MXPA97000255A - Method to identify nucleic acids that code the promoter activating proteins c- - Google Patents

Abstract

Materials and methods are described for identifying signal transduction molecules that activate promoters, such as the human c-fos proto-oncogene promoter, as well as antagonists of these molecules. Activating proteins of the human c-fos promoter and in particular novel proteins which are designated as CROC-1 and CROC-4 proteins, the nucleic acids encoding the proteins and the cells of mammals transfected with vectors containing these nucleic acids are also described.

Description

"METHOD FOR IDENTIFYING NUCLEIC ACIDS CODING THE PROMOTER ACTIVATING PROTEINS c -fos The present invention relates to materials and methods for identifying transductive signal molecules that activate the human c-fos proto-oncogene promoter and antagonists of these molecules.

BACKGROUND OF THE INVENTION Activation of cells as a result of mutation or overexpression of signal molecules, such as Ha-ras proto-oncogenes, c-fos, c-myc and c-jun, has been implicated in the aberrant cell growth which form the basis of the neoplasia. See, DeFeo, et al., Proc. Nati Acad. Sci., 78, 3328-3332 (1981); de Miller, et al., Cell, 36, 51-60 (1984); from Kelekar, and others, Mol.

Cell. Biol., 6, 7-14 (1986); and from Vogt, and others, Adv. Cancer Res., 55, 1-35 (1990). Induction of c-fos occurs in response to the activation of growth-related signal pathways after serum stimulation of 3T3 mouse cells, or in response to over-expression of normal and transformed versions of Ha -ras, respectively. It has also been shown that the constitutive expression of c-fos occurs in certain lines of human tumor. These findings suggest that the aberrant growth characteristic of the neoplastic phenotype may involve the constitutive activation of signal transduction pathways involved in the induction of proto-oncogene c-fos. See Greenberg, et al., Nature, 311, 433-438 (1984); Stacey, and others, Mol. Cell. Biol., 7, 523-527 (1987); and O'Hara, and others, Mol. Cell. Biol., 7, 2941-2946 (1987). Using the reporter genes driven by the c-fos promoter, specific enhancers have been identified in the c-fos proto-oncogene promoter that respond to activated signal transduction pathways. These enhancers include a tyrosine kinase that responds to SCM, direct repeats responsive to raf, an AP-1 site that responds to C kinase protein and a responsive serum response element. See Fujii, et al., Mol. Cell. Biol., 9 2493-2499 (1989): de Hayes et al., Proc. Nati Acad. Sci., USA, 84, 1272-1276 (1987); de Jamal et al., Nature, 344, 463-466 (1990); de Gutman, et al., Mol. Cell. Biol., 11, 5381-5387 (1991); and Fisch, et al., Mol. Cell. Biol., 9, 1327-1331 (1989). The contingent duplication systems that employ transcriptional activation of the SV40 T antigen gene to identify breeders and interact, are stably transcription factors already known. See Vasavada et al., Ind. J. Biochem.

Biophys. , 25, 488-494 (1988); Vasavada, and others, Gene, 55, 29-40 (1987); Vasavada et al., Proc. Nati Acad. Sci., 8_8, 10686-10690 (1991); and Rusconi, and others, Gene, 89, 211-221 (1990) . Due to the importance of signal molecules in the control of cell proliferation, there is a need for methods to identify molecules involved in the signal systems related to growth which in turn can be used to identify biological targets for drug discovery against tumors. There is also a need for methods of identifying agents that can interfere with these signal systems related to growth to restore normal growth when proliferation of abnormal cells is occurring.

SUMMARY OF THE INVENTION The present invention fills the aforementioned needs by providing materials and methods for identifying signal transducing molecules and antagonists thereof. More specifically, this invention provides lines of mammalian cells, the cells of which comprise: (a) a recombinant vector comprising a tissue-specific or inducible promoter operably linked to a nucleic acid encoding the large polyomavirus T antigen; and (b) a recombinant expression vector comprising a polyomavirus duplication origin and a nucleic acid that is suspected of encoding a promoter-activating protein. Preferably the promoter is the human c-fos promoter and the activating protein is an activating protein of the human c-fos promoter. The present invention further provides a method for identifying a nucleic acid encoding a promoter activating protein., which comprises: (a) culturing a mammalian cell line, the cells of which comprise: (i) a recombinant vector comprising a specific inducible or tissue promoter operably linked to the coding region of the large T antigen gene of polyomavirus; and (ii) a recombinant expression vector comprising a duplication origin of polyomavirus and a nucleic acid that is suspected of encoding a promoter-activating protein, under conditions in which nucleic acids are expressed; b) measuring the levels of the duplicate vectors 5 in the cells after a sufficient incubation period to allow duplication of the vector, whereby a nucleic acid encoding a human promoter activating protein is identified by measuring the increased levels of vectors in cells.10 Preferably, the promoter is a human c-fos promoter and the activating protein is a human c-fos promoter activating protein.A preferred recombinant vector comprising a human c-fos promoter for use herein invention 15 is the plasmid PfLAG-8 A preferred recombinant expression vector comprising a polyomavirus duplication origin is the Lalf plasmid The present invention also provides human CF promoter activator proteins having the amino acid sequences defined in Sequence Lists SEQ ID NO: 1 and SEQ ID NO: 3, or antigenic fragments thereof, and nucleic acids. that encode this protein or fragments.

In another embodiment, the present invention provides mammalian cell lines, the cells of which comprise: (a) a first recombinant expression vector comprising a reporter gene operably linked to a human c-fos promoter; and (b) a second recombinant expression vector comprising a nucleic acid encoding a human c-fos promoter activating protein. The present invention also provides a method for identifying an antagonist of a human c-fos promoter activating protein, comprising: (a) providing a mammalian cell line, the cells of which comprise: (i) a first vector of recombinant expression comprising a reporter gene operably linked to a human c-fos promoter; and (ii) a second recombinant expression vector comprising a nucleic acid encoding a human cf human promoter activating protein, and (b) contacting the cell line of step (a) with a sample that is suspected of contains an antagonist of the human c-fos promoter activating protein, and (c) measures the level of reporter gene expression, whereby an antagonist of the human c-fos promoter activating protein in the sample is identified by measurement of a reduced expression level of the reporter gene.Preferably, the second recombinant vector encodes the CROC-1 protein, the CROC-4 protein or the alpha2-macroglobulin receptor-associated protein.

