WO1997043415A9 - Facteur de liaison de la cycline de type d et emplois dudit produit - Google Patents

Facteur de liaison de la cycline de type d et emplois dudit produit

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Publication number
WO1997043415A9
WO1997043415A9 PCT/US1997/008480 US9708480W WO9743415A9 WO 1997043415 A9 WO1997043415 A9 WO 1997043415A9 US 9708480 W US9708480 W US 9708480W WO 9743415 A9 WO9743415 A9 WO 9743415A9
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WIPO (PCT)
Prior art keywords
amino acid
dmpl
acid polymer
seq
sequence
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PCT/US1997/008480
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English (en)
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WO1997043415A1 (fr
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Priority to AU31323/97A priority Critical patent/AU3132397A/en
Publication of WO1997043415A1 publication Critical patent/WO1997043415A1/fr
Publication of WO1997043415A9 publication Critical patent/WO1997043415A9/fr

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  • This invention relates generally to a novel myb-li e protein that interacts with cyclin D.
  • the interaction involves the regulation of RNA transcription.
  • the invention relates to the protein, polypeptide, including biologically active or antigenic fragments thereof, and analogs and derivatives thereof, and to methods of making and using the same, including diagnostic and therapeutic uses.
  • the invention further includes the corresponding amino acid and nucleotide sequences.
  • the cell cycle for growing cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period.
  • the cell division period is labeled the M (mitotic) period.
  • the interphase period in eucaryotes is further divided into three successive phases: Gl (gap 1) phase, which directly follows the M period; S (synthetic) phase, which follows Gl; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period.
  • Gl gap 1
  • S synthetic phase
  • G2 G2 phase
  • the cell passes a restrictive (R) point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide.
  • the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the Gl phase.
  • the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the Gl phase.
  • the Gl phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the R point late in Gl phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the R point, cells become committed to duplicating their DNA and undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous.
  • RB retinoblastoma protein
  • E2F retinoblastoma protein
  • hyperphosphorylation of RB late in Gl phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes.
  • E2F-regulated genes encode proteins that are essential for DNA synthesis
  • RB phosphorylation at the R point helps convert cells to a pre-replicative state that anticipates the actual Gl/S transition by several hours. Cells that completely lack the RB function have a reduced dependency on mitogens but remain growth factor-dependent, indicating that cancellation of the RB function is not sufficient for passage through the R point.
  • Phosphorylation of RB at the R point is initially triggered by holoenzymes composed of regulatory D-type cyclin subunits and their associated cyclin-dependent kinases, CDK4 and CDK6.
  • the D-type cyclins are induced and assembled into holoenzymes as cells enter the cycle in response to mitogenic stimulation. Acting as growth factor sensors, they are continuously synthesized as long as mitogenic stimulation continues, and are rapidly degraded after mitogens are withdrawn.
  • inhibition of cyclin D-dependent CDK activity prior to the R point either by microinjection or by scrape loading of antibodies directed against cyclin Dl or by expression of CDK4 and CDK6 inhibitors (INK4 proteins) prevents entry into S phase.
  • such manipulations have no effect in cells lacking functional RB, implying that RB is the only substrate of the cyclin D-dependent kinases whose phosphorylation is necessary for exiting the Gl phase.
  • RB-mediated controls are not essential to the cell cycle per se it is difficult to understand why mammalian cells contain three distinct D-type cyclins (Dl, D2, and D3), at least two cyclin D-dependent kinases (CDK4 and CDK6), and four INK4 proteins, all, purportedly, for the sole purpose of regulating RB phosphorylation. This apparent redundancy has been explained as a method to govern transitions through the R point in different cell types responding to a plethora of distinct extracellular signals.
  • cyclin D-dependent kinases or the cyclins alone could also be involved in the regulation of RB-independent events, perhaps linking them temporally to cell cycle controls.
  • One mechanism for this regulation could involve the direct interaction between a cyclin, such as a D-type cyclin, and a specific transcription factor, which would allow the cyclins to regulate gene expression in an RB-independent manner.
  • a cyclin such as a D-type cyclin
  • the present invention provides a new, cyclin D-associated transcription factor.
  • the transcription factor is an amino acid polymer which specifically binds D-type cyclins in vitro, specifically binds a DNA nucleotide sequence, and is involved in the regulation of genes that prevent cell proliferation.
