EP0770130A1 - PROCEDE POUR IDENTIFIER LES ACIDES NUCLEIQUES CODANT DES PROTEINES D'ACTIVATION DU PROMOTEUR c-fos - Google Patents

PROCEDE POUR IDENTIFIER LES ACIDES NUCLEIQUES CODANT DES PROTEINES D'ACTIVATION DU PROMOTEUR c-fos

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Publication number
EP0770130A1
EP0770130A1 EP95925283A EP95925283A EP0770130A1 EP 0770130 A1 EP0770130 A1 EP 0770130A1 EP 95925283 A EP95925283 A EP 95925283A EP 95925283 A EP95925283 A EP 95925283A EP 0770130 A1 EP0770130 A1 EP 0770130A1
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Prior art keywords
promoter
human
protein
fos
vector
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EP95925283A
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German (de)
English (en)
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Stanley L. Lin
Marnie L. Rothofsky
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Merck Sharp and Dohme Corp
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Schering Corp
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Publication of EP0770130A1 publication Critical patent/EP0770130A1/fr
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/22043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to materials and methods for identifying signal transducing molecules which activate the human c-fos proto-oncogene promoter and antagonists of such molecules.
  • Induction of c-fos occurs in response to the activation of growth-related signalling pathways following serum stimulation of mouse 3T3 cells, or in response to overexpression of the normal and transforming versions of rl&-ras, respectively. It has also been shown that constitutive expression of c-fos occurs in certain human tumor lines.
  • Contingent replication systems employing transcriptional activation of the SV40 T antigen gene to identify enhancers and stably interacting transcription factors are known. See, Vasavada, et al, Ind. J. Biochem. Biophys.. 25. 488- 494 (1988); Vasavada, et al. Gene. 55. 29-40 (1987); Vasavada, et al. Proc. Natl. Acad. Sci.. 88. 10686-10690 (1991); and Rusconi, et al. Gene. 89. 211-221 (1990).
  • the present invention fills the foregoing needs by providing materials and methods for identifying signal transduction molecules and antagonists thereof. More specifically, this invention provides mammalian cell lines, 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
  • a recombinant expression vector comprising a polyomavirus origin of replication and a nucleic acid suspected to encode an activating protein of said promoter.
  • 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 identifying a nucleic acid encoding a promoter activating protein, comprising:
  • a recombinant vector comprising an inducible or tissue specific promoter operatively linked to the coding region of the polyomavirus large T antigen gene;
  • 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;
  • 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
  • a preferred recombinant expression vector comprising a polyomavirus origin of replication is the plasmid
  • the present invention also provides a human c-fos promoter activating proteins having the amino acid sequences defined in the Sequence Listings SEQ ID NO:l and SEQ ID NO:3, or an antigenic fragments thereof, and nucleic acids encoding such protein or fragments.
  • the present invention provides mammalian cell lines, the cells of which comprise: (a) a first recombinant expression vector comprising a reporter gene operatively linked to a human c-fos promoter; and
  • 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 second recombinant expression vector comprising a nucleic acid encoding a human c-fos promoter activating protein
  • the second recombinant expression vector encodes CROC-1 protein, CROC-4 protein or ⁇ 2-macroglobulin receptor-associated protein.
  • LTR long terminal repeat
  • DMEM Dulbecco's modified Eagle's medium
  • SRE serum response element
  • CAT chloramphenicol acetyltransferase
  • antagonist is defined herein as a substance that blocks or inhibits the effects of a human c-fos promoter actvating protein, such as the CROC-1 protein or ⁇ 2-macroglobulin receptor-associated protein.
  • reporter gene means either a DNA molecule isolated from genomic DNA, which may or may not contain introns, or a complementary 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 radioimmunoassay (RIA).
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • Preferred reporter genes for use in the present invention include the E. coli Lac-Z gene from pCHHO (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, although many others well known in the art could be used instead.