DETAILED DESCRIPTION All references cited herein are incorporated herein in their entirety by reference. The following terms are represented herein by the abbreviations indicated: prolonged terminal repetition (LTR); Eagle Medium modified by Dulbecco (DMEM); serum response element (SRE); chloramphenicol acetyltransferase (CAT). All of the nucleic acid sequences disclosed follow the normal 5 'to 3' convention as read from left to right. Normal single-letter abbreviations are used for the nucleotide bases in the sequences (37 C.F.R. Fraction 1.822). The term "antagonist" is defined herein as a substance that blocks or inhibits the effects of the activating protein of the human c-fos promoter, such as the CROC-1 protein or the alpha2-macroglobulin receptor associated protein. The term "reporter gene" as used herein means either a DNA molecule isolated from genomic DNA, which may or may not contain introns, or a complementary DNA (cDNA) prepared using the messenger RNA as a template. In any case, the DNA encodes an expression product that is easily measurable, v. Gr, by enzymatic activity, enzyme linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). Preferred reporter genes for use in the present invention include the E. coli Lac-Z gene from pCHUO (Estratagene # 27-4508-01). The level of expression of this gene can be measured by a sensitive fluorescent substrate assay. The CAT reporter gene that will be described below is also preferred, although many others well known in the art could be used. The term "recombinant expression vector" means a vector prepared using recombinant techniques, the vector comprising an inserted nucleic acid encoding a protein such that the vector is capable of expressing the protein during transfection or transformation into an appropriate host cell. A vector comprising a nucleic acid encoding a promoter activating protein is preferred. A vector comprising a reporter gene operably linked to the human c-fos promoter is also preferred. Cells that have been "stably transformed" have the recombinant DNA incorporated into their genomic DNA. This stably incorporated DNA is retained by the transformed cells because the cells are introduced with a selection marker such as G418 resistance., which forces retention when the cells are grown in a selection medium. The present invention employs transiently transfected mammalian cell lines however, stably transformed mammalian cell lines comprising a large T antigen regulated by the c-fos promoter can also be used. The specific inducible or tissue promoters of the present invention are non-domestic promoters, that is, they are regulated and are not transcriptionally active under normal conditions, except to the extent that low base levels of constitutive expression may occur. As defined herein, the "inducible promoters" are promoters, the transcription activity 5 of which is activated or enhanced in response to changes in the cellular environment resulting in a cellular response such as stress, hormonal stimulation or differentiation . Induction occurs through the activation of a signal cascade resulting in linkage and activity improved transcription factors on the promoter side. The molecules involved in this induction include promoter promoter proteins as described herein. Inducible promoters include c-fos and c-myc promoters. Another inducible promoter is the promoter of the multiple drug resistance gene described in J. Biol. Chem., 268, 15347 to 15350 (1993). The term "tissue-specific promoter" • J- means a promoter that is active only within a subset of cell types, such as promoters that are active only in prostate cells. See Young and others, Biochem. , 31, 818-824 (1992); and Riegman, and others. Mol. Endocrinol., 5, (Number 12) 1921-1930 (1991). Other tissue-specific promoters include promoters of histone genes and regulatory element promoters of muscle, see (Genes Dev., 4, 849-859 (1990); Mol.

Cell. Biol., 9, 515-522 (1989); and Mol. Cell. Biol., 9, 2191-2201 (1989). Promoters that can be used in this invention include, but are not limited to, the promoters of the proto-oncogenes c-fos and c-myc. See, Miller, et al., Supra; and Kelekar and others, supra. Both of these protomotors regulate in gene expression, over-expression of which can lead to aberrant cell growth. The c-fos promoter is especially preferred. The term "aberrant cell growth" is defined herein as the feature of abnormal or uncontrolled cell proliferation of the neoplasms. As used herein, the term "promoter activator protein" is defined as a protein that causes transcriptional activation of one of the promoters mentioned above. Preferably, the promoter activating protein is a human c-fos promoter activating protein. Especially preferred is an activator protein having an amino acid sequence essentially identical to that of the protein associated with the alpha2-macroglobulin receptor. Also especially preferred is an activator protein having an amino acid sequence essentially identical to that of the CROC-4 protein or the CROC-1 protein, the sequences of which are defined by SEQ ID NO: 3 and SEQ ID NO: l , respectively. The considerable identifity of the amino acid sequences means that the sequence of another activation protein of the c-fos promoter was compared to the sequence defined by either SEQ ID NO: SEQ ID NO: 3 is identical or differs by one or more alterations of amino acid (deletions, additions, substitutions) that do not significantly impair the activity of transcription activation as described herein. For example, there may be alelic or interspecies variants of the sequences defined or either SEQ ID NO: SE SEQ ID NO: 3. Furthermore, it is within the skill of the art, eg, by chemical synthesis or by using modified polymerase chain reaction (PCR) primers or site-directed mutagenesis modify the DNA encoding a c-fos promoter-activating protein having the sequence defined by SEQ ID NO: SEQ ID NO: 3, to produce individual or multiple base substitutions that do not significantly impair the activity of the promoter proteins of the c-fos promoter, produced therefrom. These conservatively modified variants are within the scope of this invention.

The identity of the sequence is determined by optimizing the equalization of the residue, if necessary, and introducing spaces as required. This changes when conservative substitutions are considered as equalizations. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The homologous amino acid sequences are typically intended to include natural allelic and interspecies variations in each respective protein sequence. Typical homologous proteins or peptides will have from 25 percent to 100 percent homology (if spaces can be introduced), up to 50 percent at 100 percent homology (if conservative substitutions are included), with the amino acid sequence of the CROC-1 protein or the CROC-4 protein.

