CA2194361A1 - Method for identifying nucleic acids encoding c-fos promoter activating proteins - Google Patents

Abstract

Materials and methods are described for identifying signal transducing molecules which activate promoters, such as the human c-fos proto-oncogene promoter, as well as antagonists of such molecules. Also described are human cfos promoter activating proteins, and in particular novel proteins, designated CROC-1 protein and CROC-4 protein, nucleic acids encoding said proteins, and mammalian cells transfected with vectors containing such nucleic acids.

Description

~0 9G/01899 1~ r ~1~ /4 219431~1 ~IF,THOD FOR IDENTIFYING NUCl,F,lC ACIDS I~NCODIl~IG
c-fos PROMOTE,R ACTIVATlNG PROTEINS

The present invention relates to materials and 5 methods for identifying signal trAneducing molecules which activate the human c-fos proto-oncogene promoter and antagonists of such molecules.
BACKGROUND O~ THF, INVF:I~TION
Cell activation as a result of mutation or over-expression of signalling mnl~cules, such as the proto-oncogenes Ha-ras, c-fos, c-myc, and c-jun, has been implicated in the aberrant growth of cells that forms the basis of neoplasia. See, 15 DeFeo, etaL,Proc. Natl. Acad. Sci..78, 3328-3332 (1981); Miller, et al., Cell, 36, 51-60 (1984); Kelekar, et al., Mol. Cell. Biol.. 6. 7-14 (1986); and Vogt, et al., Adv. Cancer Res.. 55, 1-35 (1990).
Induction of cfos occurs in response to the activation of growth-relate.d signalling pathways following 20 serum stim~ tion of mouse 3T3 cells, or in response to o~G.c~ ,;,sion of the normal and transforming versions of Ha-ras, IGs~e~liYGly. It has also been shown that COI~lilulive e~pression of c-fos occurs in certain human tumor lines. These findings suggest that the aberrant growth characteristic of the 25 neoplastic phenotype can involve the constitutive activation of signal transduction pathways participating in c-fos proto-oncogene induction. See, Greenberg. et al., Nature, ~, 433-438 (1984); Stacey, et al., Mol. Cell. Biol.. ~, 523-527 (1987); and O'Hara, et al., Mol. Cell. Biol.. 7, 2941-2946 (1987).
By using c-fos promoter-driven reporter genes, specific enhancers in the c-fos proto-oncogene promoter have been identified which respond to activated signal transduction pathways. These enhancers include a tyrosine kinase responsive SCM, raf~ ollsivG direct repeats, a protein kinase WO 96/01899 ~ ~ P~.l/u,.... ~

2~ 3~

C-responsive AP-1 site, and a ras-responsive semm response element. See, Fujii, e~ al., Mol. Cell. Biol.. 2, 2493-249g (1989);
Hayes, c~al.,Proc. N~tl. A(~l Sci.. USA.~L, 1272-1276 (1987);
Jamal, et al., Nature, 344, 463466 (1990); Gutman, et aL,~, C-~.11 BjQI.. I1, 5381-5387 (1991); and Fisch, et al., J~ol. Cell. Biol 9, 1327-1331 (1989).
Contingent replication systems employing l...nsc~ ional activation of the SV40 T antigen gene to identify enhancers and stably interacting transcription factors are 10 known. See, Vasavada, er a1., Ind. J. Biochrm Bio~hys.. ~. 488-494 (1988); Vasavada, et al., (',ene, ~, 29-40 (1987~;
Vasavada, et al., Proc. Natl. Acad. Sci.. 88, 10686-10690 (1991);
and Rusconi, et ~1.,~,~, 211-221 (1990).
Because of the i~ .-hncc of signalling mnlt~cnll~s in 15 the control of cellular proliferation, there is a need for methods to identify mnlecllles involved in growth-related signaling systems which can in turn be used to identify biological targets for antitumor drug discovery. There is also a ne,-d for methods of identifying agents that can interfere with such growth-20 related signaling systems to restore normal growth whenabnormal cell proliferation is occurring.
SUMMARY OF THE INV~NT~ON

The present invention fills the foregoing needs by providing materials and methods for identifying signal transduction mnlect-l~s and antagonists thereof. More specifically, this invention provides m~nm~ n cell lines, the cells of which comprise ~a) a l~co.. ~ nt vector comprising an inducible. or tissue specific promoter operatively linked to a nucleic acid encoding polyomavirus large T antigen; and (b) a recomhin~nt expression vector comprising a polyomavirus origin of replication and a nucleic acid suspected 35 to encode an activating protein of said promoter.

~O 96101899 ~ 1 g ~L 3 6 1 PCTIUS95/07874 Preferably the promoter is the human c-fos promoter and the activating protein is a human c-fos promoter activating protein.
The present invention further provides a method for 5 identifying a nucleic acid encoding a promoter activating protein, comprising:
(a) culturing a m~mm~lion cell line, the cells of which comprise:
(i~ a recombinant vector comprising an inducible or tissue specific promoter operatively linked to the coding region of the polyomavirus large T antigen gene;
and (ii) a recombinant expression vector comprising a polyomavirus origin of replication and a nucleic acid suspected to encode an activating protein of said promoter, under conditions in which such nucleic acids are expressed; and (b) measuring the levels of replicated vectors in the cells after a period of incubation sufficient to permit vector replication;
whereby a nucleic acid encoding a human promoter activating protein is identified by mc&sui,;.--elll of increased levels of vectors in the cells.
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 in the present invention is the plasmid PfLAG-8.
A preferred recombinant expression vector comprising a polyomavirus origin of replication is the plasmid La2.
The present invention also provides a human c-f os promoter activating proteins having the amino acid sequences defined in the Sequence Listings SEQ ID NO:l and SEQ ID NO:3, or WO 9C101899 T~ 4 2 ~ 6 t an antigenic fragments thereof, and nucleic acids encoding such protein or fragments.
In another embodiment, the present invention provides mS~mm~ n cell lines, the cells of which comprise:
(a) a first recombinant expression vector ~ i"g a reporter gene operatively 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 1 0 protein.
The present invention also provides a me~hod for identifying an antagonist of a human c-fos promoter activating protein, comprising-(a) providing a ms)mm~ n cell line, the cells of which 1 5 comprise:
(i) a first recombinant expression vector c~ ;llg a reporter gene operatively lin~:ed to a human c-fos promoter; and (ii) a second recombinant expressi(m vector comprising a nucleic acid encoding a human c-fos promoter activating protein;
(b) cont~ting the cell line of step (a) with a sample suspected to contain an antagonist of the human c-fos promoter activating protein; and (c) measuring the level of exprcssion of the reporter gene;
whereby an antagonist of the human c-fos promoter activating protein in the sarnple is identified by ,..easu-c~ t of a reduced level of expression of the reporter gene.
Preferably the second recombinant expression vector encodes CROC-l protein, CROC-4 protein or o~2-macroglobulin receptor-associated protein.

