGCTTCCCGGAACGT ++ ++
38 GATTTCCCCGAAATG ++ ++
ATATTCCTGTAAGTG + n.d.
44 CATTTCTGGAAATG ++ n.d.
46 CATTTCCCGTAAATC ++ n.d.
48 ATATTACCAGAAATG + n.d.
ATTTTCCAGTAACAG + n.d.
52 CAATTTCTAAGAAAGGA ++ n.d.
56 TGCTTCTCAGAACGT ++ ++
58 TGCTTCCCCGAACGT ++ n.d.
n.d.= not d~t.,.
DOCKET NO. 016-0030.WO
29 2191 ~9 Table 2: Relative EMSA binding affinities for a series of regulatory elements ofdouble stranded ~ L~ula~iOllj differing in fianking and spacing sequences to ~ regulatory proteins activated in response to the cytokines IL-3, GM-CSF, G-CSF.
GM-Epo ~3 G-CSF G-CSF
SEQ ID No. CSF (comple~ 1) (comple~ 2) 36 ++ +
38 +++ +++
+ + ++ n.d. n.d.
~--42 + n.d. + n.d. rl.d.
44 ++ n.d. + n.d. n.d.
46 ++ + ++ ++ ++
48 + + ++ n.d. n.d.
+ n.d. + n.d. n.d.
52 ++ +++ +++ n.d. n.d.
54 - n.d. - +++
56 ++ + ++ ++
n.d.= not determined ~- - The data im Table I show that the IL-4- and IL-13-activated STAT
complexes cam bimd to all of the Gsted sequences of general structure Tl~T6AA with ~0 similar aff nity (with the exception of SEQ ID NO. 18, which was slightly lower in affinity). The ~4 and IL-13-activated STAT complexes can also bind to all ofthe hsted sequences of general structure TTNsAA with varying affinities.
A specific example of the data ~"" ,., IAI; ~ -'i in Table I can be seen with respect to the EMSA autoradiograms of FIGS. IA and IB In panel IA" A ii~
15 double-stranded ~ probes made by annealing the ~ c of SEQ
ID NOs. 14-23 were used. Lanes marked '~N7 represent c~ using extracts from untreated cells. Other lanes are marked according to the inducin=, cytokineActivated complexes can be identified by their absence in untreated eA~racts and their DOCKET NO. 016-OOiO.WO
2~ql ~89 presence m extracts treated by cytokine. The STAT complexes activated by IL4 andlL-13 bound to all of the ~ P p}obes with similar affinities (except SEQ ~
NO. 18, which bound with a slightly lower affnity), as can be seen m Lanes 2, 3, 5, 6, 8, 9, Il, 12, 14 and 15 of panel IA In parlel IB",~ A, double-st}anded ~ .rlr~ lc probes made my annealing ~ ,.. IrUl ;~lf; of SEQ ID NOs. 24-35 were used. Lanes marked 'UN' represent ~ usmg extracts from untreated cells. Other larles are marked according to the irlducing cytokine. Activated complexes carl be identified by their absence in untreated extracts and their presence in extracts - treated by cytokine. The STAT complexes activated by lL-4 and IL-13 bound to all of 10 the r l;~. ,.. ,~1.. ~1 ;~r probes with similar afr~nities, as can be seen in Lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17 and 18 of panel IB.
The data in Table 2 show that the STAT complexes activated by GM-CSF, Epo, IL-~ and G-CSF can bind to a variety of DNA sequences of geQeral structure TTNsAA with varying aftinities. rn NFS-60 cells, GCSF activated two STAT
comple.Yes that were, ~ . .;cl .~ by their differing mobilities in arl EMSA. Theslower-migrating complex (Complex 1) cornigrated with the STAT3 homodimer stimulated by LL-6 and was shown to contain STAT3 by antibody supershift ~A,u~
using a specific STAT3 antiserum (available from Upstate l~ j~tPrhnr lr oy Ll~,u~ul ~td, New York), and would selectively bind to a DNA sequence with a TTN4AA structure ` ~ 2û ~e.g., SEQ ID NO. 54). The faster-migratirlg complex (Complex 2) contained an .;.1 .l;l~ ;1 STAT complex that migrated like the STAT complexes activated by IL-3 and GM-CSF. The two G-CSF-activated complexes had markedly different affinities for some ofthe regulatûry elements (e.g. SEQID 54 vs SEQID 56).
Transient Trnnsfection Assal~s The reporter plasmids SEQID14x4-TK-LUC, SEQID 16x4-TK-LUC, SE~ID18x4-TK-LUC, SEQLD2ûx4-TK-LUC, SEQID22x4-lX-LUC, SEQL~24x4-TK-LUC, SEQL~26x4-TK-LUC, SEQlD28x4-TK-LUC, SEQlD3ûx4-lK-LUC, SEQlD32x4-TK-LUC, SEQ~34x4-TK-LUC, SEQID36x4-TK-LUC, SEQlD38x4-TK-LUC, SEQID40x4-TK-LUC, SEQ~42x4-TK-LUC, SEQlI)44x4-TK-LUC, DOCKE~NO. 016-0030.WO
2191 ~9 SEQID46x4-TK-LUC, SEQID48x4-TK-LUC, SEQID50x4-TK-LUC, SEQ~52x4-TK-LUC, SEQID52x6-TK-LUC. SEQID54x4-T~-LUC, SEQID56x4-TK-LUC, and SEQID58x4-TK-LUC contain four copies (or six copies for SEQID52x6-TK-LUC) of the same SEQ ID NOs 14-58 as those used m the EMSA's, linked to the promoter of the 5 Herpes Simplex virus thymidine kinase gene at position -35 with respect to the cap site.