The homology measurements will be at least about 50 percent, and typically at least 60 percent or more. The present invention also comprises "Antigenic fragments" of an activating protein of the human c-fos promoter. It is well known in the art that antigenic determinants (epitopes) usually contain at least about 5 amino acid residues. Ohno and others, Proc. Nati Acad. Sci. USA, 82, 2945 (1985). The antigenic fragments of the invention comprise from about 5 to about 100, and from. preferably from about 5 to about 50, amino acid residues. Whether a particular polypeptide falls within the scope of this invention can be determined by routine experiments using the methods described below. These antigenic fragments can be produced by proteolysis of the entire human c-fos promoter activating protein or by chemical or recombinant DNA synthesis. The antigenic fragments can be used to promote the production of antibodies, preferably in a mammal, by normal methods. Antibodies produced in this manner can be used to assay or purify the activating protein, using normal immunoassay or immunoadsorption methods. The present invention utilizes a recombinant vector comprising the polyomavirus T antigen gene and extends the contingent duplication system to identify proteins, the production of which leads to the transcriptional activation of the gene promoters. In contrast to the SV40 T antigen gene used by Vasavada, and others, supra, the "" * duplication and transformation properties of the polyoma T antigen gene can be separated.The separation of duplication and transformation properties is achieved by inserting a stop codon in the large 5 intron T in a region that overlaps the central coding sequences for the intermediate T antigen.This separability of the functions is important in the case of the c-fos promoter, where the prevention of T expression intermediate eliminates the possibility of transcriptional activation of the promoter through the signal systems associated with 3-kinase of c-src- and phosphatidylinositol activated with intermediate T (which are identified in the article by Talmage, et al., Cell, 59, 55-65 (1989 )) due to the base transcription of the level under the promoter. The use of the polyomavirus system allows for the extension of contingent duplication to several well-characterized murine systems. In contrast, the SV40 system T used by Vasavada and others, supra, is limited or mainly to ape (monkey) systems. Furthermore, the present system does not appear to suffer from the high frequency of truncated or rearranged insertions (approximately 25 percent) reported previously for the base system of the SV40 T antigen. Insertion disturbance occurs at a frequency of less than 2 times cent in the present system. A preferred embodiment includes the incorporation of multiple enhancers from upstream of the promoter of the large polyomavirus T antigen gene to achieve sufficient sensitivity of the promoter to allow induction of large T in response to low level expression of the encoded signal molecule with cDNA. The induction of large T in turn results in the duplication of the plasmid. Co-transfection with the cDNA library as will be described below allows the percentage of coding signal protein of the cDNA to be enriched within the population of the library through this duplication of the large T-induced plasmid. The resulting groups of plasmids from the library within a cDNA library, result in the identification of plasmids from a single library encoding the biologically active molecules that activate the promoter. The self-amplification process of the present invention provides additional sensitivity towards the detection of the coding signal molecules of the cDNAs. Duplication of the initial plasmid in response to induction, leads to the improved expression of active signal molecules due to a larger copy number of the "*" gene. This increase in signal molecules results in a greater amplification of the expression of the large T antigen, which in turn leads to duplication of larger plasmid. Preferred vectors of the present invention include novel plasmids, represented as PfLAG-8 and Lalfa2, as will be described below. The present invention further provides a method for identifying coding proteins of10 cDNAs that can activate a promoter, preferably a human promoter, and more preferably the human c-fos promoter. Particularly preferred are the cDNAs represented as CROC-1 and CROC-4, which code for the c-fos promoter activator proteins. For example, CROC-1 encodes a specific c-fos promoter activating protein represented by CROC-1 protein, having the amino acid sequence shown in SEQ ID NO: 1. Similarly, CROC-4 encodes a specific c-fos promoter activating protein represented as CROC-4 protein Having the amino acid sequence shown in SEQ ID NO: 3. The nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 are especially preferred. The present invention also provides the cDNAs encoding promoter activating proteins c-fos which are conservative mutants of the proteins encoded by CROC-1 or CROC-4. These mutants possess the binding and activation functions of the c-fos promoter of the proteins encoded by CROC-1 and CROC-4, respectively. In addition, the present invention provides compounds that are antagonists of the protein encoded by CROC-1 or CROC-4. These antagonists include proteins that are substitution or addition suppression mutants of the CROC-1 protein or the CROC-4 protein, and that bind to, but do not activate, the human c-fos promoter. It is recognized that, due to the degeneracy of the genetic code, there are many functionally equivalent nucleic acid sequences that can encode the promoter-activating proteins c-fos and activating protein antagonists of the c-fos promoter as defined herein. These functionally equivalent sequences that can be easily prepared using known methods such as chemical synthesis, PCR employing modified primers and site-directed mutagenesis, are within the scope of this invention. As used herein, the term "recombinant vector" includes both recombinant plasmids and those mentioned herein as recombinant retroviral vectors which may also be modified as described by Geller et al., Proc. Nati Acad. Sci. USA, 87, 1149 (1990). The aforementioned recombinant vectors can be used to transfect any mammalian cell capable of undergoing transfection and allow duplication of the vector, as defined herein. Even though cells from fresh tissue implantations (primary cells) could in principle be used, the use of established cell lines is preferred. Many of these cell lines are available including, eg, mouse cell line NIH 3T3 (ATCC # CRL 1658), mouse LM (TK-) (ATCC # CCL 1.3) and mouse BABL / c 3T3 Clone A31 (ATCC # CCL 163). The selection of a cell or cell line for use in the methods of the present invention will be regulated by known or determinable specificities of the vectors used. For example, murine cell lines are preferred for use with vectors comprising a recombinant vector containing the large polyomavirus T antigen gene under the control of a regulated promoter, such as the human c-fos promoter; and a mammalian recombinant expression vector comprising a polyomavirus duplication origin and a nucleic acid that is suspected to encode a human promoter activating protein such as a retroviral expression vector comprising a retroviral LTR capable of expressing the nucleic acid. Even when cells for use in the present invention were transiently transfected, stably transformed cells can also be used. Stable transformation of a mammalian cell line can be achieved using normal methods of co-transfecting the cells with one of the aforementioned recombinant vectors and with a second vector that confers resistance to the selection agent such as an antibiotic. To identify the nucleic acids encoding human c-fos promoter activating proteins using the methods of this invention, the cells are co-transfected with a recombinant vector comprising a human c-fos promoter operably linked to the large polyomavirus T antigen gene, and a cDNA library incorporated into a mammalian recombinant expression vector comprising an origin of duplication of polyomavirus. The cells are then incubated under conditions wherein the vectors containing the cDNA encoding a human c-fos promoter activating protein will stimulate the augmented vector duplication. The cells are then harvested, the plasmids are extracted and the unduplicated vectors are selectively digested with pnl. The duplicated plasmids are recovered by transforming the competent bacteria with the Dpnl-digester. Typical incubations are carried out for 2 days at 37 ° C in a humid CO2 incubator, even though the selection of conditions will be evident to those skilled in the art and will depend, eg, on the nature of the cells , the medium used and the type of the culture container. The incubation is continued for a sufficient period of time to allow the development of an intense duplicate response. The optimal time is determined by routine experiments but will typically be within the range of approximately 24 to 72 hours. A considerably increased level of vector duplication and recovery after digestion with DpnI will be detected for those vectors comprising nucleic acids encoding the human c-fos promoter proteins compared to the background resulting from the duplication of vectors lacking these nucleic acids. A considerable increase in vector duplication and recovery is typically an increase of at least about 5 times, preferably of about 8 times and especially preferably of about 20 times, above the level measured in complete absence of a plasmid that comprises a nucleic acid encoding a human c-fos promoter activating protein. The degree of increase will depend mainly on the level of background duplication. Essentially the same procedures are used to identify the nucleic acids encoding other human promoter activating proteins, using vectors comprising the promoter operably linked to a nucleic acid encoding the large polyomavirus T antigen. By selecting antagonists of the human c-fos promoter activating protein using the methods of this invention, cells are provided which are transfected simultaneously with a first vector of A recombinant expression comprising a reporter gene operably linked to a human c-fos promoter and a , second vector comprising a nucleic acid encoding a human c-fos promoter activating protein. The preferred reporter genes are the fos-CAT reporter gene Which will be described below or a reporter gene fos-lac Z. The cells are planted in an appropriate culture medium for the class of cells used. The cells are then incubated in the absence (control) or presence of variable amounts of samples containing suspect antagonists under conditions in which the gene encoding the human c-fos promoter activating protein is expressed. Under these conditions and in the absence of an antagonist, the stimulation of the human c-fos promoter will occur, resulting in the expression of the reporter gene. The samples, for example, may be aqueous solutions or miscible in water in which the isolated compounds have been dissolved, or individual fractions or collected from purification steps such as chromatographic or electrophoretic fractions. Typical incubations are carried out at a temperature of about 37 ° C in a humid CO2 incubator even when the selection of conditions will be apparent to those skilled in the art and will depend, eg, on the nature of the cells, the medium used and the type of culture container. The incubation is continued for a period of time sufficient to allow significant induction of the reporter gene at which time the level of expression of the reporter gene is measured by an appropriate assay. The optimal time to carry out the measurement is determined by routine experiments but will typically be within the range of about 24 to 72 hours, preferably about 48 hours. The highest levels of reporter gene expression will be measured in the control cultures (free of antagonist). When a culture contains an antagonist of the human c-fos promoter activating protein, a reduction in the level of reporter gene expression will be measured, the extent of which will be a direct function of the amount of the antagonist added to the medium. Antagonists present in samples added to certain cultures will be identified by measuring a significantly decreased level of reporter gene expression compared to the measured level of control cultures. A substantially decreased level of reporter gene expression is defined as a decrease of at least about 50 percent, and preferably at least about 70 percent of the level measured in the complete absence of an antagonist of a promoter-activating protein. c-fos human. Of course, the degree of decrease can be influenced by the amount of the antagonist present in the sample compared to the amount of the activating protein of the c-fos promoter used and the efficiency of the antagonist. The decreased levels of reporter gene expression due to general toxicity of the samples can be explained by transfecting a second constitutively expressed reporter gene such as lac-Z driven by a beta-actin promoter and normalizing the activity of the reporter gene c-fos to the expression lac-Z. The following non-limiting examples will serve to illustrate the present invention.