~10 g6/01899 ~ /4 3 6 ~

-s-DETAILED DESCRIPTION

All references cited herein are hereby incorporated in their entirety by reference.
The following terms are herein denoted by the indicated abbrevintions: long terminal repeat (LTR);
Dulbecco's modified Eagle's medium (DMEM); serum response element (SRE); chloramphenicol acetyltransferase ~ (CAT).
All nucleic acid sequences disclosed follow the normal 5' to 3' convention, as read from left to right.
Standard single-letter abbreviations are used for the nncl~.otitle bases in the seqllences (37 C.F.R. 1.822).
The term "antagonist" is defined herein as a 15 substance that blocks or inhibits the effects of a human c-fos promoter actvating protein, such as the CROC-l protein or c~2-macroglobulin receptor-associated protein.
The term "reporter gene" as used herein means either a DNA molecule isolated from genomic DNA, which 20 may or may not contain introns, or a comrlP.ml~nt~ry DNA
(cDNA) prepared using messenger RNA as a template. In either case, the DNA encodes an expression product that is readily measurable, e.g., by enzymatic activity, enzyme-linked immunosorbent assay (ELISA) or ra~ imml-noassay 25 (RIA). Preferred reporter genes for use in the present invention include the E. co~i Lac-Z gene from pCH110 (Stratagene #27-4508-01). The expression level of this gene can be measured by a sensitive fluorescent substrate assay.
Also preferred is the CAT reporter gene described below, 30 although many others well known in the art could be used instead.
The term "recombinant expression vector" means a vector prepared using recombinanl techniques said vector comprising an inserted nucleic acid encoding a protein such 35 that said vector is capable of expressing the protein upon wo g6,0l899 2 1 g ~ 3 6 1 ~ P~IUS95/078'~4 1~

-6~

transfection or transformation into a suitable host cell.
Preferred is a vector comprising a nucleic acid encoding a promoter activating protein. Also preferred is a vector comprising a reporter gene operatively linked to a human c-fos promoter.
Cells which have been "stably transformed" have rec---'- DNA incorporated into their genomic DNA. Such stably incol~ol~tcd DNA is retained by the transi'ormed cells because it is introduced into the cells with a selection marker, such as G418 resistance, which forces retention when the cells are grown in selection medium. The present invention employs transiently transfected m!lmmS li51n cell lines, however stably t~ ro-lllcd m~mm~liAn cell lines comprising a c-fos promoter regulated large T antigen can also be used.
The inducible or tissue specific promoters of the present invention are non-hous~keeping promoters, i.e., they are regulated and are not transcriptionally active under normal conditions, except to the extent that low basal levels of constitutive expression may occur.
As defined herein, "inducible plol~lot~l~" are ,,lllolc~ the l ~Ins~,liL~lion activity of which is activated or enhanced in response to changes in the cellular environment that results in a cellular response, such as stress, hormonal ~timnl~tion or differentiation. Induction occurs via activation of a sigr!~lling cascade resulting in the enhanced binding and activity of transcription factors at the promoter site. Molecules involYed in such induction include promoter activating proteins as descr;bed herein. Inducible promoters include the c-fos and c-myc ~)..l - Another inducible promoter is the multidrug 30 resistance gene promoter described in J. Biol. Chem,. ~, 15347-15350 (1993).
The term "tissue specific promoter" means a promoter which is active only within a subset of cell types, such as promoters which are active only in prostate cells. See. Young, ef al.,Biochem.. 31, 818-824 (1992); and Riegman, et al., ~QL

01899 ~ 14 Endocrinol..~, (No. 12) 1921-lg30 (1991). Other tissue specific promoters include promoters of late histone genes and n(IIIW~ of muscle regulatory elements. See, Genes Dev.. ~, 849-859 (1990); Mol. Cell. Biol..,~. 515-522 (1989); and Mol.
5 Cell. Biol.. 2, 2191-2201 (1989).
Promoters that can be used in this invention include but are not limited to the promoters of the proto-oncog. ~s c-fos and c-myc . See, Miller, et al., supra; and Kelekar, et al., s~pra. Both of these promoters regulate expression i~Z v~vo of 10 genes the ù~ c~ o;~ion of which can lead to aberrant cell growth. Most preferred is the c-fos promoter.
The term "aberrant cell growth" is herein defined as the abnormal or uncontrolled cell proliferation characteristic of neoplasms .
As used hereiD, the term "promoter activating protein" is defined as a protein which causes transcriptional activation of one of the above-mentioned promoters. Preferably the promoter activating protein is a human c-fos promoter activating protein. Most preferred is an activating protein 20 having an amino acid sequence subsl lnti?lly identical to that of the a2-macroglobulin receptor-associated protein. Also most preferred is an activating protein having an amino acid sequence 5~hs~?n~ 1y identical to that of the CROC-4 protein or the CROC-l protein, the S~IU~ C~ of which are defined by SEQ
25 ID NO:3 and SEQ ID NO:1, respectively. Substantial identity of amino acid ~ uellccs means that the sequence of another c-fos promoter activating protein compared to the sequence defined by either SEQ ID NO:1 or SEQ ID NO:3 is identical or differs by one or more amino acid 30 alterations (deletions, additions, substitutions) that do not subst~ntiSllly impair transcription activating activity as described herein. For example, there may be allelic or interspecies variants of the sequences defined by either S~Q ID NO: 1 or SEQ ID NO:3.