  • recombinant expression vector means a vector prepared using recombinant techniques said vector comprising an inserted nucleic acid encoding a protein such that said vector is capable of expressing the protein upon transfection or transformation into a suitable host cell.
  • a vector comprising a nucleic acid encoding a promoter activating protein.
  • a vector comprising a reporter gene operatively linked to a human c- fos promoter.
  • Cells which have been "stably transformed” have recombinant DNA incorporated into their genomic DNA. Such stably incorporated DNA is retained by the transformed 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 mammalian cell lines, however stably transformed mammalian 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-housekeeping 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.
  • inducible promoters are promoters the transcription 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 stimulation or differentiation. Induction occurs via activation of a signalling cascade resulting in the enhanced binding and activity of transcription factors at the promoter site. Molecules involved in such induction include promoter activating proteins as described herein. Inducible promoters include the c-fos and c-myc promoters. Another inducible promoter is the multidrug resistance gene promoter described in J. Biol. Chem.. 268. 15347-15350 (1993).
  • 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, et al, Biochem.. 31 . 818-824 (1992); and Riegman, et al., Mol . Endocrinol.. 5. (No. 12) 1921-1930 (1991).
  • Other tissue specific promoters include promoters of late histone genes and promoters of muscle regulatory elements. 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, et al, supra. Both of these promoters regulate expression in vivo of genes the overexpression of which can lead to aberrant cell growth. Most preferred is the c-fos promoter.
  • promoter activating protein is defined as a protein which causes transcriptional activation of one of the above-mentioned promoters.
  • the promoter activating protein is a human c-fos promoter activating protein.
  • an activating protein having an amino acid sequence substantially identical to that of the ⁇ 2-macroglobulin receptor-associated protein is also most preferred.
  • Substantial identity of amino acid sequences means that the sequence of another c-fos promoter activating protein compared to the sequence defined by either SEQ ID NO:l or SEQ ID NO:3 is identical or differs by one or more amino acid alterations (deletions, additions, substitutions) that do not substantially impair transcription activating activity as described herein.
  • amino acid alterations deletion, additions, substitutions
  • Sequence identity is determined by optimizing residue matches, if necessary, and by introducing gaps as required. This changes when considering conservative substitutions as matches.
  • 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.
  • 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 25-100% homology (if gaps can be introduced), to 50-100% homology (if conservative substitutions are included) with the amino acid sequence of the CROC-1 protein or CROC-4 protein. Homology 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) generally contain at least about 5 amino acid residues. Ohno et al.. Proc. Natl. Acad. Sci. USA. 82. 2945 (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 given polypeptide falls within the scope of this invention can readily be determined by routine experimentation using the methods described below.
  • Such antigenic fragments can be made by proteolysis of the whole human c-fos promoter activating protein or by chemical or recombinant DNA synthesis.
  • the antigenic fragments 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 immunoassay or immunoad
  • the present invention utilizes a recombinant vector comprising 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 promoters.
  • the replicating and transforming properties of the polyoma T antigen gene can be separated.
  • Vasavada, et al., supra, is limited primarily to simian (monkey) systems.
  • 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 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 smaller groups of library plasmids within a cDNA library, resulting in the identification of single library plasmids encoding biologically active molecules which activate the promoter.
  • the self-amplification process of the present invention provides additional sensitivity towards the detection of cDNAs encoding signalling molecules.
  • Initial plasmid replication in response to induction, leads to enhanced expression of active signalling molecules due to greater gene copy number. This increase in signalling molecules results in greater amplification 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 L ⁇ 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-1 and CROC-4, which encode c-fos promoter activating proteins.
  • CROC-1 encodes a specific c-fos promoter activating protein, denoted CROC-1 protein, having the amino acid sequence shown in SEQ ID NO:l.
  • CROC-4 encodes a specific c-fos promoter activating protein, denoted CROC-4 protein, having the amino acid sequence shown in SEQ ID NO:3.
  • the present invention also provides cDNAs encoding c-fos promoter activating proteins which are conservative mutants of the proteins encoded by CROC-1 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.