See FIG. 1. The reference reporter, l~-LUC (P. Lamb et al., 83 Blood 2063 (1994)), the disclosure of which is herein Ul~.Ul l)UI dL~:d by reference, is the parent vector that contains no response element. These chimeric promoters drive the expression of the ,~ structural gene for firerdy luciferase.
ME-I 80 cells were transfected with the reporter plasmids of above by calcium phosphate Cu~ d~iOIl. Cells were seeded at 1-4X105/ml the day before .r~ 11 Cells were exposed to a calcium phosphate precipitate containing the above reporter plasmids (10-20 llglml) and the ~ -C~ aill~ plasmid pCHl 10 (5 llg/ml, Cul~ .; lI,y available from Pharmacia Biotech, Piscataway, NJ) for 15 IZ h. The medium was then changed and the cells allowed to recover for 16-18 h.
~ -nmhinAnt cytokines were then added prediluted in growth medium and the cellsharvested after 6 h. Cells were Iysed and luciferase and l3~ AI~ IrISj~ - activities determined using standard techniques. See, e.~. J. R De Wet et al., 7 Mol. CeU. Biol.
725 (1987) and S_mbrook et al., Molecular ('.llmir~g A Laboratorv Manual 2nd ed., ~ 20 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). For each sample the normalized response was determined by dividing light units obtamed from the luciferase assay with the ~ SAt~ if tA`r- activity in the same Iysate as detern~ined using a ~L. UlllO~ substrate. The results of these l l A 11~ are shown below in Table 3 .
Numbers given are the mean fold inductions ~A-fold induction' is defined as the normalized 25 response in a cytokine-treated sample divided by the normalized response in an untreated sample). The value in parentheses is the number of i" t l, ,rt, ,l c.~ a included to calculate the mean.
TF-l cells were transfected with the reporter plasmids of above by the DEAE-dextran method as described (J. Suzow and A.D. Friedman, 13 Mol. Cell. ~iol.
2141 (1993)) with the following ".. ~ test reporter constructs we~e added to a DOCKE-l NO. 016-0030.WO
of 3 .~glmL during the trAn~rtinn~ pMsvcAT yector was not added to the L~ art~.Liùll rnixtures, the growth medium used was as described above for TF-1 cells, and cytokine inductions were carried out for 4-5 h. Cells were Iysed and luciferase activity deterrrined using standard techniques. T~ r~ were performed in a batch,S and identical numbers of transfected cells were then separately induced with cytokine for the 4-5 h induction period. The results of these L~ rr~ are shown below in Table4. Numbers given are the mean fold i~ductions ~Afold induction' for TF-1 l, O~r~- l;ol~ is defined as the luciferase response in a cytokine-treated sample divided by the lucrferase 5 ~ response in an untreated sample). The value in ~ .ILIle~ is the number of 10 ;"~ I ` included to calculate the mean.
NFS-60 cells were transfected with the reporter plasmids described above by the DEAE-dextran method as described in the preceding para~raph with the following .~ test reporter constructs were added to a ~,UllCcllLl4Liull of 6 ,ug/mL during the Ll~art~_iiull, and cytokine inductions were carried out for 2.5h. The 15 results of these l . A~ ~rr~ are shown below in Table 5.
Stable Transfection of NFS-60 Cells Stable Ll Allarr,~,Liull of NFS-60 cells was A- ~ U' ~ If ~l as described by H. Pahl et al., 19 Exp. Hematol.. 1038-1041 (1991). In brief, 1.4 x 107 factor~ ..J~ 'FS-60cells were washed twice with RPMI-1640 and then ~calla~ ded in RPMI-1640 (0.5 ~~ 20 mL); linearized reporter DNA (16 llg) and EcoRI-digestcd pSV2NEO plasmid (4 llg, Cu~ ly available from ATCC) were then added. Cells and plasmids were incubated for 5 min at RT in a 0.4 cm ~ 1IU~UIdLiUI~ cuvette (Bio-Rad), then subjected to a 330V, 960 ~F pulse, using a Bio-Rad Gene Pulser. Cells were il~ ,diALtly incubated on ice for 15 min, then placed in normal growth medium (10 mL). Two days later, G418 (300 ~Lg/mL, Boehringer-Mannheim, Illdi~di)olia, IN) was added to the culture. Stablely transfected cells were cloned by limiting dilution. Al.yluAilllALely 400 clones were screened for G-CSF-inducible luciferase actiYit,Y, and 16 positive clones were identified and .,Il,_~.Lt~ further. The results for four of the positiYe clones are . d in Table 6. The number of; ~ . ..l CA~)t:lilll~,.l~ included to calculate0 the mearl is indicated iri y~ c~ ,scs.