W0 ~6/0~89g ~ /Q I4 ~
gl ~lA~~ i l~ld~Vl FullL~ o~c. it is well within the skill of the art.
e.g., by chernical synthesis or by the use of modi~led polymerase chain reaction (PCR) primers or site-directed m~-t~gPn~cic to modify DNA encoding a c-fos promoter 5 activating protein having the sequence defined by either SEQ
ID NO:] or SEO 11~ NO-3, to produce single or multiple base substitutions which do not suhst~n~i~lly impair the activity of c-fos promoter activating proteins produced therefrom.
Such c-~- vdtiYely modified variants are within the scope 10 of this invention.
Sequence identity, is det~ incd by optimizing residue matches, if necessary, and by introducing gaps as required. This changes when considering conservative subsfitnlione as matches. Conservative substitutions typically 15 include substitutions within the ~ollowing groups: glycine, alanine; valine, icol -, leucine; aspartic acid, glutamic acid;
asparagine, ~lut~minP; serine, threonine; Iysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences are typically intended to include natural allelic and intc.~ ie;s 20 variations in each respective protein sequence. Typical homologous proteins or peptides will have from 25-100%
homology (if gaps can be introduced), to 50-100% homology (if conservative subQ~it~ltiong are included) with the amino acid sequence of the CROC-1 protein or CROC-4 protein. Homology 25 measures will be at least about 50%, and typically at least 60%
or more.
The present invention also comprises "antigenic fragments" of a human c-fos promoter activating protein. It is well known in the art that antigenic determinants ~epitopes) 30 generally contain at least about 5 amino acid residues. Ohno et al., Proc. Natl Acad. Sci. USA. 82, Zg45 (1985). The antigenic fragments of the invention comprise from about 5 to about 100, and preferably about 5 to about 50, amino acid residues.
Whether a ~iven polypeptide falls within the scope of this ~f096/01899 ~ 361 ~ vr~4 g invention can readily be det~-rmin~d by routine e ~ nt~tion using the methods described below.
Such antigenic fragm~rlte can be made by proteolysis of the whole human c-fos promoter activating 5 protein or by chemical or recomhin~lnt DNA synthesis. The antigenic fragml~n~s can be used to elicit production of antibodies, preferably in a mammal, by standard methods. The antibodies thus produced can be used to assay for or purify the activating protein, using standard in munoassay or 10 immunoadsorption methods.
The present invention utilizes a recombinant vector co-llplisi,lg the polyomavirus T antigen gene and extends the system of contingent replication to identify proteins the production of which leads to transcriptional activation of gene 15 promoters. In contrast to the SV40 T antigen gene used by Vasavada, et al., supra, the replicating and transforming properties of the polyoma T antigen gene can be separated.
Separation of the replirating and transforming properties is ~rComrlich~d by inserting a stop codon in the large 20 T intron in a region overlapping the central coding se4u~,..ccs for middle T antigen. This separability of functions is important in the case of the c-fos promoter, where prevention of middle T
expression eliminates the possibility of transcriptional activation of the promoter via the rniddle T-activated c-src- and 25 phosphatidylinositol 3-kinase-associated signalling systems (identified in Talmage, et al., Clell, ~i!, 55-65 (1989)) due to low level, basal transcription from the promoter.
Use of the polyomavirus system enables the extension of contingent replication to several well-characteri~ed 30 murine systems. In contrast, the SV40 T system used by Vasavada, et al., supra, is limited primarily to simian (monkey) ~ systems. In addition, the present system does not appear to suffer the high frequency of truncated or rearranged inserts (approximately 25 percent) previously reported for the SV40 T

W096101899 v r~ a,..~ /1"4--21~3~1 antigen-based system. Alteration of inserts occurs at a frequency of less than 2 percent in present system.
A preferred embodiment includes the incorporation of multiple enhancers from the promoter upstream of the polyomavirus large T antigen gene to achieve sufficient sensitivity of the promoter to permit large T induction in response to low level expression of a cDNA-encoded signalling molecule. Large T induction in turn results in plasmid replication. Co-transfection with a cDNA library as described below allows the percentage of cDNAs encoding signalling proteins to be enriched within the library population. through such large T-induced plasmid replication. The resulting enrichment permits successive screening of increasingly srnaller groups of library plasmids within a cDNA library, resulting in the i~l~ntific~tica of single library plasmids encoding biologically active mnh~cl~les which activate the promoter.
The self-~mplifi~ l~ion process of the present invention provides additional sensitivity towards the detect;on of cDNAs encoding signalling molecules. Initial plasmid replication, in response to induction, leads to enhanced expression of active signalling mr~lPc~ s due to greater gene copy number. This increase in signalling molecules results in greater :~mp~ific~til: of large T antigen expression, which in turn leads to greater plasmid replication.
Preferred vectors of the present invention include novel plasmids, denoted PfLAG-8 and Lo~2, as described below.
The present invention further provides a method for identifying cDNAs encoding proteins which can activate a promoter, preferably a human promoter, and more preferably the human c-fos promoter. More preferred are the cDNAs, denoted CROC-I and CROC-4, which encode c-fos promoter activating proteins. For example CROC-I encodes a specific c-fos promoter activating protein, denoted CROC-I protein, having the amino acid sequence shown in SEQ ID NO:l. Similarly, CROC-4 encodes a specific c-fos promoter activating protein, denoted ~0 96/01899 PCT/IJS95/U7874 21~4~61 CROC-4 protein, having the amino acid sequence shown in SEQ
ID NO:3. Most preferred are the nnrleotidP se~ ncts shown in SEQ ID NO:I and SEQ ID NO:3.
The present invention also provides cl~NAs encoding 5 c-fos promoter activating proteins which are conservative mutants of the proteins encoded by CROC-l or CROC-4. Such mutants possess the binding and c-fos promoter activating functions of the proteins encoded by CROC-1 and CROC-4, respectively .
In addition, the presenl invention provides compounds which are antagonists of the protein encoded by CROC-I or CROC-4. These antagonists include proteins which are deletional, substitutional or ~ ditionnl mutants of the CROC-l protein or CROC-4 protein, and which bind to, but do not 15 activate, the human c-fos promoter.
It is recognized that, because of the dtge~ of the genetic code, there are many functionally equivalent nucleic acid sequences that can encode c-fos promoter activating proteins and c-fos promoter activating protein antagonists as 20 defined herein. Such functionally equivalent sequences, which can }eadily be prepared using known methods such as chemical synthesis, PCR employing modified primers, and site-directed mn~gen~oeiC~ are within the scope of this invention.
As used herein, the term "recombinant vector"
25 includes both rec~-mhin~nt plasmids such as those mPn~ ned herein and rec~mhin~n~ retroviral vectors, which can also be engineered as described by Geller et al., Proc. Natl. Acad. Sci.
USA, 87, 1149(1990).
The foregoing recombinant vectors can be used to 30 transfect any m~mm~ n cell capable of undergoing transfection and permitting vector replication, as herein defined. Although cells from fresh tissue explants (primary cells) could in principle be used, the use of ~st~hlichcd cell lines is preferred. Many such cell lines are available including, e.g., 35 NIH 3T3 mouse (ATCC# CRL1658),L-M(TK-) mouse (ATCC# CCL

WO Y61V189~ P~ /4 21 9~

1.3) and BALB/c 3T3 Clone A31 mouse (ATCC# CCL 163~ cell lines.
The choice of a cell or cell line for use in the methods of the present invention will be dictated by the known 5 or ~Pt~rminol~lP specifiritipc- of the vectors used. For example, the murine cell lines are preferred for use with vectors CO~ illg a recnmhinqnt vector containing the polyomavirus large T antigen gene under the control of a regulated promoter, such as the human c-fos promoter; and a mqmmalian 10 recombinant expression vector co~ lising a polyomavirus origin of replication and a nucleic acid suspected ~o encode a human promoter activating protein, such as a retroviral expression vector ctmrrieing a retroviral LTR capable of expressing the nucleic acid.
Although cells for use in the present invention were transiently transfected, stably-transformed cells can also be used. Stable transformation of a mqmmqliqn cell line can be ~cr mrlich~ d by using standard methods to co-b-ansfect the cells with one of the above-mPntion~d recombinant vectors and 20 with a second vector which confers resistance to a selection agent such as an antibiotic.
To identify nucleic acids encoding human c-fos promoter activating proteins using the methods of this invention, cells are co-l~"src~t~d with a lecol-lbillallt vector 25 comprising a human c-fos promoter operatively linked to pol~ul~vi-us large T antigen gene, and a cDNA library incol~u.~t~d into a n.qmmqliqn recombinant expression vector comprising a polyomavirus origin of replication. The cells are then incubated under conditions in which vectors containing 30 cDNA encoding a human c-fos promoter activating protein will stimulate increased vector replication. The cells are then harvested, the plasmids extracted and unreplicated vectors selectively digested with Dpnl. Replicated plasmids are recovered by transforming competent bacteria with the Dpnl 35 digest.