EXAMPLES General Materials and Methods: Unless specified otherwise, the percentages given below for solids in solid mixtures, liquids in liquid mixtures and solids in liquids are on a weight / weight, volume / volume and weight / volume basis, respectively. Sterile conditions are maintained during cell culture. Normal recombinant methods were used, such as those described in the Sambrook article, and others, "Molecular Cloning, A Laboratory Manual, 2 ed", Cold Spring Harbor Laboratory Press (1989). Dpnl is a known restriction endonuclease isolated from Diplococcus pneumoniae and can be obtained commercially from ICN Biomedicals, Sigma Chemical Company or New England BioLabs, Inc. The restriction endonucleases Asel, BamHI, BglII, BstXl, Clal, Fspl, Hincl, Narl, Notl, SacI I, Sali, Seal, Xibal and Xhol are known and can be obtained commercially, eg, from Sigma Chemical Company. The restriction endonucleases BamHI, BssHII, BstXI, HincII, SalI, Seal and Xbal are known and can be obtained commercially, v.gr, from ICN Biomedicals. The restriction endonucleases AflIII, Asel, BamHI, BglII, BssHII, BstXI, Clal, FspI, HincII, Nael, NarI, NotI, SacI, SalI, Seal, XbaI and XhoI are known and '- * can be obtained commercially, v. gr., from? ew England 10 BioLabs, Inc. Saul restriction endonuclease is known and can be obtained commercially, v.gr, from Boehringer Mannheim. The nuclease of the enzyme "mung" bean is known and can be obtained commercially from? Ew Englan Biolabs, Ine or Sigma Chemical Company. The synthetic polylinker used to prepare the Lalfa2 vector was obtained from? Ew England Biolabs, Inc. and has the sequence shown in SEQ ID? O: 2. The linker Ncol, d (pAGCCATGGCT) is known and commercially available from? Ew England Biolabs, Inc. (catalog # 1150). The vector pUC19 (ATCC 37254, GenBank Accession #: X02514) can be obtained commercially from? Ew England Biolabs, Inc. or IC? Biomedicals. The nucleotide sequence and the restriction sites of pUCl9 are described in Yasnisch-Perron, et al., In Gene, 33, 103-119 (1985). The following DNA, used to prepare the plasmids of the present invention, is publicly available: The polyoma virus strain A2 (ATCC # 45017); and human c-fos genomic c-fos (ATCC # 41042). In addition, the DNA sequence of strain A2 of polyoma virus is disclosed in DNA Tumor Viruses, ed. Tooze, J. (1980) (Cold Spring Harbor Press), pages 834 to 838. The construction of the retroviral vector pMV7 is described by Kirschmeier, et al., DNA, 7, 219-225 (1988), starting from the plasmids pPyori and pMV (ATCC # 37190). The vector pMV7 is well known in the art and is freely and extensively distributed in many laboratories. In addition, retroviruses similar to pMV7 that could otherwise be used in this invention are readily available, such as pV-mos (ATCC # 41037). The construction of the fos-CAT reporter gene that was described below was prepared using the commercially available pCAT basic vector (Promega catalog # E1041). Mouse monoclonal antibodies directed against the hemagglutinin epitope and fluorescein-conjugated rabbit anti-mouse IgG are commercially available from Boehringer Mannheim.

For selection of a cDNA library, a unidirectional cDNA library of poly A RNA from the human brain (Clontech, Palo Alto, CA) was prepared using the Superscript GIBCO cloning kit (Grand Island, NY) and inserted into the sites. Sall / Notl in the plasmid Lalfa2. The separation and visualization of nucleic acids was carried out as described in the Sambrook article, and others, supra, by electrophoresis in agarose gels and visualization with ethidium bromide. The entire nucleotide sequence was carried out using the dideoxy-mediated chain termination method described in the article by Sanger et al., Proc. Nati Acad. Sci. USA, 74, 5463-5467 (1977). To obtain the sequences of CROC-1 and CROC-4, the DNA sequence was carried out on both strands. Co-transfection of the cells with PfLAG and Lalfa2 containing a cDNA encoding a biologically active signal molecule causes the activation of the c-fos promoter, resulting in the production of the large T antigen. The production of the large T antigen stimulates the intracellular duplication of the plasmids containing the polyomavirus duplication origin. The plasmids are recovered from the cultures of the transfected cell by "Hirt extraction" using the methods described in Hirt, J. Mol. Biol., 26, 365-369 (1967). The unduplicated plasmids are selectively destroyed by restriction with Dpnl. The duplicated plasmids are then recovered by transformation in competent bacteria. NIH 3T3 mouse fibroblasts from early passage (ATCC # CRL 1658) and rat 2 fibroblasts (ATCC # CRL 1764) were grown in DMEM supplemented with 10 percent bovine calf serum and 50 micrograms per milliliter of gentamicin sulfate. The E. coli DH10B used in the present invention can be obtained commercially from GIBCO.

Construction of plasmids: Two basic plasmids were constructed for use in the present invention. The first one (represented as PfLAG) comprised a large polyomavirus T antigen gene regulated by a human promoter that served as a source of the large T antigen during promoter activation, and which was based on the human c-fos promoter. The second plasmid (represented as Lalfa2) was a retroviral cDNA expression vector containing the polyomavirus duplication origin.