DOCKET~NO. 016-0030.WO
~ ~ 219~189 Table 3: T, ~ iU~ induction in ME-I80 cells of reporter constructs ,UI~)UI clLlllg multiple copies of test STAT regulatory elements. The values given are mean fold inductions in response to the irldicated cytokine. The value in the p~LlCll~ .S~S is the number of S included to calculate the mean.
Reporter CoreElement IL 4 IL-13 TK-LUC none û.9 (3) I.û (3) SEQ~14x4TK-LUC CACTTCCCAAGAACAG 22 (3) 9.7 (3) SEQD~16x4TK-LUC TGCTTCCCCGGAACGT 1.3 (3) I.û (3) SEQn~18x4TK-LUC TGCTTCCCCAGAACGT 1.2 (3) 1.1 (3) SEQrD20x4TK-LUC TGCTTCCCAAGAACGT 1.5 (3) 1.2 (3) SEQII)22~s4TK-LUC CACTTCCCCGGAACAG 2.7 (3) 1.6 (3) SEQrD24x4TK-LUC CACTICCCCAGAACAG 8.0 (3) 3.0 (3) SEQrD26x4TK-LUC CACTTCCCAGGAACAG lû (3) 6.4 (3) SEQrD28x4TK-LUC TACTTCCCAAGAACAT 3.0 (3) 1.5 (3) SEQID30x4TK-LUC CGCTTCCCAAGAACGG 1.5 (3) 1.3 (3) SEQrD32x4TK-LUC CACTTCTTAAGAACAG 7.3 (3) 3.3 (3) SEQlD34x4TK-LUC CACTTTCCA.~GAACAG 1.7 (3) 1. I (3) SEQrD36x4TK-LUC TGCTTCCCGGAACGT I . I (3) n.d.
SEQrD38x4TK-LUC GATTTCCCCGAAATG 0.8 (3) n.d.
SEQn~40x4TK-LUC ATATTCCTGTAAGTG 1.2 (3) n.d.
SEQrD44x4TK-LUC CATTTCTG&AAATG 1.1 (3) n.d.
SEQrD46x4TK-LUC CATTTCCCGTAAATC 1.0 (3) n.d.
SEQD~48x4TK-LUC ATATTACCAGAAATG 12 (3) n.d.
SEQrD50x4TK-LUC ATTTTCCAGTAACAG 1.0 (3) n.d.
SEQrD52x4TK-LUC CAATTTCTAAGAAAGGA 0.8 (3) n.d.
SEQrD56x4TK-LUC TGCTTCTCAGAACGT 1.7 (3) n.d.
SEQlD58x4TK-LUC TGCTTCCCCGAACGT 1.2 (3) n.d.
n.d.= not d~t~
21911~9 Table 4 T ~ 1 induction in TF-I cells of reporter constructs ~ICul yOI ~Lilg mu~tiple copies of test STAT regulatory elements The values given are mean fold i~ductions in response to the indicated cytokine The value in the ~-,~1l.,~ is the number of l ,~1 ; ,t~
included to calculate the mean Reporter 11,4 GM-CSF Epo ~3 TK-LUC 0 7 (2) 0 8 (2) 0 8 a) û 7 a) SEQID36x4TK-LUC n.d. 3 4 (2) 18 (2) 2 8 (2) SEQID38x4TK-LUC 14 (2) 9 6 (4) 3 1 (4) 6 8 (3) - SEQID40x4TK-LUC n.d. n.d. 12 (2) 13 (2 SEQID42x4TK-LUC n.d. n.d. 10 (2) 12 (2 SEQID44x4TK-LUC n.d. n.d. 18 (3) 0 9 SEQID46x4TK-LUC n.d. n.d. 0 8 (2) 0 9 (2 SEQID48x4TK-LUC n.d. n.d. , 12 (2) 1 I (2) SEQID50x4TK-LUC n.d. n.d. 15 (2) 3 1 (2) SEQlD52x6TK-LUC n.d. 7 6 (2) 3 5 (2) 7 3 (2 SEQlD14x4TK-LUC 3 3 (3) 13 (2) 0 8 (2) 13 (2 n.d.= not d~t~A ' ,-?:._ 10 Table 5 T, . ~ 1 induction in NFS-60 cells of reporter constructs UI LJUI dLillg multiple copies of test STAT regulator,v elerllents The values given are mean fold inductions in response to the indicated cytol~ne The value in the pcu t~lth~ .s is the number of ~ 1, ,", , included to calculate the mean Reporter Core Element G-CSF ~3 IL,6 SEQID54x4TK-LUC TTCCCGAA 14 (2) 10 (V 41 (2) SEQID36x4TK-LUC TTCCCGGAA 24 (2) 3 5 (2) 4 8 (2) SEQID56x4TK-LUC TTCTCAGAA 3 1 (2) 14 (2) 17 (2) SEQlD38x4TK-LUC TTCCCCGAA 24 (2) 61 (2) 6 9 (2) DOCKET NO. 016-0030.WO
~ ~ 2191189 Table 6: TIA~IC~ AI induction in NFS-60 cells stablely transfected with the SEQID38x4TK-LUC reporter plasmid. The values given are mean fold inductions in response to the indicated cytokine. The value in the ~,~cl~L~ ses is the number of ~ included to calcuiate the 5 mean.