  • the present invention provides compounds which are antagonists of the protein encoded by CROC-1 or CROC-4. These antagonists include proteins which are deletional, substitutional or additional mutants of the CROC-1 protein or CROC-4 protein, and which bind to, but do not activate, the human c-fos promoter.
  • nucleic acid sequences that can encode c-fos promoter activating proteins and c-fos promoter activating protein antagonists as defined herein.
  • Such functionally equivalent sequences which can readily be prepared using known methods such as chemical synthesis, PCR employing modified primers, and site-directed mutagenesis, are within the scope of this invention.
  • recombinant vector includes both recombinant plasmids such as those mentioned herein and recombinant retroviral vectors, which can also be engineered as described by Geller et al, Proc. Natl. Acad. Sci. USA, SI, 1149 (1990).
  • the foregoing recombinant vectors can be used to transfect any mammalian cell capable of undergoing transfection and permitting vector replication, as herein defined.
  • cells from fresh tissue explants could in principle be used, the use of established cell lines is preferred.
  • Many such cell lines are available including, e.g., NIH 3T3 mouse (ATCC# CRL 1658), L-M (TK-) mouse (ATCC# CCL 1.3) and BALB/c 3T3 Clone A31 mouse (ATCC# CCL 163) cell lines.
  • a cell or cell line for use in the methods of the present invention will be dictated by the known or determinable specificities of the vectors used.
  • the murine cell lines are preferred for use with vectors comprising a recombinant vector containing the polyomavirus large 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 origin of replication and a nucleic acid suspected to encode a human promoter activating protein, such as a retro viral expression vector comrpising a retroviral LTR capable of expressing the nucleic acid.
  • stably-transformed cells can also be used. Stable transformation of a mammalian cell line can be accomplished by using standard methods to co-transfect the cells with one of the above-mentioned recombinant vectors and with a second vector which confers resistance to a selection agent such as an antibiotic.
  • cells are co-transfected with a recombinant vector comprising a human c-fos promoter operatively linked to polyomavirus large T antigen gene, and a cDNA library incorporated into a mammalian recombinant expression vector comprising a polyomavirus origin of replication.
  • the cells are then incubated under conditions in which vectors containing 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 digest. Typical incubations are carried out for 2 days at 37°C in a humidified CO 2 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. Incubation is continued 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.
  • a substantially 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 substantial increase in vector replication and recovery is typically an increase of at least about 5-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 dependent upon the level of background replication.
  • cells are provided which are simultaneously transfected with a first recombinant expression vector comprising a reporter gene operatively 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 /05-lac Z reporter 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 suspected antagonists under conditions in which the gene encoding the human c-fos promoter activating protein is expressed. Under such conditions, and in the absence of an antagonist, stimulation of the human c-fos promoter will occur, resulting in reporter gene expression.
  • the samples can be, e.g. aqueous or water-miscible solutions in which isolated compounds 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 a humidified CO2 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.
  • Incubation is continued for a period of time sufficient to permit significant 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 determined by routine experimentation but will typically be in 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 (antagonist free) cultures.
  • a reduction in the level of reporter gene expression 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 measuring a substantially decreased level of reporter gene expression, compared to the level measured in the control cultures.
  • a substantially 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 antagonist of a human c-fos promoter activating protein.
  • the degree of decrease may be influenced 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.
  • reporter gene expression due to general toxicity of samples can be accounted 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.
  • 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.
  • Dpnl is a known restriction endonuclease isolated from Diplococcus pneumoniae and is commercially available from ICN Biomedicals, Sigma Chemical Company or New England BioLabs, Inc.
  • restriction endonucleases Asel, BamW, Bglll, BstXl, Clal, Fspl, Hindi, Narl, Notl, Sacll, Sail, Seal, Xbal and Xhol are known and are commercially available, e.g. from Sigma
  • Sail, Seal, Xbal and Xhol are known and are commercially available, e.g. from New England BioLabs, Inc.