The Lalfa2 vector of retroviral cDNA was prepared in the following manner. Strain A2 of polyomavirus DNA was digested with Ba Hl / Narl and the resulting 750 bp fragment was ligated into the JBamHI / Narl sites in pUC19 to provide a plasmid designated pOri. The retroviral vector pMV7 was digested with FspI / AfJIII and the resulting 4 kb band containing two Moloney murine sarcoma LTR viruses was ligated into the J? IncII / AflIII fragment of pOri, to provide a plasmid called pMV7-2. A neomycin resistance gene present between the two murine sarcoma virus Moloney LTR and pMV7-2 was removed by digestion of Saul / Clal and replaced by a synthetic polylinker (described above) to provide the plasmid pMV7-3. To allow blue-white selection, the polylinker in pUC19 was replaced by a? -col linker and then the 360 bp region of lac Z was removed by digestion with Asel / Narl, terminated with "mung" bean nuclease and ligated in the polylinker pMV7-3. The resulting plasmid named Lalfa2 was 4.5 kb and contained unique SalI and NotI sites at the 5 'and 3' ends respectively of the lac Z gel. An initial translation codon followed by a sequence of AD? encoding a histidine hexamer, was inserted 5 'with respect to the AD? c insertion site to ensure expression of the AD? c-encoded protein from truncated AD? c inserts lacking initial codons and to assist to the subsequent protein purification. Plasmid PfLAG was prepared by the following procedure. The large polyomavirus T antigen under the control of the human c-fos promoter was introduced by digesting the 5.9 kb BamHI fragment from pcfos-1, disclosed by Curran et al., Mol. Cell. Biol., 3, 914-921 (1983), with Nael to remove the entire coding region of the c-fos gene and insert the 2.8 kb band of BstXI / i? IncII from the polyomavirus encoding the polyoma T antigen. The intermediate and small T expression was removed by inserting a stop codon at the Seal site by placing at position 605 of the AD? of polyomavirus disclosed by Tooze, supra. The resulting construct was named PfLAG-1 (for the _fos promoter / large T antigen). A third vector, called HEL, was prepared to be used to identify the intracellular locations of CROC-1. The coding sequences of the histidine hexamer of Lalfa2 were removed by digestion of BglII / Sall and replaced with coding sequences for the nine amino acid-influenced viruses of the HAI epitope described in the article by Field et al., Mol. Cell. Biol., 8, 2159-2165 (1988). The duplication origin of SV40 was then inserted into the unique Xbal site between the origin of polyoma duplication and 5 'LTR to provide HEL. A fourth vector was prepared to be used to confirm the ability of the activating proteins of the human c-fos promoter suspected to stimulate the c-fos promoter. The fos-CAT reporter gene described by Deschamps et al., In Science, 233, 1174-1177 (1985) was prepared by inserting the human c-fos promoter from -735 (BamHl site) to +42 (Nael site) on top of the CAT gel. bacterial in the basic vector pCAT (Promega).

Determining the upgrading requirements for contingent duplication that is dependent on PfLAG: For purposes of the present invention, the human c-fos promoter in PfLAG must remain transcriptionally silent in the still cells, but be sensitive enough to respond to the low level expression of the molecules by active signals encoded with AD? C producing sufficient T antigen to cause duplication of the plasmid. The sensitivity and level of gene induction from the promoter can be increased by incorporation of additional enhancer elements in the promoter. The multiple enhancer elements were incorporated into PfLAG-1 by isolating a fragment of approximately 500 bp or more of XhoI / BssHII (blunt end) containing the c-fos enhancing elements and ligating the enhancer region at the Xhol / SacI l site ( of blunt end) of the previous PfLAG. To determine the number of breeders required to exhibit contingent duplication, a series of PfLAG containing 1, 2, 4 and 8 enhancer regions were constructed. The following experiments were then carried out to define the upgrading requirements for the contingent duplication that depends on PfLAG. Muramatsu and others, Mol. Cell. Biol., 9, 831-836 (1989) have shown that the expression of the catalytic domain of protein kinase C includes the c-fos promoter. The deduced amino acid nucleotide sequence of the C-betal rat protein kinase is described in the article by Housey et al., Cell, 52, 343-354 (1988). The catalytic domain of the C-betal rat protein kinase was incorporated into Lalfa2 to produce a construct called pMVPkCAbetal. The co-transfection of a mixture of pMVPkC / \ betal / Lalfa2 with PfLAG containing 1, 2, 4 or 8 enhancer regions, would therefore provide a means to test the sensitivity of each PfLAG. A detection threshold sensitivity about a plasmid of forty for cDNA selection was that which was used. Therefore, a ratio of 1:40 (w / w) of pMVPkC / \ betal / Lalfa2 for co-transfection with each of PfLAG to NIH 3T3 cells was used in the procedure described below. The cells were incubated for forty-eight hours after transfection. The plasmids were extracted and examined after digestion with Dpnl for high recovery of the plasmid indicative of contingent duplication. The results obtained under these conditions are presented in Table 1. These data demonstrate that eight enhancer regions (PfLAG-8) were required for significant activation of plasmid duplication allowing an eight-fold increase in plasmid recovery relative to the antecedents resulting from the co-transfection of PfLAG-8 with the vector alone. Induction with PfLAG-8 varied from 6 fold to more than 20 times increases in plasmid recovery, depending mainly on the background level.

Table 1. Requirement of the human c-fos enhancer to activate the activated contingent duplication of the large polyomavirus T antigen. * Construction Number of Plasmid Total Number of Plasmid Co-transfected Regions of Colonies Enhancers PfLAG-1 pMV7-Z PfLAG-1 pMV7PkCAbetal Lalfa2 PfLAG-2 pMV7-Z 33 PfLAG-2 pMV7PkCAbetal 22 Lalfa2 PfLAG-4 pMV7-Z 109 PfLAG-4 pMV7PkCAbetal 101 Lalfa2 PfLAG-8 pMV7-Z 363 PfLAG-8 pMV7PkC / \ betal 1915 pMV7PkCAbetal Lalfa2 * 2 micrograms of PfLAG were co-transfected with 18 micrograms of either pMV7-Z or a 1:40 (w / w) ratio of pMV7PkCAbetal / Lalfa2. The results presented are the average of two experiments.

The effect of the concentration of plasmids encoding a promoter-activating protein in the recovery of pMVPkCAbetal within a total population of plasmids is determined by varying the concentration of pMVPkCAbetal in a mixture of pMVPkC / \ betal / Lalfa2 before c-transfection with PfLAG-8. Because Lalfa 2 has a modified lac Z gene derived from pUC19, bacteria transformed with Lalfa2 will turn blue, while bacteria transformed with pMVPkCAbetal will remain white when tested on agar plates containing ampicillin, X-gal and IPTG. The percentage of pMVPkCAbetal is determined by expressing the number of white colonies as a percentage of total colonies formed after bacterial transformation of Hirte extracts digested with Dpnl. Experiments were carried out by co-transfecting PfLAG-8 with mixtures of pMV7PkC / \ betal / Lalfa2 starting at a ratio of 1:80 (w / w), then diluting to a ratio of 1: 400 using the methods to be described then. The competent E. coli DH10B was transformed into extracts of Hirt digested with DpnI and tested on agar containing ampicillin, X-gal and IPTG. The percentage of pMV7PkC / \ betal in the colonies recovered was determined by the number of white colonies in relation to the total colonies.

To ensure that the white colonies resulted from the transformation of pMVPkCAbetal, the plasmids recovered and the restriction was presented on a map. All white colonies showed the restriction pattern of 5 pMVPkCAbetal correct. The results presented in Table 2 show that even when the number of colonies recovered is reduced to the background levels at high dilution of pMV7PkAbetal, the actual percentage of < "colonies of pMV7Pk / \ betal increases, indicating that they can transfect a minimum of 400 library colonies with PfLAG-8 to enrich a population of cDNA library for signal transduction molecules encoding cDNA. The selection of the initial cDNA library, therefore, was carried out with plasmid groupings that comprise of 400 or more plasmids in order to acquire a library population enriched with coding activators of the c-fos promoter cDNAs.