  • the cyclin D-associated transcription factor is a substrate of cyclin D2-CDK4 kinase.
  • the transcription factor consists of about 760 amino acids.
  • the present invention includes an amino acid polymer that has a binding affinity for one or more D-type cyclins, and one or more of the following characteristics in addition to the property described above: (I) The relative binding affinity of the amino acid polymer for cyclin D2, as compared to that for a cyclin D2 mutant that is disrupted in an amino-terminal LEU-X- CYS-X-GLU pentapeptide, is minimally less disparate than the relative binding affinity of retinoblastoma protein for cyclin D2 as compared to that for the same cyclin D2 mutant.
  • the amino acid polymer remains able to detectably interact with a cyclin D2 mutant, containing substitutions in the amino-terminal LEU-X-CYS-X-GLU pentapeptide, under conditions where the binding of retinoblastoma protein to that same cyclin D2 mutant is essentially undetectable.
  • the amino acid polymer binds preferentially to a specific DNA nucleotide sequence.
  • the amino acid polymer is a substrate of the cyclin D2-CDK4 complex.
  • the amino acid polymer contains three atypical tandem myb repeats.
  • D-type cyclins bind less avidly to the amino acid polymer than to retinoblastoma protein, both in vitro and in Sf9 cells.
  • Cyclin D-CDK4-dependent phosphorylation of retinoblastoma protein proceeds to a much higher stoichiometry than the comparative phosphorylation of the amino acid polymer under standard conditions for cyclin D-CDK4 kinase reactions.
  • Cyclin D-dependent kinases phosphorylate the amino acid polymer at an atypical recognition sequence.
  • the amino acid polymer binds preferentially to nucleic acids containing the nonamer sequence CCCGTATGT.
  • a catalytically-inactive CDK4 does not enter into a stable ternary complex with cyclin D and the amino acid polymer under conditions where retinoblastoma protein, cyclin D and the identical catalytically-inactive CDK4 form stable ternary complexes.
  • the amino acid polymer activates transcription more readily in quiescent cells lacking cyclin D expression, than in proliferating cells containing cyclin D.
  • Enforced expression of cyclin D-CDK4 does not influence the stability of the amino acid polymer.
  • Enforced expression of cyclin D-CDK4 does not influence the ability of the amino acid polymer to preferentially localize to the nucleus of transfected mammalian cells.
  • the amino acid polymer binds preferentially to a DNA nucleotide sequence, termed herein the cyclin D-associated transcription factor binding site or the DMPl binding site.
  • the binding site has the core trinucleotide sequence GTA.
  • the nucleotide sequence contains a nonamer consensus sequence CCCG(G/T)ATGT.
  • the nucleotide sequences contain multiple concatamers of the nonamer consensus sequence.
  • the nucleotide sequence contains the nonamer consensus sequence CCCGTATGT.
  • the present invention provides an isolated amino acid polymer obtained from animal cells, produced recombinantly, or prepared by chemical synthesis.
  • the amino acid polymer is mammalian.
  • the amino acid polymer is human.
  • the amino acid polymer is obtained from a murine cell and has the sequence of SEQ ID NO: l .
  • the amino acid polymer is obtained from a human cell and contains the amino acid sequence of SEQ ID NO:24.
  • the isolated amino acid polymer is obtained from a human cell, is encoded on human chromosome 7 at a position which corresponds to 7 q 21 , and contains about 760 amino acids including the 262 amino acids of SEQ ID NO:24.
  • the present invention relates to the identification and elucidation of a direct interaction between D-type cyclins and a novel myb-like transcription factor termed herein DMPl .
  • This novel factor has been found to specifically interact with cyclin D2.
  • This present invention also describes the regulation of gene expression by D-type cyclins, and other related methods of use, in an RB-independent manner.
  • DMPl includes a central DNA-binding domain containing three atypical myb repeats flanked by highly acidic segments located at its amino- and carboxylterminal ends.
  • the present invention includes amino acid sequences coding for DMPl, including amino acid sequences containing conservative substitutions of such amino acids.
  • the present invention also includes a peptide that corresponds to the DNA-binding domain of the amino acid polymer of the present invention.
  • the peptide has an amino acid sequence of SEQ ID NO: 16.
  • the peptide has an amino acid sequence of SEQ ID NO: 16 having conservative amino acid substitutions.