  • the restriction endonuclease Saul is known and is commercially available, e.g. from Boehringer Mannheim.
  • the enzyme mung bean nuclease is known and is commercially available from New England Biolabs, Inc or Sigma.
  • the synthetic polylinker used in preparing the vector L ⁇ 2 was obtained from New England Biolabs, Inc. and has the sequence shown in SEQ ID NO:2.
  • the Ncol linker d(pAGCCATGGCT) is known and is commercially available from
  • nucelotide sequence and restriction sites of pUC19 are described by Yanisch-Perron, et al, in Gene.
  • Polyomavirus DNA strain A2 (ATCC # 45017); and human genomic c-fos (ATCC # 41042).
  • Retroviral vector pMV7 Construction of the retroviral vector pMV7 is described by Kirschmeier, et al. DNA . 7. 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 widely distributed in many laboratories.
  • retroviruses similar to pMV7 which could be used instead in this invention are readily available, such as pV-mos (ATCC# 41037).
  • the fos-CAT reporter gene construct described below was prepared using the commercially available pCAT-basic vector (Promega catalog # El 041).
  • 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) Superscript cloning kit, and inserted into the SalllNotl sites in plasmid L ⁇ 2.
  • nucleic acids 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
  • Co-transfection of cells with PfLAG and L ⁇ 2 containing a cDNA encoding a biologically active signalling molecule causes activation of the c-fos promoter, resulting in the production of large T antigen.
  • the production of large T antigen stimulates intracellular replication of plasmids containing the polyomavirus origin of replication. Plasmids are recovered from the 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 Dpnl. Replicated plasmids are then recovered by transformation into competent bacteria.
  • NIH 3T3 mouse fibroblasts ATCC# CRL 1658
  • Rat 2 fibroblasts ATCC# CRL 1764
  • DMEM fetal calf serum
  • bovine calf serum 50 ⁇ g/ml gentamycin sulfate.
  • the DH10B E. coli used in the present invention are commercially available from GIBCO.
  • the first (denoted P f LAG) 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 L ⁇ 2) was a retroviral cDNA expression vector containing the polyomavirus origin of replication.
  • the retroviral cDNA vector L ⁇ 2 was prepared as follows. Polyomavirus DNA strain A2 was digested with BamHl/ Narl and the resulting 750 bp fragment was ligated into the BamHl/Narl sites in pUC19 to give a plasmid denoted pOri.
  • the retroviral vector pMV7 was digested with Fspl/Afl ⁇ l and the resulting 4 kb band containing the two Moloney murine sarcoma virus LTRs was ligated into the Hincll/Afllll 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 SaullClal digestion and replaced by a synthetic polylinker (described above) to give the plasmid pMV7-3.
  • the polylinker in pUC19 was replaced with a Ncol linker, then the 360 bp lac Z region was removed by Asel/Narl digestion, blunt ended with mung bean nuclease, and ligated into the pMV7-3 polylinker.
  • the resultant plasmid, denoted L ⁇ 2 was 4.5 kb and contained unique Sail and Notl sites at the 5' and 3' ends, respectively, of the lac Z gene.
  • the P f LAG plasmid was prepared via the following procedure.
  • the polyomavirus large T antigen under the control of the human c-fos promoter was introduced by digesting the 5.9 kb BamHl fragment of pc/ ⁇ s-l, disclosed by Curran, et al, Mol. Cell. Biol.. 3.
  • a third vector denoted HEL, was prepared for use in identifying the intracellular locations of CROC-1.
  • the histidine hexamer coding sequences of L ⁇ 2 were removed by Bglll/Sall digestion and replaced with coding sequences for the nine amino acid influenza virus HA1 epitope described in Field, et al, Mol. Cell. Biol.. 8. 2159-2165 (1988).
  • the SV40 origin of replication was then inserted at the unique Xbal site between the polyoma origin of replication and the 5' LTR, to give HEL.