96tO1899 ~ 3 ~ 14 Typical incubations are carried out for 2 days at 37~C in a h-~mitlifif~d C02 incubator, although the choice of e ~ "l'O~c will be apparent to those skilled in the art and will depend, e.g., upon the nature of the cells, the medium used and the type of culture container. Tr~llh~tinn is c~ntimled for a period of time sufficient to permit development of a strong replicative response. The optimal time is determined by routine experimentation but will typically be in the range of about 24 to 72 hours.
0 A s~hs~:mti~lly increased level of vector replication and recovery after Dpnl digestion will be detected for those vectors comprising nucleic acids encoding human c-fos promoter activating proteins as compared to background resulting from replication of vectors lacking such nucleic acids.
A su~hst~nti~l increase in vector replication and recovery is typically an increase of at least about ~-fold, preferably about 8-fold, and most preferably about 20-fold, above the level measured in the complete absence of a plasmid comprising a nucleic acid encoding a human c-fos promoter activating protein. The degree of increase will be primarily dep~ndent upon the level of background replication.
SUbst~nti~lly the same procedures are used for identifying nucleic acids encoding other human promoter activating proteins, by utilizing vectors comprising the promoter operatively linked to a nucleic acid encoding polyomavirus large T antigen.
In screening human c-fos promoter activating protein antagonists using the methods of this invention, cells are provided which are sim--lt~r~ously transfected with a first rec--mhin~nt expression vector comprising a reporter gene opel~Li~ly linked to a human c-fos promoter and a second vector comprising a nucleic acid encoding a human c-fos promoter activating protein. Preferred reporter genes are the fos-CAT reporter gene described below or a fos-lac Z reporter WO961018~9 21~436L ~ 4 ~

gene. The cells are planted in a culture medium appropriate to the kind of cells used.
The cells are then incubated in the absence (control) or presence of varying quantities of samples containing 5 suspected antagonists under conr~ nQ in which the gene encoding the human c-fos promoter activating protein is expressed. Under such conditions, and in the absence of an antagonist, stimnl:ltion of the human c-fos promoter will occur, resulting in reporter gene expression. The samples can be, e.g.
10 aqueous or water-miscible solutions in which isolated cnmpounf~ have been dissolved, or individual or pooled fractions from purification steps such as chromatographic or electrophoretic fractions.
Typical incubations are carried out at about 37~C in 15 a hnmidifi~od C02 incubator, although the choice of conditions will be apparent to those skilled in the art and will depend, e.g., upon the nature of the cells, the medium used and the type of culture container.
ub~tion is continued for a period of time 20 sufficient to permit signific~nt reporter gene induction, at which time the level of expression of the reporter gene is measured by an appropriate assay. The optimal time for making the measurement is det~rmi -d by routine experimentation but will typically be in the range of about 24 to 72 hours, 25 preferably about 48 hours.
The highest levels of reporter gene expression will be measured in the control (antagonist free) cultures. V~here a culture contains a human c-fos promoter activating protein antagonist, a reduction in the level of reporter gene expression 30 will be measured, the degree of which will be a direct function of the quantity of antagonist added to the medium. Antagonists present in the samples added to some of the cultures will be identified by rn~Curing a ~uhst~nti~lly decreased level of reporter gene expression, compared to the level measured in 35 the control cultures.

~1096101899 r.,~
~@3~ ~

A subsrAnti~lly decreased level of reporter gene expression is defined as a decrease of at least about 50%, and preferably at least about 70%, of the level measured in the complete absence of an arltagonist of a human c-fos promoter activating protein. Of course, the degree of decrease may be intl~ ced by the quantity of antagonist present in the sample compared to the quantity of human c-fos promoter activating protein used and the efficiency of the antagonist.
Decreased levels of reporter gene expression due to general toxicity of samples can be ~co~lntPd for by transfecting a second constitutively expressed reporter gene, such as lac-Z
driven by a ~-actin promoter and normalizing c-fos reporter gene activity to lac-Z expression.
The following non-limiting Examples will serve to illustrate the present invention.
FxAMpl F~~
Materials and Gener~ql Me~hods-Unless otherwise specified, percentages given below for solids in solid mixtures, liquids in liquid mixtures, and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, l~o}Jccli~cly~ Sterile conditions are r~qin~qinPd during cell culture.
Standard recornbinant methods were used throughout, such as those described in Sambrook, e~ al.
"Molecular Cloning. A Laboratory Manual, 2 ed.", Cold Spring Harbor Laboratory Press (1989~.
Dpnl is a known restriction endonuclease isolated from Diplococcus pneumoniae and is commercially available from ICN Bi~nlPAicqlc~ Sigma Chemical Company or New England BioLabs, Inc.
The restriction endonl~clPq~es Asel, ~amHI, Bglll, BstXI, Clal, Fspl, ~incll, Narl, Notl, Sacll, Sall, Scal, X~aI and wo s6/ols~s ;~ PCSIUS9~107874 --Xhol are known and are co~ ially available, e.g. from Sigm~
Chemical Company.
The restriction endonucleases BamHI, BssHII, BsfXI, HlncII, Sall, Scal and Xbal are known and are commercially available, e.g. from ICN Biom~ al~.
The restriction en~onl~r!~cps AfllII, Asel, BamHI, ~gllI, Bs~HII, BstXI, Clal, FspI, Hincn, Nael, Narl, NorI, SaoII, Sall, ScaI, XbaI and Xhol are known and are cornrnercially available, e.g. from New England BioLabs, Inc.
The restriction endonuclease Saltl is known and is cul~ c~ially available, e.g. from Boehringer h~nnh~im The enzyme mung bean nuclease is known and is commercially available from New England Biolabs, Inc or Sigma Chemical Company.
The synthetic polylinker used in preparing the vector La2 was obtained from New England Biolabs, Inc. and has the sequence shown in SEQ ID NO:2. The NcoI linker d(pAGCCATGGCT) is known and is cormmercially available fiom New England Biolabs, Inc. (catalog $t 1150).
The vector pUCI9 (ATCC 37254, GenBank Accession #: X02514~ is colll~ ;ially available from New England Biolabs, Inc or ICN Bi-mPdic~llg The nucelotide sequence and restriction sites of pUCl9 are described by Yanisch-Perron, et al., in Gene, 33, 103-119 (1985).
The following DNA, utilized in preparing the plasmids of the present invention, is publicly available:
Polyollu.~,ilu6 DNA strain A2 (ATCC # 45017); a~1d human genomic c-fos (ATCC # 41û42).
In addition, the DNA sequence of polyomavirus strain A2 is reported in DNA Tumor Viruses. ed. Tooze, J. (1980) (Cold Spring Harbor Press), pp. 834-838.
Construction of the retroviral vector pMV7 is described by Kirschmeier, ~t al., PNA, 1., 219-225 (1988~, starting from plasmids pPyori and pMV (ATCC# 37190). The vector pMV7 is well known in the art and has been freely and ~o 96,0l89g ~ 1 9 4 3 6 1 ~ 14 .