Table 2. Dependence on the concentration of 20 pMV7PkC / \ betal in the recovery of the plasmid. * Ratio of Colony Percentage Number pMV7PkC / \ betal Colonies of per / Lalfa2 Blue White pMV7PkC / \ Beta Co-transfected vessel 1:80 206 4 1.9 210 1: 160 146 7 4.6 153 1: 240 134 6 4.3 '150 , / 1: 320 97 24 19.8 121 1: 400 90 24 21. .1 114 * Co-transfection with PfLAG-8 and Lalfa2 alone provided a background of 102 colonies / vessel in this experiment 20 Cell Culture and Transfection: For transfections, 8 x 10 ^ 3T3 cells were planted in the growth medium in 100-millimeter vessels and allowed to settle overnight. The next day, the transfections were carried out by the method of igler et al., Cell, 11, 223-232 (1977) using calcium phosphate. After the 4 hour exposure to the calcium phosphate precipitate, the cells were raised twice with phosphate-stabilized saline, re-fed with DMEM supplemented with 0.5 percent bovine calf serum and incubated at 37 ° C for from 40 to 48 hours. The cells were harvested and the plasmids were extracted by the procedure of Hirt, supra. The extracted plasmids were digested with Dpnl for a minimum of 24 hours. The Dpnl digestives were extracted with phenol and precipitated in ethanol. The DNA was resuspended in 20 microliters of TE (1 mM EDTA + 10 mM Tris, pH 8.0) and transformed into competent DH10B bacteria (GIBCO). The co-transfections were carried out through the aforementioned procedure at a cDNA / PfLAG ratio of 9: 1 (w / w) using 20 micrograms of DNA per vessel.

Selection of cDNA Library Using Contingent Duplication: A human brain cDNA library was co-transfected with PfLAG-8 in NIH 3T3 cells through the methods described above. The plasmid deposits comprised approximately 30 to 40 plasmids and were co-transfected with PfLAG-8 and examined for a minimum 5-fold increase in plasmid recovery. The plasmids of the active deposits were recovered and sub-divided into secondary deposits of four plasmids each and examined in a similar manner for activation of the contingent duplication. The plasmids of each active secondary deposit were then examined individually for contingent application. From approximately 1,400 initially selected plasmids, two plasmids named CROC-1 and CROC-2 (for contingent doubling of cDNA) consistently provided high plasmid recovery when co-transfected with PfLAG-8. The nucleotide sequence for CROC-1 is shown in SEQ ID NO: 1. A third plasmid, designated CROC-4 was identified by additional plasmid selection. Plasmid CROC-4 also consistently provided high plasmid recovery when co-transfected with PfLAG-8. The nucleotide sequence for CROC-4 is shown in SEQ ID NO: 3.

Promoter Activation Confirmation c-fos Using a fos-CAT Reporter Gen: Certain extraneous factors could also cause the high plasmid recovery observed in the contingent duplication test. For example, incomplete bacterial methylation of Dpnl sites will confer Dpnl resistance to differences in transfection efficiency or transformation.To eliminate these possibilities, each CROC-1, CROC-2 and CROC-4 was transfected. with a fos-CAT reporter gene and tested for CAT activity elevation in the following manner: Rat 2 cells were co-transfected with 18 micrograms of cDNA expressed with Lalfa2 (i.e., CROC-1, CROC-2 or CROC- 4) + 2 micrograms of fos-CAT for 4 hours and then fed with DMEM + 0.5 percent calf serum. Cells were harvested 72 hours after transfection and CAT assays were carried out through the procedure of Gorman et al., Mol. Cell. Biol., 2, 1044-1051 (1982). 15 CAT activity was significantly induced by CROC-1, CROC-2 and CROC-4 ^ indicative of activation of the c-fos promoter. The degree of activation was approximately 50 percent of the activation caused by co-transfection with pMVPkCAbetal. In contrast, the vector alone did not induce significant CAT activity nor did it randomly select the plasmids from the cDNA library isolated from the same plasmid deposits as CROC 1, 2 and 4, but did not activate the contingent duplication. These results confirm that the The high plasmid recovery observed during co-transfection of CROC 1, 2 or 4 with PfLAG-8 was due to the activation of the c-fos promoter in contingent duplication mediated with PfLAG.

Analysis of c-fos Activating Proteins The sequence revealed that CROC-2 encodes the protein associated with the newly identified alpha2-macroglobulin receptor (AMRAP) disclosed in the article by Strickland et al., J. Biol. Chem., 266, 13364-13369 (1991). The insertion is almost full length and extends from the starting codon that is framed with the start codon of the internal vector to the tail of poly A. A sequence of 347 base pairs corresponding to nucleotides 555-897 of CROC -4 has been submitted to GenBank (Accession number Z40809) as an expression sequence tag. The CROC-1 cDNA encodes 19 kd of protein with an amino acid terminal half and a basic carboxy terminus as shown in SEQ ID NO: l. The protein includes a kinase target domain that contains phosphorylation sites for a variety of kinases involved in signal transduction. Specifically, the kinase target region comprises adjacent potential nearby phosphorylation sites for: (a) tyrosine kinases (motif RXXXEXXXY, amino acids 81-89), Cooper et al., J. Biol. Chem., 259, 7835 -7841 (1984); casein kinase 2 (TIYE motif, amino acids 82-85), Kuenzel et al., J. Biol. Chem .; 262, 9136-9140 (1987); protein kinases dependent on cAMP (motif RIYS, amino acids 87-90) Glass et al. J. Biol. Chem., 261, 2987-2993 (1986) and Kishimoto et al., J. Biol. Chem., 260 12492-12499 (1985); and C protein kinase (SLK motif, amino acids 90-92), Kishimoto et al., supra. The kinase target domain of the CROC-1 protein is a stretch of twelve amino acids placed at the beginning of the basic domain. The known transactivation capacity of the acidic domains in general, which is combined in the potential of the basic domains to bind the DNA, suggests that CROC-1 could function as a transcriptional activator whose activity is regulated by phosphorylation of the kinase target domain . The phosphorylation would cause an additional increase in the acidity of the region, thus improving its potential for transcriptional activation, as well as to cause a change in the structural conformation of the protein. The tissue length and distribution of CROC-1 mRNA was determined by Northern analysis of the RNA containing poly A isolated from various human tissues, using the 1.8 kb Sall / Notl insert of CROC-1 as a test probe. The CROC-1 mRNA was approximately 2.3 kb in length, approximately 0.5 kb larger than our cDNA insert, and present in all tissues examined at the highest levels expressed in the brain, skeletal muscle and kidney. In comparison, the 1.5 kb of CROC-2 mRNA was present in all tissues examined, but with the highest levels being expressed in the heart, placenta and kidney. No additional transcript signals were found as a result of alternative splicing or multiple sets of transcription-termination-polyadenylation signals, as disclosed for CROC-2 by Strickland et al., Supra. The intracellular localization of the CROC-1 protein was determined by cloning CROC-1 in HEL and electroporating the resulting plasmid into COS-7 cells (ATCC # CRL 1651). Incorporation of the CROC-1 nucleic acid into the HEL vector allows fusion of the hemagglutinin epitope on the CROC-1 protein. The intracellular location of the CROC-1 protein was then determined by immunofluorescence microscopy using the anti-mouse monoclonal body directed against the hemagglutinin epitope. The electroporation of CROC-1 in HEL resulted in intense nuclear fluorescence. In contrast, HEL electroporation only resulted in a general cytoplasmic fluorescence indicating that nuclear localization is an inherent property of the CROC-1 protein. The present invention encompasses modifications and variations that will be apparent to those skilled in the art. The specific embodiments described herein are representative examples only, the scope of the present invention having been defined by the claims.