  • 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 human c-fos promoter in P f LAG must remain transcriptionally silent 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 can be increased by the incorporation of additional enhancer elements into the promoter.
  • a threshold sensitivity of detecting about one plasmid out of forty for cDNA screening was used. Therefore a 1:40 (wt/wt) ratio of pMVPkC ⁇ i/L ⁇ 2 for co-transfection with each of the PfLAGs into NIH 3T3 cells was utilized in the procedure described below. Cells were incubated for forty-eight (48) hours following transfection. The plasmids were extracted and examined, following Dpnl digestion, for elevated plasmid recovery indicative of contingent replication. The results obtained under these conditions are presented in Table 1.
  • PfLAG is co-transfected with 18 ⁇ g of either pMV7-Z or a 1:40 (wt/wt) ratio of pMV7PkC ⁇ i/L ⁇ 2.
  • the results presented are the average of two experiments.
  • the effect of the concentration of plasmids encoding a promoter actvating protein on the recovery of pMVPkC ⁇ i within a total population of plasmids is determined by varying the concentration of pMVPkC ⁇ i in a pMVPkC ⁇ i /L ⁇ 2 mixture prior to co-transfection with PfLAG-8.
  • L ⁇ 2 has a modified lac Z gene derived from pUC19
  • bacteria transformed with L ⁇ 2 will turn blue
  • bacteria transformed with pMVPkC ⁇ i will remain white, when plated on agar plates containing ampicillin, X-gal, and IPTG.
  • the percentage of pMVPkC ⁇ i is determined by expressing the number of white colonies as a percentage of total colonies formed after bacterial transformation of pnl-digested Hirt extracts.
  • transfections 8 x 10 ⁇ 3T3 cells were planted in growth medium in 100 mm dishes and allowed to attach overnight. The following day, transfections were performed by the method of Wigler, et al. Cell. 1 1 . 223-232 (1977), using calcium phosphate. After a 4-hour exposure to the calcium phosphate precipitate, cells were washed twice with phosphate buffered saline, re-fed with DMEM 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 Dpnl for a minimum of 24 hours. Dpnl digests were phenol extracted and ethanol precipitated. DNA was resuspended in 20 ⁇ L TE (1 mM EDTA +10 mM Tris, pH8.0), and transformed into competent DH10B bacteria (GIBCO).
  • a human brain cDNA library was co-transefected with PfLAG-8 into NIH 3T3 cells via the methods described 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 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-1 and CROC-2 (for contingent replication of cDNA), consistently gave elevated plasmid recovery when co-transfected with PfLAG-8. The nucleotide sequence for CROC-1 is shown in SEQ ID NO:l.
  • CROC-4 A third plasmid, denoted CROC-4, was identified by further plasmid screening. Plasmid CROC-4 also consistently gave elevated plasmid recovery when co-transfected with P f LAG-8.
  • the nucelotide sequence for CROC-4 is shown in SEQ ID NO:3.
  • CROC-1, CROC-2 and CROC-4 were co-transfected with a fos-CAT reporter gene and tested for elevation of CAT activity as follows. Rat 2 cells were co-transfected with 18 ⁇ g Loc2-expressed cDNA (i.e., CROC-1, CROC-2 or CROC-4) + 2 ⁇ g fos-CAT for 4 h, then refed with DMEM + 0.5% calf serum. Cells were harvested 72 hours after transfection and CAT assays performed via the procedure of Gorman, et al. Mol. Cell. Biol.. 2. 1044-1051 (1982).
  • CAT activity was significantly induced by CROC-1, CROC-2 and CROC-4, indicative of c-fos promoter activation.
  • the extent of activation was approximately 50% of the activation caused by co-transfection with pMVPkC ⁇ i .
  • vector alone did not induce substantial CAT activity, nor did randomly chosen cDNA library plasmids isolated from the same plasmid pools as CROCs 1, 2 and 4, but which did not activate contingent replication.
  • CROC-2 encodes the recently identified ⁇ 2-macroglobulin receptor-associated protein (AMRAP) disclosed in Strickland, et al, J. Biol. Chem.. 266. 13364-13369 (1991).