widely distributed in many laboratories. In addition, l~,hu~ cs similar to pM'V7 which could be used instead in this invention are readily available, such as pV-mos (ATCC#

4 1 037).
The fos-CAT reporter gene construct 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 cDNA library screening a unidirectional cDNA
library was made from human brain poly A RNA (Clontech, Palo Alto, CA) using the GIBCO (Grand Island, NY) Su~ l cloning kit, and inserted into the SalllNotl sites in plasmid L~2.
Separation and visualization of nucleic acids was carried out as described in Sambrook, et al., supra, by electrophoresis on agarose gels and visualization with ethidium bromide. All nucleotide sequencing was performed using the dideoxy-mediated chain termination method described in Sanger, et al., Proc. Natl. Acad. Sci. IJSA.l~, 5463-5467 (1977).
To obtain the sequences of CROC-I and CROC-4, DNA sequencing was performed on both strands.
Co-transfection of cells with PfLAG and La2 containing a cDNA encoding a biologically active ~ign~lling molecule causes activation of the c-fos promoter, resulting in the production of large T antigen. The production of large T
antigen stim~ tes intracellular replication of plasmids c~nt~ining the polyull~a\~ s origin of replication. Plasmids are recovered from tl e transfected cell cultures by "Hirt extraction"
using the methods described in Hirt, J. Mol Biol.. 26, 365-369 (1967). Unreplicated plasmids are selectively destroyed by restriction with DpnI. Replicated plasmids are then recovered by transformation into competent bacteria.

WO Y6101899 2 L 9 ~ 3 5 1 . ~ 4 ~

Early passage NIH 3T3 mouse fibroblasts (ATCC#
CRL 1658) and Rat 2 fibroblasts (ATCC# CRL 17641 were grown in DMEM ~u~ with l0'3'c bovine calf serum and 50 ~lg/ml gentamycin sulfate.
The DHIOB E. coli used in the present invention are commercially available from GIBCO.
Construction of Plagmi-l~

Two basic plasmids were constructed for use in the prescnt invention. The first (denoted PfLAG) comprised a human promoter-regulated polyomavirus large T antigen gene which served as a source of large T antigen upon activation of the promoter, and was based on the human c-fos promoter. The second plasmid (denoted La2) was a retroviral cDNA expression vector co~~ining the polyomavirus origin of replication.
The retroviral cDNA vector La2 was prepared as follows. Polyomavirus DNA strain A2 was digested with BamHIlNarI and the resulting 750 bp fragment was ligated into the BamHllNarI sites in pUCl9 to give a plasmid denoted pOri.
The retrovira] vector pMV7 was digested with F spl/AflllI and the resulting 4 kb band conlAining the two Moloney murine sarcoma virus LTRs was ligated into the HincIIlAfllII fragment of pOri, to give a plasmid denoted pMV7-2. A neomycin resistance gene present between the two Moloney murine sarcoma virus LTRs in pMV7-2 was removed by Saul/ClaI
digestion and replaced by a synthetic polylinker (described above) to give the plasmid pMV7-3. To enable blue-white screening, the polylinker in pUCl9 was replaced with a NcoI
linker, then the 360 bp lac Z region was removed by AseI/lVarl digestion, blunt ended Witll mung bean nuclease, and ligated into the pMV7-3 polylinker. The resultant plasmid, denoted La2, was 4.5 kb and contained unique Sall and Norl sites at the 5' and 3' ends, ~ ,uc~Li~ly~ of ehe ~ac Z gene. A translaeional start codon, followed by a DNA sequence encoding a histidine ~V0 961018gg P~ 14 21~43~1 hexamer, was inserted 5' to the cDNA insertion site to insure expression of cDNA-encoded protein from truncated cDNA
inserts lacking start codons, and to aid in ~ubse~lLFrt protein purification.
The PfLAG plasmid was prepared via the following procedure. The polyomavirus large T anligen under the control of the human c-fos promoter was introduced by digesting the 5.9 kb BamHI fragment of pcfos-l, disclosed by Curran, et al., Mol. Cell. Biol.. ~, 914-921(1983), with Nael to remove the 1 0 entire coding region of the c-fos gene and inserting the 2.8 kb Bs~XII~incll band from polyomavirus, encoding the polyoma T
antigen. Middle and small T expression was t~limin~ d by inserting a stop codon in the ScaI site located at position 605 of the polyomavirus DNA sequence reported in Tooze, s~pra. The 15 resulting construct was denoted PfLAG-1 (for promoterfO5/large T antigen).
A third vector, denoted HEL, was prepared for use in identifying the intracellular locations of CROC-I. The histidine hexamer coding sequl~nres of Lo~2 were removed by BglII/Sall 20 digestion and replaced witll coding scqucnccs for the nine amino acid influenza virus HAI epitope described in Field, e~ aL, Mol. Cell. Biol.. 8, 2159-2165 (1988). The SV40 origin of replir~ti~ n was then inserted at the unique Xbal site between the polyoma origin of replication and the 5' LTR, to give HEL.
2~ A fourth vector was prepared for use in confirming the ability of suspected human c-fos promoter activating proteins to stimulate the c-fos promoter. The fos-CAT reporter gene described by Desch~mrs. et al., in ~cience, 233, 1174-1177 (1985), was prepared by inserting the human c-fos promoter 30 from the -735 (Bam~ll site) to +42 (~ael site) in front of the bacterial CAT gene in the pCAT basic vector (Promega).