Clone G-CSF ~3 I~6 r l ~.
IEII 17.2 (3) 16.5 (3) 3.4 (3) 6G8 17.3 (3) 20.6 (3) 3.6 (3) IB10 16.8 (3) 27.6 (3) 2.7 (3) 4C2 12.8(3) 20.1 (3) 2.3 (3) The data ~Ill,,.r.A. ,~d in Table 3 when compared to the in vitro binding 10 data described above clearly d~ ollOlldLe that in vitro binding is not predictive of Ll f..~c.il,~ivl.dl activity. Thus, all of the DNA elements that were il.~ Ltd as multimers into the reporter vectors bound the STAT complexes activated by lL4 and IL-13 with a similar affinity; however, oUI1.1lioill~l~ not all could mediate ~
induction in response to lL-4 and lL-13 (defined as greater than a 2-fold induction).
- ~ 15 Although it has been reported that a sequence element found in the promoter of the FcRIIb gene (SEQID52) is necessary for the IL4 It~/O~ o:l ofthis gene and can bind the STAT complex activated by IL4 in vitro (I. Kohler et al., 345 FEBS Lett. 187 (1994)), it is clear from the data in Table 3 (SEQID52x4TK-LUC entry) that this element is not sufticient on its own to mediate IL-4 I~ e~ further "...~. . 5f ~20 the .l ~ .,... lf l ;...l between in vitro binding data and functional, l . A ,~ u~Al activity The data 5111~ I in Table 4 when compared to the in vitro binding data described above again clearly d~ that in vitro binding cannot be relied on to be predictive of ~ AI activity. Thus, many of the DNA elements that were ih~c~ oldL~d as multimers into the reporter vectors bound the STAT complexes 25 activated by IL4, GM-CSF, Epo and ~-3 with v~ing affnities (Table 2); however, DOCKET NO. 016-00~0.WO
2~ 8~
most could not mediate a ~ induction in response to these c-ytokines (defined as greater than a 2-fold induction).
The data ~"".., - ;,. .l in Table S again show that in vitro binding data do notreliablycorrelatewiththeabilityoftheelementstomediateall""~. .;1,1;.~.~,.1 5 induction. As described above, G-CSF activates two complexes in NFS-60 cells, one containing STAT3 and one containing an ~ STAT protein resembling the complex activated by IL-3 in NFS-60 cells. Response elements that could bind to both G-CSF-activated complexes, such as SEQID38 and SEQID36, mediated a strong . . ,~ l induction in response to G-CSF, and the response element that bound ~ ~ 10 strongly only to the STAT3-containing, G-CSF-activated complex (SEQ~54) was activated by G-CSF (though not quite as strongly as were the sequences that bound both complexes). However, ~ul~Jliaiul~ly, the response element that bound only the IL-3-activated complex (SEQID56) was not activated in response to IL-3 (def~ned as less than a 2-fold induction) and was only weaklyr activated by G-CSF.
The data ~.. ,,~1~, ;,. ~1 in Table 6 show that the NFS-60 clones stablely transfected with SEQID38x4TK-LUC respond well to both G-CSF, IL-3 and IL-6 (though the response to IL-6 was slightly lower than what was obtained in the transient Also, compared to the transiently transfected NFS-60 cells, the stable NFS-60 clones appear to respond more robustly to IL-3. N~ , in general, the 20 transient ~. ~. ,~ data are a good indicator of what to expect when the reporter is stablely tr_nsfected into the NFS-60 cells.
It has previously been reported that many cytokines, including IL-3, GM-CSF, Epo, aCSF, IL~ and IL-13 activate STAT or STAT-like complexes that bind to DNA sequence elements related to the GAS element that was first .1"., ". I rl 1 ~ in the 25 promoters of IFNy-responsive genes. The data in Example I .,~ ,lu ,;~ ~,Iy show that, ~u~ , in vitro binding is not predictive of L.~.~ ,L;ol~cl activation for the cytokines IL-3, IL4, IL-13, GM-CSF, G-CSF and Epo. One can certainly not therefore assume that the in vitro STAT complex binding observed in previously published work is directly ~. c.~lcL~l~ into a functional reporter assay. To date there has been no 30 reported d~ ollaLI CLi.,.l that the DNA sequences reported to bind to the STAT or . ~
DOCKE~T NO 016-OOiO WO . . - -2 ~ 9 STAT-lilce compleYes activated by IL-3, IL ~1, L-13, GM-CSF, G-CSF or Epo can mediate ~ 1 induction in response to those cytokines~ Because it is not possible to eYtrapolate from in vitro binding data that a g,iven sequence will be functional, the d "" ~ of functional activity such as that shown in the eYample 5 above is absolutely critical.