  • AMRAP ⁇ 2-macroglobulin receptor-associated protein
  • a 347 base pair sequence corresponding to nucleotides 555-897 of CROC-4 has been submitted to GenBank (Accession # Z40809) as an expression sequence tag.
  • CROC-1 cDNA encodes a 19 kd protein with an acidic amino terminal half and a basic carboxy terminus, as shown in SEQ ID NO:l.
  • the protein includes a kinase target domain which contains phosphorylation sites for a variety of kinases involved in signal transduction.
  • 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.
  • the kinase target domain of the CROC-1 protein is a twelve amino acid stretch located at the start of the basic domain.
  • CROC-1 mRNA The length and tissue distribution of CROC-1 mRNA was determined by Northern analysis of poly A-containing RNA, isolated from various human tissues, using the 1.8 kb Sall/Notl insert of CROC-1 as a probe.
  • CROC-1 mRNA was approximately 2.3 kb in length, about 0.5 kb longer than our cDNA insert, and present in all tissues examined, with the highest levels being expressed in brain, skeletal muscle, and kidney.
  • the 1.5 kb CROC-2 mRNA was present in all tissues examined, but with the highest levels being expressed in heart, placenta, and kidney. 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, et al, supra.
  • Intracellular localization of the CROC-1 protein was determined by cloning CROC-1 in HEL and electroporating the resultant plasmid into COS-7 cells (ATCC# CRL 1651). Incorporation of CROC-1 nucleic acid into the HEL vector enables the in frame fusion of the hemagglutinin epitope to the CROC-1 protein. The intracellular location of CROC-1 protein was then determined by immunofluorescence microscopy using mouse monoclonal antibody directed against the hemagglutinin epitope. Electroporation of CROC-1 in HEL resulted in intense nuclear fluorescence. In contrast, electroporation of HEL alone resulted in general cytoplasmic fluorescence, indicating that nuclear localization is an inherent property of the CROC-1 protein.
  • AGT AAA AGT CCC TCG CAA TTT CGA CTG TTG GAA GAA CTC GAA GAA GGC 144 Ser Lys Ser Pro Ser Gin Phe Arg Leu Leu Glu Glu Leu Glu Glu Gly 35 40 45
  • 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

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Abstract

Cette invention se rapporte à des substances et à des procédés qui servent à identifier des molécules transductrices de signal qui activent des promoteurs, tels que le promoteur de proto-oncogène c-fos humain, ainsi que des antagonistes de ces molécules. Cette invention décrit également des protéines activant ce promoteur c-fos humain, et en particulier de nouvelles protéines, appelées protéine CROC-1 et protéine CROC-4, des acides nucléiques codant ces protéines, ainsi que des cellules de mammifères transfectées au moyen de vecteurs contenant de tels acides nucléiques.
EP95925283A 1994-07-08 1995-07-05 PROCEDE POUR IDENTIFIER LES ACIDES NUCLEIQUES CODANT DES PROTEINES D'ACTIVATION DU PROMOTEUR c-fos Withdrawn EP0770130A1 (fr)

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US7364894B2 (en) 1995-09-15 2008-04-29 Centelion Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy
US7279313B2 (en) 1995-09-15 2007-10-09 Centelion Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy
US6048693A (en) * 1996-10-16 2000-04-11 Bittech, Inc. Phenotypic assays of cyclin/cyclin-dependent kinase function
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US6063578A (en) * 1998-10-22 2000-05-16 Signal Pharmaceuticals, Inc. Dual reporter system and methods of use therefor
CA2621083C (fr) 2005-08-30 2017-04-11 University Of Miami Immunomodulation des agonistes, des antagonistes et des immunotoxines du recepteur 25 du facteur de necrose tumorale (tnfr25)
AU2010279637B2 (en) 2009-08-03 2012-08-23 University Of Miami Method for in vivo expansion of T regulatory cells
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