W0961018~9 ~1943~1 r~ 4 --Deterrninin~ Enh~ncer Re,luh. .~..ts for P~LAG-de,pendent Contingent Rep]ication:

For purposes of the present invention, the human 5 c-fos promoter in PfLAG must remain llans~ ionally s;lent in quiescent cells, but be sensitive enough to respond to the low level expression of active, cDNA-encoded signalling molecules by producing sufficient T antigen to cause plasmid replication.
The sensitivity and level of gene induction from the promoter 10 can be increased by the incorporation of additional enhancer elements into the promoter. Multiple enhancer elements were incorporated into PfLAG-I by isolating an approximately 500 bp or more Xhol/BssHIl(blunt-ended) fragment containing the c-fos enhancer elements, and ligating the enhancer region into 15 the XhoI/Sacll (blunt-ended) site of the previous PfLA&.
To determine the number of enh~r~,-rc required to display contingent replication, a series of PfLAGs, containing 1,2, 4 and 8 enhancer regions, were col~ The following experiments were then conducted to define the enhancer 20 l~quilcll.~,ots for PfLAG-dependent contingent replication.
Muramatsu, et al., Mol. Cell. Biol.. 2, 831-836 (1989) have shown that expression of the catalytic domain of protein kinase C induces the c-fos promoter. The nucleotide and deduced amino acid sequence of rat protein kinase C-~ I are 25 described in Housey, et al., Cell, 52, 343-354 (1988). The catalytic domain of rat protein kinase C-~l was incorporated into La2 to m~ke a construct, denoted pMvpkc~
Co-transfection of a pMVPkC~ /Lc~2 mixture with a Pfl.AG
containing 1, 2, 4 or 8 enhancer regions would therefore provide 30 a means of testing the sensitivity of each PfLAG.
A threshold sensitivity of detecting about one plasmid out of forty for cDNA screening was used. Therefore a 1:40 (wt/wt) ratio of pMypkc~llLo~2 for co-transfection with each of the PfLAGs into NIH 3T3 cells was utilized in the 35 procedure described below. Cells were incubated for forty-eight VOg6/01899 r~J~
~9~3~

(48) hours following transfection. The plasmids were extracted and e~r -~, following ~pnl digestion, for elevated plasmid recovery indicative of contingent replication. The results obtained under these conditions are presented in Table 1.
5 These data show that eight enhancer regions (PfLAG-8) were required for significant activation of plasmid replication, permitting an eight-fold increase in plasmid recovery over background resulting from co-transfection of PfLAG-8 with vector alone. Induction with PfLAG-8 ranged from 6-fold to 10 greater than 20-fold increases in plasmid recovery, dep~ntling primarily on the level of background.
Table 1. Human c-fos enhancer requirement to activate polyomavirus large T antigen-activated contingent 15replicatior.~
Plasmid No. of Co-transfecte~ Total Construct Enhancer Plasmid Number of Re~ions Colonies PfLAG-I 1 pMV7-Z 8 PfLAG-I I pMV7PkC~31 5 LoL2 PfLAG-2 2 pMV7-Z 3 3 PfLAG-2 2 pMV7PkC~l~l 2 2 Lo~2 PfLAG-4 4 pMV7-Z 109 PfLAG-4 4 pMV7PkC~ 101 Lo~2 PfLAG-8 8 pMV7-Z 363 PfLAG-8 8 pMV7PkC~31 1915 Lor2 - - pMV7PkC~,B1 7 L~2 2 llg PfLAG is co-transfected with 18 ~,lg of either pMV7-Z or a 1:40 (wt/wt) ratio of pMV7PkC~,BI/Loc2.
The results presented are the average of two experiments.

WO96/01899 2Ig~36~ .t/~/4--The effect of the concentration of plasmids encoding a promoter actvating protein on the recovery of pMVPkC~p l within a total population of plasmids is de~-~minPd by varying the concentration of pMVPkC~l in a pMVPkC~l/La2 mixture 5 prior to co-transfection with PfLAG-8. Because Lc~2 has a modified lac Z gene derived from pUCl9, bacteria transformed with L~2 will turn blue, whereas bacteria transformed with pMVPkC~ will remain white, when plated on agar plates c~n~aining ampicillin, X-gal, and IPTG. The percentage of 10 pMVPkC~l is d~t- ...;.fd by expressing the number of white colonies as a percentage of totnl colonies formed after bacterial transformation of Dpnl-digested Hirt extracts.
Lxperiments were conducted by co-transfecting PfLAG-8 with the pMV7PkCI~l/La2 mixtures beginning at a 1:80 ratio (wtlwt), then diluting down to a 1400 ratio, using the methods described below. Competent DHlO13 E. coli were transformed with Dpn I-digested Hirt extracts and plated on agar cont:~ining ~lmpicillin X-gal, and IPTG. The percent of pMV7PkC~I in the recovered colonies was determined by the 2G number of white colonies over the total colonies.
To insure that the white colonies resulted from transformation of pMVPkC~ l, plasmids were recovered and restriction mapped. All white colonies showed the correct pMVPkC~I restriction pattern. The results presented in Table 25 2 show that although the number of recovered colonies is reduced to background levels at high pMV7Pk~ dilution, the actual percentage of pMV7Pk~ colonies increases; i~lica~inE~
that a minimum of 400 library colonies can be transfected with PfLAG-8 to enrich a cDNA library population for cDNA encoding 30 signal transducing molecules. Initial cDNA library screening was therefore performed with plasmid pools comprised of four hundred or more plasmids in order to acquire a library population enriched in cDNAs encoding activators of the c-fos promoter.

~,~O gC/01899 P~
~9~3$l Table 2. Conc~ .tion dependence of pMV7PkC~ ~1 on plasmid r covery,~
Ratio of co- Number of Percent Colonies transfected Colonies per Dish pMV7PkC~1 Blue V~Thite pM~T7PkC~
/Lc~2 1:8~ 206 4 I.g 210 0 146 7 4.o 153 1:2'0 134 6 4.3 150 I :3' 0 97 24 19.8 1 21 1:4()0 90 24 21.1 1 14 ~Co-transfection with PfLAG-8 and Lc~2 alone gave a background of 102 colonies/dish in this experiment.

Cell Culture and Transfection:

For transfections. 8 x 105 3T3 cells were planted in 10 growth medium in 100 mm dishes and allowed to attach overnight. The following day, transfections were performed by the method of Wigler, et a/., Cell, I l, 223-232 (1977), using calcium ph~5rhatP. After a 4-hour exposure to the calcium phosphate precipitate, cells were washed twice with phosphate 15 buffered saline, re-fed with DMIEM supplemented with 0.5%
bovine calf serum, and incubated at 37~C for 40-48 hours. Cells were harvested and the plasmids were extracted by the procedure of Hirt, supra. The extracted plasmids were digested with DpnI for a minimum of 24 hours. Dpnl digests were 20 phenol extracted and ethanol precipitated. DNA was res~lcrenrlPd in 20 IlL TF, (I mM EDTA +10 mM Tris, pH8.0), and transformed into c~ l DHIOB bacteria (GIBCO).
Co-transfections were performed via the above procedure at a cDNA/PfLAG ratio of 9:1 (wt/wt), using 20 llg 25 DNA per dish.