SEQUENCE LIST (1. GENERAL INFORMATION: (i) APPLICANT (A) NAME: Schering Corporation Patent Department K-6-1 (1990) (B) STREET: 2000 Galloping Hill Road (C) CITY: Kenilworth (D) STATE: New Jersey (E) COUNTRY: United States of America (F) ZIP CODE: 07033-0530 (G) TELEPHONE: 908- 298-5150 (H) TELEFAX: 908-298-5388 (I) TELEX: (ii) TITLE OF THE INVENTION: Method for Identifying Nucleic Acids Encoding Activator Proteins of the c-fos Promoter. (iii) SEQUENCE NUMBER: 3 (iv) READILY FORM ON THE COMPUTER: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Macintosh - • * (C) OPERATING SYSTEM: Macintosh 7.1 (D) SOFTWARE: Microsoft Word 5.1a (v) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: PCT / US95 / (B) SUBMISSION DATE: June 1995 (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 272,412 0 (B) SUBMISSION DATE: July 8, 1994 (2) INFORMATION FOR SEQ ID NO: l: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 1930 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear 0 (ii) TYPE OF MOLECULE: cDNA (iii) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l ATG GAT CTC AGG CCT AGA TCT CAT CAC CAT CAC CAT CAT TGG TGC CAG 48 Met Asp Leu Arg Pro Arg Ser His His His His His His His Trp Cys Gln 5 10 15 TGT GCT GGT CGA CCC ACG CGT CCG GAT GGC AGC CAC CAC GGG CTC GGG 96 Cys Wing Gly Arg Pro Thr Arg Pro Asp Gly Ser His His Gly Leu Gly 20 25 30 AGT AAA AGT CCC TCG CAA TTT CGA CTG TTG GAA GAA CTC GAA GAA GGC 144 Ser Lys Ser Pro Ser Gln Phe Arg Leu Leu Glu Glu Glu Lelu Glu Glu Glu 35 40 45 CAO AAA GOA GTA GGA GAT GGC ACA GTT AGC TGG GGT CTA GAA GAT GAC 192 Gln lys Gly Val Gly Asp Gly Thr Val Ser Trp Gly Leu Glu Asp Asp 50 55 60 GAA GAC ATG ACÁ CTT ACÁ AGA TGG ACÁ GGG ATG ATA ATT GGG CCT CCA 240 Glu Asp Met Thr Leu Thr Arg Trp Thr Gly Met lie lie Gly Pro Pro 65 70 75 80 AGA ACÁ ATT TAT GAA AAC CGA ATA TAC AGC CTT AAA ATA GAA TGT GGA 288 Ring Thr He Tyr Glu Asn Aro lie Tyr Ser Leu Lvs He Glu Cys Gly 85 90 95 CCT AAA TAC CCA GAA GCA CCC CCC TTT GTA AGA TTT GTA ACÁ AAA ATT 336 Pro Lys Tyr Pro Glu Wing Pro Pro Phe Val Arg Phe Val Thr Lys He 100 105 110 AAT ATG AAT GGA ATA AGT TCT AAT GGA GTG GTG GAC CCA AGA GCC 384 Asn Met Asn Gly Val Asn Ser Ser Asn Gly Val Val Asp Pro Arg Wing 115 120 125 ATA TCA GTG CTA GCA AAA TGG CAG AAT TCA TA ^ AGC ATC AAA GTT GTC 432 He Ser Val Leu Wing Lys Trp Gin Asn Ser Tyr Ser He Lys Val Val - - 130 135 140 CTG CAA GAG CTT CGG CGC CTA ATG ATG TCT AAA GAA AAT ATG AAA CTC 480 Leu Gln Glu Leu Arg Arg Leu Met Met; Being Lys Glu Asn Met Lys Leu 145 150 155 160 CCT CAG CCG CCC GAA GGA CAG TGT TAC AGC AAT TAA TCA AAA AGA AAA 528 Pro Gln Pro Pro Glu Gly Gln Cys Tyr Ser Asn *** 165 170 ACC ACA GGC CCT TCC CCT TCC CCC CAA TTC GAT TTA ATC AGT CTT CAT 576 TTT CCA CAG TAG TAA ATT TTC TAG ATA CGT CTT GTA GAC CTC AAA GTA 624 CCG GAA AGG AAG CTC CCA TTC AAA GGA AAT TTA TCT TAA GAT ACT GTA 672 AAT GAT ACT AAT TTT TTG TCC ATT TGA AAT ATA TAA GTT GTG CTA TAA 720 CAA ATC ATC CTG TCA AGT GTA ACC ACT GTC CAC GTA GTT GAA CTT CTG 768 GGA TCA AGA AAG TCT ATT TAA ATT GAT TCC CAT CAT AAC TGG TGG GGC 816 ACA TCT AAC TCA ACT CTG AAA AGA CAC ATC ACÁ CAA TCA CCT TGC TGC 864 TGA TTA CAC GGC CTG GGG TCT CTG CCT TCT CCC TTT ACC CTC CCG CCT 912 CCC ACC CTC CCT GCA ACÁ ACÁ GCC CTC TAG CCT GGG GGG CTT GTT AGA 960 GTA GAT GTG AAG GTT TCA GGT CGC AGC CTG TGG GAC TAC TGC TAG GTG 1008 TGT GGG GTG TTT CGC CTG CAC CCC TGG TTC CTT TAA GTC TTA AGT GAT 1056 GCC CCT TCC AAA CCA TCA TCC TGT CCC CAC GCT CCT CCA CTC CCG CCC 1104 TTG GCC GAA GCA TAG ATT GTA ACC CCT CCA CTC CCC TCT GAG ATT GGC 1152 TTC GGT GAG GAA TTC AGG GCT TTC CCC ATA TCT TCT CTC CCC CCA CCT 1200 TTA TCG AGG GGT GCT GCT TTT T T CCC TCC TCC TCA AGT TCC TTT TTG 1248 CAC CGT CAC CAC CCA ACÁ CCT TCC ATG ACÁ CTT CCT TGC TTT GGC CAG 1296 AAG CCA TCA GGT AAG GTT GGA AAG AGC CTC TGA CCT CCC TTG TTT AGT 1344 TTT GGA ACC ATA CTC ACT CAC TCT CCA CCA GCC TGG GAA ATG AAT ATT 1392GGG TCC TCA GCC CTG CCA CCC TCT GCT GTC ATC AGC TGA TGC ATT GTT 1440 TTT AGC TCA GGT TTT GAT AAG GTG AAA AGA ATA GTC ACC AGG GTT ACT 1488 CAG ACC TGC CAG CTC TCG GAG TCC TTG GTG GTT GAA CTT GGA GAA AGA 1536 CCG CAT GAA GAT ACT TGT AAG CAC ACÁ TGA TCC CTC TGA ATT GTT TTA 1584 CTT TCC TGT AAC TGC TTT TGC TTT TAA AAA TTG AAG AAG TTT TAA ACÁ 1632 GGG CTT TCA TTT GGT CAT CCT TGC AAT CCA TTG GGG TCT AGT TTG GAA 1680 TCT GAC AAC TGG AAC AAA AAG AAC CTT GAA TCC GGT GCA TGC CTT GGT 1728 TTT GGT GCT GCT GCT GCT TCC CAA GAT CCT CAG CAG GGA TTA AGA AGG 1776 AAC CCG GTG TGC ACA GCA GAT CCC CGA AAT TGG TGG GCT TGA CCT CCT 1824 GGC AAA TTG CTG CGT CTT TCC ACT TGC TGA GCA CCA CTA AAT GCG 1872 AAA TGT GGA TGC ATA CCG AAA TAA AAG CAA TTC ATT GTG TAC TAA AAA 1920 AAA AAA AAA TO 1930 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE. »Double 15 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA? *. (iii) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: 20 CAC GTG AAT TCA AGA TCT CTG CAG AAG CTT TCC GGA CCG GGC CGC GTA 48 GCA CGC GTA ATA ATT ATC GAT 69 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 925 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 GTCGACCCAC GCGTCCGCTC CTCACAGAAG CCTGGAGCTG GGCATCCAAG AAGAAGCAGC 60 CTCATTTGTT TTCTGGTGTC ATCGTAGGTG GCCACCTATG GCTTTTGGGA ATGTAAAAAG 120 GGCAGCTCTC TGGC ATG TTC CTG ACT GAG GAT CTC ATA ACA TTT AAC TTG 170 Met Phe Leu Thr Glu Asp Leu He Thr Phe Asn Leu 5 10 AGG AAC TTC CTC CTT TTC CAG CTT TCG GAG TCA AGC TTC TCA CCT GGG 218 Arg Asn Phe Leu Leu Phe Gln Leu Trp Glu Ser Ser Phe Ser Pro Gly 15 20, 25 GCG GGT GGG TTC TGC ACC ACC CTC CCA CCC TTC TTC CTC CGT GTG GAC 266 Wing Gly Gly Phe Cys Thr Thr Leu Pro Pro Ser Phe Leu Arg Val Asp 30 35 40 GAT AGA GCC ACA TCC AGC ACC ACG GAC AGC TCC CGG GCG CCT TCA TCT 314 Asp Arg Wing Thr Ser Ser Thr Thr Asp Ser Ser Arg Wing Pro Ser Ser 45 50 55 60 CCT CGT CCT CCA GGC AGC ACA AGC CAT TGT GGA ATC TCC ACC AGG TGT 362 Pro Arg Pro Pro Gly Ser Thr Ser His Cys Gly He Ser Thr Arg Cys 65 70 75 ACÁ GAA CGG TGC CTC TGC GTC CTG CCA CTC AGG ACC TCT CAA GTC CCC 410 Thr Glu Arg Cys Leu Cys Val Leu Pro Leu Arg Thr Ser Gln Val Pro 80 85 9C GAT GTG ATG GCT CCT CAG CAT GAT CAG GAG AAA TTC CAT GAT CTT GCT 458 Asp Val Met Ala Pro Gln His Asp Gln Glu Lys Phe His Asp Leu Wing 95 100 105 TAT TCC TGT CTT GGG AAG TCC TTC TCC ATG TCT AAC CAA GAT CTA TAT 506 Tyr Ser Cys Leu Gly Lys Ser Phe Ser Met Ser Asn Gln Asp Leu Tyr 110 115 120 GGC TAT AGC ACC AGC TCT TTG GCT CTT GGC TTG GCA TGG CTA AGT TGG 554 Gly Tyr Ser Thr Ser Ser Leu Wing Leu Gly Leu Wing Trp Leu Ser Trp 125 130 135 140 GAG ACC AAA AAG AAG AAT GTA CTT CAT CTG GTT GGG CTG GAT TCC CTC 602 Glu Thr Lys Lys Lys Asn Val Leu His Leu Val Gly Leu Asp Ser Leu 145 150 155 TGATAAGCCT TCCCAGTTGA CTGAAAGATG AGGCTAGGCT CTAGCAAGTT GAAGTCAAAC 662 * * * CAGCTCCTTC AAGAAGCTTT GAGCAGAATG AAGTGGGGAG GACCCAGCTT CCAGCCCAGG 722 AAGCCCACTG TACCTGGAGC CATCTGGGAT AAGACTTTGA CCCATGACTC CCATATCCAC 782 AGCCTGTCCA TCCTAGCCCA TCCCAGTTTA TCCTGTATCA TTTGAGCTGG GATTCCCACA 842 TCCTCTGAGT TGGAAGTCCC ATCTCAAGTC TTCAATAAAG ACTCTTGAAT ATTGAAAAAA 902 925 CGC AAAAAAAAAA AAAGGGCGGC