While in accordance with the patent statutes, description of the preferred weight fractions, and processing conditions have been provided, the scope of theinvention is not to be limited thereto or thereby. Various ~ and alterations of the present invention will be apparent to those skilled in the art without departing ` ~ 10 from the scope and spirit of the present invention.
C~ y, for an u~ , of the scope of the present invention, reference is made to the following claims Pa~. Docket No. 016-0030.W0 ~ 2~91 1~9 SEQUE~CE LI-SI~G
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SYNTHETIC DNA"
40 (xi) SEQUENCE DESCR~PTION: SEQ ID NO: 1:
TTCNN~.GAA 9 Pat` Docket No. 016-0030.WO
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, . . .. . . . .
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2191 ~89 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
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5/23 2191 ~89 (2) INFORMATION FOR SEQ ID NO: 10:
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(A) LENGTH: 9 base pairs S (B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE mE: other ~ucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICACID
SYNTHETIC DNA"
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20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinole 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRrPTION: Idesc = ~OTHER NUCLEIC ACID, SYNTHETIC DNA"
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35 (2) ~IFORMATION FOR SEQ ID NO: 12:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs (B) TYPE: nucleic acid 40 (C) STRANDEDNESS: sin_le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~
SYNTHETIC DNA"
Pat Docket No. 01~-0030.WO
6/23 21 9~ 1 ~9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
TTCTAAGAA g (2) INFORMATION FOR SEQ ID NO: 13:
) SEQUENCE CHARACTERISTICS:
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25 (i) SEQUENCE CHARACTERISTICS:
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35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
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(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Par. Docket No. 01-6-0030.~0 7l23 2 7 9 ~
i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
5 (~) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
10 (2) INFORMATION FOR SEQ ID NO: 16:
Cl) SEQUENCE CHARACTERISTICS:
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(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Pat~ Doclcet No. 016-0030~WO
. ~ .
8l23 2 ~ 9 ~
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 20 base pairs 5 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
~.
(2) INFORMATION FOR SEQ ID NO: 19:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE. other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AClD
SYNTHETIC DNA"
30 (Xl~ SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GATCACGTTC TG&GGAAGCA 20 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
PaC. DocketNo. 016-0030.WO
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(2) LNFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (13) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear _ (ii) MOLECULE TYPE: other nucleic acid .. 15 (A)DESCRIPTION: /desc="OTHERNUCLEICACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
r ('') INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE C~IARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear ~,. (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~D, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat. DocketNo. 016-0030.WO
-- 10/23 21qll89 (u) MOLECULE TYPE: other nucleic acid (A) DESCRrPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRlPTION: SEQ ID NO: 23:
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(i) SEQUENCE CHARACTERISTICS:
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SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
= 30 (A) LENGTH: 21 base pairs ~_- (B) TYPE. nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ lD NO: 25:
Pat. Docket No. 016-0030.WO
11/23 2~91~89 ~2) lNFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENOE DESCRIPTION: SEQ ID NO: 26:
~3' ~_'.T 15 GATCCACTTC CCAGGAACAG A 21 (2) INFORMATION FOR SEQ ID NO: 27:
20 (i) SEQUENCE CHARACTER~STICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
,, 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
35 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid 40 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc= "OTHER~IJCLEIC ACID
S~IIC DNA"
Pat: Docket No. 016-0030.WO
2191 ~8~
(xi) SEQUENOE DESCRIPTION: SEQ ID NO: 28:
(2) lNFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 21 base pairs ~) mE: nucleic acid (C) STRANDEDNESS: single . (D) TOPOLOGY: linear , r (ii) MOLECULE mE: other nucleic acid ~: - 15 (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
(2) INFORMATION FOR SEQ ID NO: 30:
25 (i) SEQUENCE CHARACTERTSTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~-~~ (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
(2) INFORMATION FOR SEQ ~ NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat`DocketNo. 016-0030.WO
(ii) MOLECULE TYPE: other nucleic acid (A) DESCF~IPTION: Idesc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ lD NO: 31:
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQ~ENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEO ID NO: 32:
(Z) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTE~TIC DNA"
Pat`DocketNo. 016-0030.WO
2 1 9 1 1 8q (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
(2) INFORMATION FOR SEQ lD NO: 34:
(~) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
(2) INFORMATION FOR SEQ ~ NO: 35:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRA.~DEDNESS: sin~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid f -' (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTEI: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat. Docket No. 016-0030.WO
i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
10 (V ~FORMATION FOR SEQ ID NO: 37:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid 15 (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SY~THETIC DNA"