  • the present invention also includes a peptide that corresponds to the transactivation domain of the amino acid polymer of the present invention.
  • the peptide has an amino acid sequence of SEQ ID NO: 18.
  • the peptide has an amino acid sequence of SEQ ID NO: 18 having conservative amino acid substitutions.
  • the peptide has an amino acid sequence of SEQ ID NO:20.
  • the peptide has an amino acid sequence of SEQ ID NO:20 having conservative amino acid substitutions.
  • the peptide has an amino acid sequence consisting of SEQ ID NO: 18 and SEQ ID NO:20.
  • the present invention further includes a peptide that corresponds to the D-type cyclin binding domain of the amino acid polymer of the present invention.
  • the peptide has an amino acid sequence of SEQ ID NO:22.
  • the peptide has an amino acid sequence of SEQ ID NO: 22 having conservative amino acid substitutions.
  • DNA and RNA nucleotide sequences that encode for the amino acid polymers of the present invention, and methods of use thereof are also included.
  • One method of the invention includes the use of DMPl as a transcription factor due to its specificity in binding to oiigonucleotides containing the nonamer consensus sequence CCCG(G/T)ATGT.
  • a recombinant expression vector comprising the foregoing consensus sequence operably associated with a gene for expression can be prepared.
  • DMPl activates the transcription of a heterologous gene including reporter genes driven by a minimal promoter containing concatamerized DMPl binding sites.
  • the invention provides for expression of DMPl with the foregoing expression vector in order to enhance DMPl -mediated transcription from the expression vector.
  • Another aspect of the present invention includes GST-DMP1 fusion proteins that bind directly to D-type cyclins in vitro, including radiolabeled D-type cyclins.
  • complexes between full-length DMPl and D-type cyclins readily form in intact Sf9 insect cells engineered to co-express both proteins under baculovirus vector control.
  • a further aspect of the invention includes the use of detectable labels, such as but not limited to a protein including an enzyme, a radioactive element, a bioluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore.
  • detectable labels such as but not limited to a protein including an enzyme, a radioactive element, a bioluminescent, a chromophore that absorbs in the ultraviolet and/or visible and/or infrared region of the electromagnetic spectrum; and a fluorophore.
  • the present invention includes an amino acid polymer labeled with such a detectable label.
  • the present invention also includes reporter genes encoding proteins that contain detectable labels, such as green fluorescent protein, or an 35 S-labeled protein, can interact with a label such as a labeled antibody or can catalyze a reaction that gives rise to a detectable signal, such as the bioluminescence catalyzed by
  • the present invention also includes antibodies to all of the amino acid polymers of the instant invention.
  • the antibodies of the present invention may be either polyclonal or monoclonal. Either type of antibody can further comprise a detectable label described above.
  • the present invention provides nucleic acids that contain nucleotide sequences or degenerate variants thereof, which encode the amino acid polymers of the present invention.
  • the nucleotide sequence can be a DNA sequence of SEQ ID NO:2 or an RNA sequence corresponding to SEQ ID NO:3; or a DNA sequence encoding a full length human DMPl containing the nucleic acid sequence SEQ ID NO:25 or an RNA sequence encoding a full length human DMPl containing the nucleic acid sequence SEQ ID NO: 26.
  • the nucleic acid encodes a full length human DMPl containing the amino acid sequence of SEQ ID NO:24.
  • the nucleic acid encodes an isolated amino acid polymer which is encoded on human chromosome 7 at a position which corresponds to 7 q 21, and contains about 760 amino acids, including the 262 amino acids of SEQ ID NO:24.
  • the present invention also includes a nucleic acid encoding a peptide that corresponds to the DNA-binding domain of the amino acid polymer of the present invention.
  • the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 16 having conservative amino acid substitutions.
  • the nucleic acid sequence is SEQ ID NO: 17.
  • the present invention also includes a nucleic acid encoding a peptide that corresponds to the transactivation domain of the amino acid polymer of the present invention.
  • the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:18,, or SEQ ID NO: 18 having conservative amino acid substitutions.
  • the nucleic acid sequence is SEQ ID NO: 19. In yet another specific embodiment of this type, the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:20, or SEQ ID NO:20 having conservative amino acid substitutions. In one specific embodiment of this type, the nucleic acid sequence is SEQ ID NO:21. In yet another specific embodiment of this type, the nucleic acid encodes a peptide having an amino acid sequence consisting of SEQ ID NO: 18 and SEQ ID NO:20 or consisting of an amino acid sequence of SEQ ID NO: 18 and SEQ ID NO:20 having conservative amino acid substitutions.