Claims (18)

CLAIMS:

1. A mammalian cell line, the cells of which comprise: (a) a recombinant vector comprising a specific inducible promoter or tissue operably linked to a nucleic acid encoding the large polyomavirus T antigen; and (b) a recombinant expression vector comprising a polyomavirus duplication origin and a nucleic acid that is suspected of encoding a promoter-activating protein.

2. A mammalian cell line according to claim 1, wherein the promoter is the human c-fos promoter.

3. A mammalian cell line according to claim 2, wherein the recombinant vector is the PfLag-8 plasmid.

4. A mammalian cell line according to claim 1, wherein the expression vector is the Lalfa2 plasmid.

5. A vector comprising a human c-fos promoter operably linked to a nucleic acid encoding the large polyomavirus T antigen.

6. A vector according to claim 5, wherein the plasmid is pfLag-8.

7. A recombinant expression vector comprising a polyomavirus duplication origin and a nucleic acid that is suspected of encoding a promoter-activating protein.

8. A vector according to claim 7 which is the plasmid Lalfa2.

9. A method for identifying a nucleic acid encoding a promoter activator protein comprising: (a) culturing a mammalian cell line according to claim 1, under conditions in which these nucleic acids are expressed; and (b) measuring the levels of the duplicate vectors in the cells after a sufficient incubation period to allow duplication of the vector; whereby, a nucleic acid encoding a promoter activating protein is identified by measuring the increased levels of vectors in the cells.

10. A method according to claim 9, wherein the promoter is the human c-fos promoter.

11. A human c-fos promoter activating protein having an amino acid sequence as defined by SEQ ID NO: 1 or SEQ ID NO.3, or an antigenic fragment thereof.

12. A nucleic acid encoding a protein according to claim 11.

A nucleic acid according to claim 12, having essentially the same sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3

14. A mammalian cell line, the cells of which comprise: 0 (a) a first recombinant expression vector comprising a reporter gene operably linked to the human c-fos promoter; and (b) a second recombinant expression vector comprising a nucleic acid encoding a human c-fos promoter activating protein.

15. A mammalian cell line of "according to claim 14, comprising NIH 3T3 mouse cells.

16. A mammalian cell line of 0 according to claim 14, wherein the second recombinant expression vector encodes the CROC-1 protein, the CROC-4 protein, or a protein associated with the alpha2-macroglobulin receptor.

17. A recombinant expression vector comprising a reporter gene operably linked to a human c-fos promoter.

18. A method for identifying an antagonist of a human c-fos promoter activating protein comprising: (a) providing a mammalian cell line according to claim 14; (b) contacting the cell line of step (a) with a sample suspected of containing an antagonist of the human c-fos promoter activating protein; and (c) measuring the expression level of the reporter gene; whereby an antagonist of the human c-fos promoter activating protein is identified by measuring a reduced level of expression of the reporter gene.