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(2) INFORMATION FOR SEQ ~) NO: 38:
(i) SEQUENCE CHARACTERISTICS:
30 (A) LENGTH: 19 base pairs - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
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Q
Pat. Docket No. 016-0030.WO
21 9~ 1 89 (2) ~FORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRAMDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 10 (A) DESCRIPTION: /desc = "OTHERNUCIEIC ACID, SYNTHETIC DNA"
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'~- ~ 15 GATCCATTTC GGGGAAATC 19 (2) ~FORMATION FOR SEQ ID NO: 40:
20 (i) SEQUENCE CHARACTERISTICS:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lg base pairs (13) TYPE: nucleic acid 40 (C~ STRANDEDNESS: sin~le (D) TOPOLOGY: linear ~ui) MOLECULE TYPE: other rlucleic acid (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID, SYNTHETIC DNA"
Pat'Docket No. 016-0030.WO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
(2) INFORMATION FOR SEQ lD NO: 42:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) MOLECULE mE: other nucleic acid (A)DESCRIPTION: /desc="OTHERNUCLEICAClD, SYNTHETIC DNA"
(xi) SEQUENCE DESCRrPTION: SEQ ID NO: 42:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) T~PE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .~ (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
(2) lNFORMATION FOR SEQ ID ~O: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairS
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear Pat.~DocketNo. 016-00~0.WO
2' 91 ~ ~9 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTE~R NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ~ NO: 44:
(2) INFORMATION FOR SEQ ID NO: 45:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs , tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTEIETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
(2) INFORMATION FOR SEQ ID NO: 46:
~1) SEQUENCE CHARACTERISTICS:
(A) LENGTH 19 base pairs (B) TYPE: nudeic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other ~ucleic acid (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYN~HETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Pat. Docket No. 016-0030.WO - -l9/23 2 1 9 1 1 8 9 (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHERNUCLEIC ACID
SYNTHETIC DNAN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
(2) INFORMATION FOR SEQ ID NO: 48:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs ~B) mE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GATCATATTA CCAGAAATG l9 (2) ~FORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGl~I: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
Pat` Docket No. 016-0030.WO
2~91 '~9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ~: 15 (A)DESCRrPTION: /desc="OTHERNUCLEICACID, SYNTEIETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
(2) INFORMATION FOR SEQ lD NO: 51:
25 (i) SEQUENCE CIIARACTERlSTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
35 (xi) SEQUENCEDESCRIPTION: SEQIDNO: 51:
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERrSTICS:
(A) LENGTH: 21 base pairs (~3) TYPE: nuc~eic acid (C) STRANDEDNESS: single (D) TOPOLOGY: lin~r Pa~. Docket No. 016-0030.WO
21/23 219~189 (ii) MOLEC~LE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
10 (2) INFORMATION FOR SEQ ID NO: 53:
) SEQUENCE CEIARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid t,_" 1s (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID
20 . SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ lD NO: 53:
(2) INFORMATION FOR SEQ ID NO: 54:
~I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 35 ~li) MOLECU~E TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC ACID, SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Pat. Oocket No. 01~-003~.WO
2V23 2~91 18q (2) ~FORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A)DESCr~lPTION: /desc="OTHERNUCr,EICACrD
SYNTHETIC DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
t~ 15 GATCACGTTC GGGAAGCA 18 (2) rNFORMATION FOR SEQ ~ NO: 56:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear i) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OT~R NUCLEIC ACID
SYNTHETIC DNA"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
(2) INFORMATION FOR SEQ ID NO: 57:
) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (u) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OTHER NUCLEIC AC~
SYNTHETIC DNA"
, . . . . . . .
. Pat. Docket No. 016-00~0.WO
. ~ .
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(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear IOLECULE TYPE: other nucleic acid ~- .. 15 (A) DESCRIPTION: /desc = "OTHERNUCLEIC ACID
SYNTHETIC DNA"
(~) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
(2? INFORMATION FOR SEQ ID NO: 59:
25 (i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~- (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: Idesc = "OTHER NUCLEIC ACID
SYNTHETIC DNA"
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Claims (22)
1. A DNA construct comprising:
(a) an oligonucleotide sequence comprising a regulatory element of the nucleotide sequence TTNxAA, operably linked to (b) a promoter, operably linked to (c) a heterologous gene, wherein N is independently selected from A, T, C or G and x is 4, 5, 6 or 7, and wherein the DNA construct is operably linked in such a manner that the heterologous gene is under the transcriptional control of the promoter and oligonucleotide sequence when the oligonucleotide sequence is bound by a STAT
protein activated in response to IL-2, IL-3, IL-4, IL-7, IL-9, IL-13, IL-15, G-CSF, GM-CSF, Epo or Tpo.
(a) an oligonucleotide sequence comprising a regulatory element of the nucleotide sequence TTNxAA, operably linked to (b) a promoter, operably linked to (c) a heterologous gene, wherein N is independently selected from A, T, C or G and x is 4, 5, 6 or 7, and wherein the DNA construct is operably linked in such a manner that the heterologous gene is under the transcriptional control of the promoter and oligonucleotide sequence when the oligonucleotide sequence is bound by a STAT
protein activated in response to IL-2, IL-3, IL-4, IL-7, IL-9, IL-13, IL-15, G-CSF, GM-CSF, Epo or Tpo.