  • the nucleic acid sequence consists of SEQ ID NO: 19 and SEQ ID NO:21.
  • the present invention further includes a nucleic acid encoding a peptide that corresponds to the D-type cyclin binding domain of the amino acid polymer of the present invention.
  • the nucleic acid encodes a peptide having an amino acid sequence of SEQ ID NO:22, or SEQ ID NO:22 having conservative amino acid substitutions.
  • the nucleic acid sequence is SEQ ID NO:23.
  • nucleic acids containing sequences complementary to these sequences, or nucleic acids that hybridize to any of the foregoing nucleotide sequences under standard hybridization conditions are also part of the present invention.
  • the nucleic acids hybridize to the foregoing nucleotide sequences under stringent conditions.
  • the nucleic acid is a recombinant DNA sequence that is operatively linked to an expression control sequence.
  • Another aspect of the invention includes methods for detecting the presence or activity of the amino acid polymer of the invention in a biological sample that is suspected to contain the amino acid polymer. These methods include steps of contacting a biological sample with a nucleotide probe under conditions that allow binding of the nucleotide probe to the amino acid polymer to occur, and then detecting whether that binding has occurred.
  • the nucleotide probe contains the sequence CCCGTATGT. The detection of the binding indicates the presence or activity of the amino acid polymer in the biological sample.
  • the nucleotide probe may be labeled with a detectable label as described above.
  • the nucleotide probe has a detectable label containing the radioactive element, 32 P, and the detecting step includes performance of an electrophoretic mobility shift assay.
  • the DMPl binding site may be used to isolate a DMPl amino acid polymer by specific affinity binding. More particularly, the CCCGTATGT nonanucleotide may be used to isolate a mammalian DMPl polypeptide.
  • Another aspect of the present invention includes methods of activating selective transcription of a heterologous gene operably associated with a DNA sequence to which the present transcription factor binds in mammalian cells. These methods include the step of recombinantly fusing a control unit comprising the nucleotide sequence, e.g. ,
  • the endogenous transcription factor of the invention in the mammalian cell will be sufficient to activate selective transcription of the heterologous gene.
  • the basal level of the amino acid polymer in the mammalian cells used will be insufficient to activate detectable transcription of the recombinant heterologous gene.
  • the amino acid polymer of the present invention may be added to the mammalian cell, e.g. , by microinjection or transfection, with an expression vector comprising the transcription factor gene into the cells, thereby activating transcription of the selected gene.
  • the present invention also includes the use of an oligonucleotide comprising the DMPl binding site, e.g., the nonamer sequence CCCGTATGT, as a competitive inhibiter for blocking the activation of selective transcription by the amino acid polymer.
  • an oligonucleotide comprising the DMPl binding site, e.g., the nonamer sequence CCCGTATGT, as a competitive inhibiter for blocking the activation of selective transcription by the amino acid polymer.
  • the present invention also includes an antisense nucleic acid against an mRNA coding for the amino acid polymer of the present invention and is therefore capable of hybridizing to the mRNA.
  • the antisense nucleic acid may be either an RNA or a DNA, preferably containing a phosphodiester analog.
  • the present invention provides a transgenic animal comprising the expression vector which provides for increased or "super-" expression of the cyclin D- associated transcription factor homologously recombined in a chromosome or a cyclin D- associated transcription factor that no longer binds a cyclin D, such as cyclin Dl.
  • the present invention provides a transgenic animal in which the gene encoding an amino acid polymer of the present invention, such as murine DMPl , has been disrupted so as to be unable to express a functional transcription factor.
  • Disruption of expression can be achieved by (i) knocking out the gene; (ii) introducing a null or nonsense mutation in the gene; (iii) deleting the regulatory sequences necessary for effective transcription of the gene; and (iv) introducing a mutation into the gene that results in expression of an inactive protein, e.g. , a protein which fails to bind to DNA, to the DMPl binding site on DNA, to transactivate genes under the control of a DMP1- responsive promoter, or any combination of the foregoing.
  • an inactive protein e.g. , a protein which fails to bind to DNA, to the DMPl binding site on DNA, to transactivate genes under the control of a DMP1- responsive promoter, or any combination of the foregoing.