wo g6/01899 2 ~ 9 ~ 3 ~ ,Ol4 cDNA Library Screenin~ usin~ Contin~ent Replication h human brain cDNA library was co-transefected with PfLAG-S into NIH 3T3 cells via the methods described 5 above. Plasmid pools, comprised of approximately 30-40 plasmids, were co-transfected with PfLAG-8 and examined for a minimum 5-fold increase in plasmid recovery. Plasmids from active pools were recovered and subdivided into secondary pools of four plasmids each. and similarly examined for 10 activation of contingent replication. Plasmids from each active secondary pool were then examined individually for contingent replication. From approximately 1400 plasmids screened initially, two plasmids, denoted CROC-I and CROC-2 (for cl~nfingPnt leplication of cDNA), consistently gave elevated 15 plasmid recovery when co-transfected with PfLAG-8. The nucleotide sequence for CROC-I is shown in SEQ ID NO:l.
A third plasmid, denoted CROC-4, was identified by further plasmid screening. Plasmid CROC-4 also co~ ff~ntly gave elevated plasmid recovery when co-transfected with 20 P~LAG-8. The nucelotide sequence for CROC-4is shown in SEQ ID NO:3.
Confirmation of c-fos Promoter Activation usin~ a fo~s-CAT
Reporter Gen~-Certain extraneous factors could also cause theelevated plasmid recovery observed in the contingent replication assay. For example, incomplete bactelial methylation of the Dpnl sites, which will confer Dpr~l resistance.
30 or differences in transfection or transformation efficiency. To eliminate these p~c~ihilifi~s, each of CROC-I,CROC-2 and CROC-4 was co-l~ sf~ d with a fos-CAT reporter gene and tested for elevation of CAT activity as follows. Rat 2 cells were co-transfected with 18 ~,lg Lo~-e~pressed cDNA (i.e., CROC I, CROC-2 or CROC-4~ + 2 ,ugfos-CAT for 4 h, then refed with D~IEM

~0 96/01899 2 ~ ~ ~ 3 6 1 PCTIUS9~107874 + 0.5% calf serum. Cells were harvested 72 hours after transfection and CAT assays performed via the procedure of Gorman, etal.,Mol. Cell. Biol..~, 1044-1051 (1982).
CAT activity was cignificln~ly induced by CROC-l, 5 CROC-2 and CROC-4, indicative of c-fos promoter activation. The extenl of activation was approximately 50% of the activation caused by co-transfection with pMVPkC~ . In contrast, vector alone did not induce snhst:mfi~l CAT activity, nor did randomly chosen cDNA library plasmids isolated from the same plasmid 10 pools as CROCs 1, 2 and 4, but which did not activate c~-ntinge replication. These results confirm that the elevated plasmid recovery observed upon co-transfection of CROCs 1, 2 or 4 with PfLAC-8 was due to activation of the c-fos promoter in PfLAG-mediated contingent replication.
An~ysis of c-fos Activatin~ Proteins:

Sequencing revealed that CROC-2 encodes the recently i~lel-tified a2-macroglobulin receptor-associated 20 protein (AMRAP) disclosed in Strickland, et al., J. Biol. Chem..
266, 13364-13369 (1991). The insert is nearly full length and extends from the start codon, which is in frame with the internal vector start codon, to the poly A tail.
A 347 base pair sequence corresponding to 25 nucleotides 555-897 of CROC-4 has been submit~d to GenBank (Accession # Z40809) as an expression sequence tag.
CROC-I cDNA encodes a 19 kd protein with an acidic amino terminal half and a basic carboxy terminus, as shown in SEQ ID NO:I. The protein includes a kinase target domain which 30 contains phosphorylation sites for a variety of kinases involved in signal transduction. Specifically, the kinase target region is comprised of adjacent proximal potential phosphorylation sites for: (a) tyrosine kinases (RXXXEXXXY motif, amino acids 81-89), Cooper, et al., J. Biol. Chem.. 259, 7835-7841 (1984); casein 35 kinase 2 (TIYE motif, amino acids 82-85), Kuenzel, et al., J. Biol.

WO 96/01899 ~ 4 Çhem., ~, 9136-9140 (1987); cAMP-dependent protein kinases (RIYS motif, amino acids 87-90) Glass, et al., J. Biol.
~h~m,.. ~L. 2987-2993 (1986) and Kichim-tc. et al., J. Biol.
Chem., ~1, 12492-12499 (1985); and pro~ein kinase C (SLK
5 motif, amino acids 90-92), Kishimoto, ef al., supra.
The kinase target domain of the CROC-I protein is a twelve amino acid stretch located at the start of ~he basic domain. The known transactivating ability of acidic domains in general, combined with the potential of basic dormains to bind DNA, suggests that CROC-l could function as a transcriptional activator whose activity is regulated by phosphorylation of the kinase target domain. Phosphorylation would cause a further increase in the acidity of the region, thereby enh:~ncing its potential for trPnc~riptictn~l activation, as well as cause a change 15 in the structural conformation of the protein.
The length and tissue distribution of CROC-I mRNA
was determined by Northern analysis of poly A-containing RNA, isolated from various human tissues, using the 1.8 kb Sall/~otl insert of CROC-1 as a probe. CROC-1 mRNA was approximately 20 2.3 kb in length, about 0.5 kb longer than our cDl~A insert, and present in all tissues eY~min~d, with the highest levels being expressed in brain, skeletal muscle, and kidney. In CU~ I.DVU, the 1.5 kb CROC-2 mRNA was present in all tissues ç~ rnin~tl but with the highest levels being expressed in heart, placenta, 25 and kidncy. No evidence was found for additional transcripts, as a result of alternative splicing or multiple sets of transcription-termination-polyadenylation signals, as reported for CROC-2 by Strickland, e~ al., s~pra.
Intracellular localization of the CROC- I protein was 30 detprminpd by cloning CROC-1 in HEL and electroporating the resultant plasmid into COS-7 cells (ATCC# CRL 1651).
Incorporation of CROC-I nucleic acid into the MEL vector enables the in frame fusion of the hemagglutinin epitope to the CROC-I
protein. The intracellular location of CROC-I prolein was then 3~ determined by immunofluorescence microscopy osing mouse ~0 96/01899 - PC~/IJS9~/07874 ~9~3~1L

monoclonal antibody directed against the hemagglutinin epitope. Elc~l..,po.dtion of CROC-I in HEL resulted in intense nuclear fluorescence. In cw~trast, elc~,tlopo,.,tion of HEL alone resulted in general cytoplas [nic fluorescence, ind~ ting that 5 nuclear loc~li7~tion is an inherent property of the CROC-I
protein .
The present invention encompasses modifications and variations which will be evident to those skilled in the art.
The specific embodiments described herein are representative 10 examples only, the scope of the present invention being defined by the claims.

wo g6,0l899 2 1 9 ~ 3 6 1 . ~ 14 ;;

SEQUENOE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: Schering Corporation Patent Depart" ,el ,l K-6-1 (1990) (B) STREET: 2000 Galloping Hill Road (C) CITY: Kenilworth 1 0 (D) STATE: New Jersey (E) COUNTRY: U.S.A.
(F) POSTAL CODE ~ZIP): 07033-0530 (G) TELEPHONE: 908-298-5150 (H) TELEFAX: 908-298-5388 (I) TELEX:

(ii) TITLE OF INVENTION: Method for Identifying Nucleic Acids Encoding c-fos Promoter Activating Proteins ~iii) NUMBER OFSEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh 7.1 (D) SOFTWARE: Microsoft Word 5.1 a (v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/
(B) FILING DATE: -June-1995 (vi) PRIOR APPUCATION DATA
(A) APPLICATION NUMBER: US 081272,412 (B) FILING DATE: 8-JUL-1994 ~ 096l0l899 219 4 ~ 6 1 r~ ,4 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1930 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) SEOUENCE DESCRIPTION: SEQ ID NO:I:

15 ATG GAT CTC AGG CCT AGA TCT C'AT CAC CAT CAC CAT CAT TGG TGC CAG 48 Met Asp Leu Arg Pro Arg Ser His His His His His His Trp Cys Gln TGT GCT GGT CGA CCC ACG caT CCG GAT GGC AGC CAC CAC GGG CTC GGG 96 20 cys Ala Gly Arg Pro Thr Arg Pro Asp Gly Ser His His Gly Leu Gly Ser Lys Ser Pro Ser Gln Phe Arg Leu Leu Glu Glu Leu Glu Glu Gly CAG AAA GGA GTA GGA GAT GGC ACA GTT AGC TaG GGT CTA GAA GAT GAC 132 Gln lys Gly Val Gly Asp Gly Thr Val Ser Trp Gly Leu Glu Asp Asp Glu Asp Met Thr Leu Thr Arg Trp Thr Gly Met Ile Ile Gly Pro Pro Ara Thr Ile TYr Glu Asn Ara Ile Tvr Ser Leu Lvs Ile Glu Cys Gly 40 Pro Lys Tyr Pro Glu Ala Pro Pro Phe Val Arg Phe Val Thr Lys Ile 100 105 llO

Asn Met Asn Gly Val Asn Ser Ser Asn Gly Val Val Asp Pro Arg Ala Ile Ser Val Leu Ala Lys Trp Gln Asn Ser Tyr Ser Ile Lys Val Val WO 96/OlD99 ~ ' r l/L /D/.4 21 ~

Leu Gln Glu Leu Arg Arg Leu Met Met Slr Lys Glu Asn Met Lys Leu Pro Gln Pro Pro Glu Gly Gln Cy~ Tyr Ser Asn TTT CCA CAG TAG TAA ATT TTC TAG ATA CGT CTT GTA GAC CTC AAA CTA 6a4 GGA TCA AGA AAG TCT ATT TAA ATT GAT TCC CAT CAT AAC TGG TC~7 GGC 816 TGT GGG GTG m CGC CTG CAC CCC TGG TTC CTT TAA GTC TTA AGT GAT 1056 CTT TCC TGT AAC TGC m TGC TTT TAA AAA TTG AAG AAG TTT TAA ACA 1632 ~0~6~ 21~ 1 r~l/u. ~4 TCT GAC AAC TGG AAC AAA A~G AAC CTT GAA TCC GGT GCA TGC CTT GGT 1728 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 69 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) SEQUENCE DESCRIPI'ION: SEQ ID NO:2:

CAC GTG AAT TCA AGA TCT CTG CAG AAG CTl' TCC GGA CCG GGC CGC GTA 48 GCA CGC GTA ATA ATT ATC GAT 6~

W 096~1899 ~ 1 9 4 ~ 6 ~ /4 -(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 926 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: iinear (xi) SEQUENCE DESCRlPTiON: SEQ ID NO:3:
GTCGACCCAC ~o~..u~,- CTCACAGAAG CCTGGAGCTG GGCATCCAAa ~ Ar~A~ 60 CTCATTTCTT il~G.~l~ ATCGTAGCTG GCCACCTATG 4U~ ,A ATGTA~AAAG 120 - GGCAGCTCTC TGGC ATG TTC CTG ACT GAG GAT CTC ATA ACA TTT AAC TTa 170 Met Phe Leu Thr Glu Asp Leu Ile Thr Phe Asn Leu Arg Asn Phe Leu Leu Phe Gln Leu Trp Glu Ser Ser Phe Ser Pro aly ~5 15 20 25 Ala Gly Gly Phe Cys Thr Thr Leu Pro Pro Ser Phe Leu Arg Val Asp A~p Arg Ala Thr Ser Ser Thr Thr Asp Ser Ser Arg Ala Pro Ser Ser Pro Arg Pro Pro Gly Ser Thr Ser His Cy~ Cly Ile Ser Thr Arg Cys ACA GAA CGG TCC CTC TGC GTC CTG CCA CTC AGC. ACC TCT CAA GTC CCC 410 Thr Glu Arg Cy~ Leu cy5 Val Leu Pro Leu Arg Thr Ser Gln Val Pro ~30 85 90 GAT GTG ATG GCT CCT CAG CAT GAT CAG GAG AAA TTC CAT GAT CT~ GCT 458 Asp Val Met Ala Pro Gln Hi8 A~p Gln Glu Lys Phe His A~p Leu Ala gs loo loS

Tyr Ser CYs Leu Gly Lys Ser Phe Ser Met Ser Asn Gln Asp Leu ~yr ~ 096l018g9 ~ 1 9 ~ 3 ~ I r~

Gly Tyr Ser Thr Ser Ser Leu Ala Leu Gly Leu Ala Trp Leu Ser Trp '~ 5 12~ 130 135 140 Glu Thr Lys Lys Lys As~ Val Leu ~is Leu Val Gly Leu Asp Ser Leu AGCCTGTCCA TCCTAGCCCA TCCCAG~TTA l~Ul~l~'l~A TTTGAGCTGG GATTCCCACA ~342 ~ A~AAAA AbArr~r~rr-rr CGC 925

Claims (18)

We Claim:

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

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

3. A mammalian cell line of claim 2 wherein the recombinant vector is the plasmid PfLAG-8.

4. A mammalian cell line of claim 1 wherein the expression vector is plasmid L.alpha.2.

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

6. A vector of claim 5 which is the plasmid PfLAG-8.

7. A recombinant expression vector comprising a polyomavirus origin of replication and a nucleic acid suspected to encode a promoter activating protein.

8. A vector of claim 7 which is the plasmid L.alpha.2.

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

10. A method of 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 of claim 11.

13. A nucleic acid of claim 12 having substantially 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:
(a) a first recombinant expression vector comprising a reporter gene operatively 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 .

15. A mammalian cell line of claim 14 comprising NIH
3T3 mouse cells.

16. A mammalian cell line of claim 14 wherein the second recombinant expression vector encodes the CROC-1 protein, the CROC-4 protein, or .alpha.2-macroglobulin receptor-associated protein.

17. A recombinant expression vector comprising a reporter gene operatively 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 of claim 14;
(b) contacting the cell line of step (a) with a sample suspected to contain an antagonist of the human c-fos promoter activating protein; and (c) measuring the level of expression of the reporter gene;
whereby an antagonist of the human c-fos promoter activating protein is identified by measurement of a reduced level of expression of the reporter gene.