2. A DNA construct according to claim 1, wherein the STAT protein comprises STAT5 protein and/or STAT6 protein.
3. A DNA construct according to claim 2, wherein the STAT protein is STAT6 protein.
4. A DNA construct according to claim 1, wherein the oligonucleotide sequence is selected from the group consisting of TTCNNNGAA and TTCNNNNGAA, where N is independently selected from A, T, C or G.
5. An DNA construct according to claim 4, wherein the oligonucleotide sequence is selected from the group consisting of TTCCCGGAA (SEQID NO. 10), TTCCCCGAA (SEQ ID NO. 11), TTCTAAGAA (SEQ ID NO. 12), TTCTCAGAA (SEQ ID NO. 13), and their double stranded complements.
6. A DNA construct according to claim 1, wherein the oligonucleotide sequence is double stranded.
7. A DNA construct according to claim 1, wherein the oligonucleotide sequence comprises a multimer of the regulatory element of claim 1.
8. A DNA construct according to claim 1, wherein the promoter is selected from the group consisting of the gene promoter of the Herpes simplex virus thymidine kinase, the adneovirus Elb and yeast alcohol dehydrogenase, and the heterologous gene is selected from the group consisting of the gene for luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase, secreted placental alkaline phosphatase, human growth hormone, t-PA, green fluorescent protein and interferon.
9. A cytokine-responsive host cell transfected with the DNA
construct of claim 1.
construct of claim 1.
10. A cytokine-responsive host cell according to claim 9, wherein the cell comprises a HepG2 cell, U937 cell, ME-180 cell, a TF-1 cell or NFS-60 cell.
11. A method for measuring the ability of a compound to act as an agonist of cytokine-mediated gene transcription comprising:
(a) contacting the compound with a host cell according to claim 9 under conditions in which the heterologous gene is capable of being expressed inresponse to the compound; and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
(a) contacting the compound with a host cell according to claim 9 under conditions in which the heterologous gene is capable of being expressed inresponse to the compound; and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
12. A method for measuring the ability of a compound to act as an antagonist of cytokine-mediated gene transcription comprising:
(a) contacting the compound with a host cell according to claim 9 in the presence of a predetermined amount of a cytokine under conditions in which the heterologous gene is capable of being expressed in response to the cytokine; and(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound.
(a) contacting the compound with a host cell according to claim 9 in the presence of a predetermined amount of a cytokine under conditions in which the heterologous gene is capable of being expressed in response to the cytokine; and(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound.
13. A DNA construct comprising:
(a) an oligonucleotide sequence comprising a regulatory element of the nucleotide sequence ANTTCNNNNGAANA (SEQ ID NO. 3), or its double stranded complement, operably linked to (b) a promoter, operably linked to (c) a heterologous gene, wherein N is independently selected from A, T, C or G, and wherein the DNA construct is operably linked in such a manner that the heterologous gene is under the transcriptional control of the promoter and oligonucleotide sequence when the oligonucleotide sequence is bound by a STAT6 protein activated in response to a STAT6-activating cytokine.
(a) an oligonucleotide sequence comprising a regulatory element of the nucleotide sequence ANTTCNNNNGAANA (SEQ ID NO. 3), or its double stranded complement, operably linked to (b) a promoter, operably linked to (c) a heterologous gene, wherein N is independently selected from A, T, C or G, and wherein the DNA construct is operably linked in such a manner that the heterologous gene is under the transcriptional control of the promoter and oligonucleotide sequence when the oligonucleotide sequence is bound by a STAT6 protein activated in response to a STAT6-activating cytokine.
14. A DNA construct according to claim 13, wherein the cytokine is selected from the group consisting of IL-4, IL-7, IL-9, IL-13 and IL-15.
15. A DNA construct according to claim 13, wherein the oligonucleotide sequence is selected from the group consisting of ACTTCCCAAGAACA (SEQ ID NO. 4), ACTTCCCCGGAACA (SEQ ID NO. 5), ACTTCCCCAGAACA (SEQ ID NO. 6), ACTTCCCAGGAACA (SEQ ID NO. 7), ACTTCCTAAGAACA (SEQ ID NO. 8), ACTTCTTAAGAACA (SEQ ID NO. 9), and their double stranded complements.
16. A DNA construct according to claim 13, wherein the oligonucleotide sequence is double stranded.
17. A DNA construct according to claim 13, wherein the promoter is selected from the group consisting of the gene promoter of the Herpes simplex virus thymidine kinase, the adneovirus Elb and yeast alcohol dehydrogenase, and the heterologous gene is selected from the group consisting of the gene for luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase, secreted placental alkaline phosphatase, human growth hormone, t-PA, green fluorescent protein and interferon.
18. A DNA construct according to claim 13, wherein the oligonucleotide sequence comprises a multimer of the regulatory element of claim 12.
19. A cytokine-responsive host cell transfected with the DNA
construct of claim 13.
construct of claim 13.
20. A cytokine-responsive host cell according to claim 19, wherein the cell comprises a HepG2 cell, U937 cell, ME-180 cell, a TF-1 cell or NFS-60 cell.