  • the present invention also includes methods of identifying genes that are under the control of DMPl -responsive promoters. Such genes play an important role in cell regulation, and more particularly in hindering the proliferation of the cell.
  • the present invention also includes drug assays for identifying drugs that antagonize or agonize the effect of DMPl on genes under the control of a DMPl-responsive promoter.
  • One such method is for identifying a drug that inhibits the transactivation of a gene by DMPl in situ, comprising cotransfecting a cell with a first expression vector containing a reporter gene under the control of a promoter responsive to DMPl , and a second expression vector encoding DMPl , or a fragment thereof capable of transactivating the promoter.
  • a potential drug is then contacted with the cell, and the expression of the reporter gene is detected.
  • a drug is identified when the expression of the reporter gene is decreased. In preferred embodiments of this type, the identified drug prevents the detectable expression of the reporter gene.
  • the second expression vector encodes an amino acid polymer having the amino acid sequence of SEQ ID NO: l .
  • the second expression vector encodes a fragment of DMPl having an amino acid sequence of SEQ ID NO: 18, or SEQ ID NO: 18 having conservative substitutions.
  • the promoter is an artificial DMPl -responsive promoter.
  • the artificial promoter consists of 8X BS2 (CCCGTATGT) inserted into the Xhol-Smal sites of pGL2 (Promega) 5' to a minimal simian virus 40 (SV40) early promoter driving the reporter gene.
  • the reporter gene is firefly luciferase.
  • the cell is a mammalian cell, such as a mouse NIH-3T3 fibroblast.
  • the mammalian cell is a human cell.
  • the potential drug may be selected by rational design, such as an analog of a cyclin, or an analog to the DNA-binding domain of DMPl, as described herein.
  • the potential drug can be randomly obtained from a drug library, including from one described herein.
  • the present invention also includes in vitro assays to identify drugs that will bind to the cyclin binding domain of DMPl.
  • the cyclin binding domain has an amino acid sequence of SEQ ID NO:22, or SEQ ID NO:22 having conservative substitutions.
  • Such drugs can either inhibit DMPl by acting as an analog of the cyclins; or alternatively, the drug can prevent the inhibition of the cyclin-dependent inhibition of DMPl by preventing a cyclin from binding to DMPl while not interfering with the transactivation properties of DMPl.
  • the method comprises placing the cyclin binding domain of DMPl on a solid support, contacting the cyclin binding domain of DMPl with a potential drug that is labeled, washing the solid support, and detecting the potential drug associated with the cyclin binding domain of DMPl.
  • a potential drug is identified as a drug if it is detected with the cyclin binding domain of DMPl .
  • the method can further comprise a step of washing the solid support with an excess of a cyclin, such as cyclin D2, prior to the detection step.
  • a potential drug is identified as a drug, if washing with cyclin hinders or prevents the detection of the labeled drug with cyclin binding domain of DMPl .
  • the potential drug may be selected by rational design, such as an analog of a cyclin, or alternatively the potential drug can be randomly obtained from a drug library, including from one described herein.
  • An identified drug can then be assayed in situ, as described above to determine whether it enhances or diminishes the transactivation of a reporter gene under the control of a DMPl -responsive promoter.
  • a drug is selected as an antagonist of DMPl when the expression of the reporter gene is decreased.
  • a drug is selected as an agonist of DMPl when the expression of the reporter gene is increased.
  • the method can further comprise coexpressing a cyclin, such as cyclin D2, and DMPl in a cell and determining whether the drug prevents the inhibitory effect of the cyclin.
  • a drug is selected as an agonist of DMPl , if it can hinder and/or prevent the inhibitory effect of the cyclin.
  • An additional embodiment includes a method of determining the effect of the drug on a CDK comprising contacting the identified drug with a CDK and performing a cyclin-CDK kinase assay on an appropriate substrate, such as retinoblastoma protein (as described herein) in the absence of a cyclin, wherein a drug is selected if the kinase assay is negative.
  • the cyclin-CDK kinase assay is next performed with cyclin, the CDK, appropriate substrate and an excess of the drug.
  • a drug is selected which does not interfere with the phosphorylation of the appropriate substrate by the cyclin-CDK.
  • Figures 1A-1B show the Amino Acid Sequence of murine DMPl .