21. A method for measuring the ability of a compound to act as an agonist of cytokine-mediated gene transcription comprising:
(a) contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed inresponse to the compound; and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
(a) contacting the compound with a host cell according to claim 19 under conditions in which the heterologous gene is capable of being expressed inresponse to the compound; and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
22. A method for measuring the ability of a compound to act as an antagonist of cytokine-mediated gene transcription comprising:
(a) contacting the compound with a host cell according to claim 19 in the presence of a predetermined amount of a cytokine under conditions in which the heterologous gene is capable of being expressed in response to the cytokine; and(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound.
(a) contacting the compound with a host cell according to claim 19 in the presence of a predetermined amount of a cytokine under conditions in which the heterologous gene is capable of being expressed in response to the cytokine; and(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/411,020 | 1995-03-27 | ||
| US08/411,020 US5712094A (en) | 1995-03-27 | 1995-03-27 | Methods for detecting modulators of cytokine action |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2191189A1 true CA2191189A1 (en) | 1996-10-03 |
Family
ID=23627230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002191189A Abandoned CA2191189A1 (en) | 1995-03-27 | 1996-03-25 | Methods and associated reagents for detecting modulators of cytokine action |
Country Status (6)
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| US (1) | US5712094A (en) |
| EP (1) | EP0760853A1 (en) |
| JP (1) | JPH10510999A (en) |
| AU (1) | AU5429796A (en) |
| CA (1) | CA2191189A1 (en) |
| WO (1) | WO1996030515A1 (en) |
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| US5821053A (en) * | 1995-02-10 | 1998-10-13 | Center For Blood Research, Inc. | LIL-Stat DNA binding sites and methods for identifying inhibitory binding agents |
| US6509313B1 (en) * | 1996-02-28 | 2003-01-21 | Cornell Research Foundation, Inc. | Stimulation of immune response with low doses of cytokines |
| JP2001503982A (en) * | 1996-11-01 | 2001-03-27 | スミスクライン・ビーチャム・パブリック・リミテッド・カンパニー | Method for detecting a compound that modulates the effect of obesity (OB) protein |
| US7001733B1 (en) | 1998-05-12 | 2006-02-21 | Rigel Pharmaceuticals, Inc. | Methods and compositions for screening for modulations of IgE synthesis, secretion and switch rearrangement |
| WO2000006696A2 (en) * | 1998-07-30 | 2000-02-10 | University Of South Florida | Method for the modulation of function of transcription factors |
| WO2001079555A2 (en) | 2000-04-14 | 2001-10-25 | Millennium Pharmaceuticals, Inc. | Roles of jak/stat family members in tolerance induction |
| CN1322736A (en) * | 2000-05-09 | 2001-11-21 | 上海博德基因开发有限公司 | New polypeptide husan stat 2 gene 11 and its encoding polynucleotides |
| WO2002096943A1 (en) * | 2001-05-25 | 2002-12-05 | Asahi Kasei Kabushiki Kaisha | Stat6-activating genes |
| CA2528281A1 (en) * | 2003-06-30 | 2005-01-06 | Biovitrum Ab | Methods for identifying agents, which regulate cytokines |
| US7892733B1 (en) * | 2004-04-22 | 2011-02-22 | Amgen Inc. | Response element regions |
| ES2543381T3 (en) * | 2008-03-04 | 2015-08-18 | Centre National De La Recherche Scientifique | Test with indicator gene, cells and kit to perform said test |
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| EP0487298A3 (en) * | 1990-11-21 | 1992-12-16 | Schering Corporation | Human gamma interferon antagonist/agonist screen |
| AU1536392A (en) * | 1991-01-18 | 1992-08-27 | Oncogene Science, Inc. | Methods of transcriptionally modulating expression of oncogenes and tumor suppressor genes |
| WO1995008001A1 (en) * | 1993-09-15 | 1995-03-23 | New York University | Dna sequence which binds transcriptional regulatory proteins activated in response to various cytokines and uses thereof |
| EP0722497A1 (en) * | 1994-04-14 | 1996-07-24 | Ligand Pharmaceuticals, Inc. | Dna spacer regulatory elements responsive to cytokines and methods for their use |
| AU681025B2 (en) * | 1994-04-14 | 1997-08-14 | Ligand Pharmaceuticals Incorporated | DNA regulatory elements responsive to cytokines |
| US5591825A (en) * | 1994-07-05 | 1997-01-07 | Tularik, Inc. | Interleukin 4 signal transducers |
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1996
- 1996-03-25 CA CA002191189A patent/CA2191189A1/en not_active Abandoned
- 1996-03-25 EP EP96911398A patent/EP0760853A1/en not_active Withdrawn
- 1996-03-25 JP JP8529557A patent/JPH10510999A/en active Pending
- 1996-03-25 WO PCT/US1996/004012 patent/WO1996030515A1/en not_active Application Discontinuation
- 1996-03-25 AU AU54297/96A patent/AU5429796A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0760853A1 (en) | 1997-03-12 |
| AU5429796A (en) | 1996-10-16 |
| JPH10510999A (en) | 1998-10-27 |
| US5712094A (en) | 1998-01-27 |
| WO1996030515A1 (en) | 1996-10-03 |
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