  • Figure 1 A shows the DMPl protein sequence. The three myb repeats are underlined with the first (residues 224-273) and third (residues 334-392) repeats demarcated by italics. Ser-Pro and Thr-Pro doublets are in bold face type, and acidic residues clustered at the amino- and carboxyterminal ends of the protein are indicated by double underlining.
  • Figure IB shows the three myb repeats within mouse DMPl (top) and c-myb (bottom) are aligned with identical positions indicated by vertical bars.
  • Figure 2 is a gel showing the binding in vitro of D-type cyclins to RB and DMPl fusion proteins.
  • [ 35 S]methionine-labeled D-type cyclins prepared by in vitro transcription and translation are mixed with the bacterially produced GST fusion proteins or GST controls as indicated above the figure. Proteins bound to glutathione-Sepharose beads are washed, denatured, and separated on gels. Lanes 1, 5, and 9 show aliquots of input radioactive proteins corresponding to 25% of that actually used in each of the subsequent binding reactions. The mobilities of the three different D-type cyclins are denoted at the right. All protein inputs and exposure times are matched.
  • Figure 3 is a gel showing the binding of D-type cyclins to DMPl in insect Sf9 cells. Insect cells coinfected with baculovirus vectors encoding DMPl , D-type cyclins (Dl , D2, D3), wild-type CDK4 (K4), or a catalytically inactive CDK4 mutant (M) as indicated at the top of each panel of the figure are metabolically labeled with [ 35 S]methionine.
  • Figure 3A Lysates are divided in half, and proteins in one aliquot are separated directly on denaturing gels.
  • Figure 3B The
  • the remaining proteins are precipitated with immune serum to the DMPl C-terminus (denoted by I at the bottom of Figure 3B) or with nonimmune serum (N), and the washed precipitates are electrophoretically separated in parallel. Positions of DMPl isoforms, 78 and 54 kDa products (arrows, see text), D-type cyclins, and CDK4 are indicated at the right of each panel of the figure and those of molecular weight markers are shown at the left of Figure 3 A. Exposure times are 18 hours.
  • Figures 4A-4D are gels showing the phosphorylation of DMPl .
  • Figure 4A Lysates from Sf9 cells coinfected with wild-type baculovirus (lanes 1 and 5) or with vectors encoding the indicated D-type cyclin and CDK4 (other lanes) are used as sources of kinases to phosphorylate the GST fusion proteins indicated at the bottom of the panel.
  • Figure 4B SF9 cells are coinfected with recombinant baculoviruses encoding DMPl , cyclin D2, and CDK4 (4) or CDK6 (6) as indicated at the top of the panel of the figure.
  • Cells are metabolically labeled with either [ 35 S]methionine (lanes 1-8) or 32 P-orthophosphate (lanes 9-12) and half of the [ 35 S]methionine- labeled lysates are treated with calf intestinal phosphates (lanes 5-9). All lysates are then precipitated with an antiserum to the DMPl C-terminus, and DMPl is resolved on denaturing gels.
  • Figure 4C
  • Sf9 cells are coinfected with the indicated baculovirus vectors encoding DMPl, D-type cyclins (Dl, D2, D3), cyclin E, CDK2 (2), CDK4 (4), or a catalytically inactive CDK4 mutant (M), and cells labeled with [ 35 S]methionine are lysed, precipitated with antiserum to DMPl , and the protein resolved on denaturing gels.
  • Figure 4D Lysates used for the experiment shown in Figure 4C are assayed for protein kinase activity, using either a GST-RB fusion protein (lanes 1-10) or histone HI (lanes 11-13) as the substrate. Autoradiographic exposure times are 8 hours for Figure 4 A and 18 hours for Figures 4B-4D.
  • Figures 5A-5B show DMPl oligonucleotide binding sequences.
  • Figure 5A The sequences of 27 oiigonucleotides selected via repeated rounds of DMPl binding and PCR amplification are determined. The frequency of bases at 13 positions are shown at the top with a 9 base consensus defined below.
  • Figure 5B Six oiigonucleotides, all containing identical flanking sequences as indicated, are synthesized and used either as probes or competitors in the electrophoretic mobility shift assays shown in Figures 6-8.
  • Figures 6A-6C show the oligonucleotide binding specificity of recombinant DMPl and ETS2 proteins.
  • Figure 6A Sf9 cell lysates containing approximately 4 ng recombinant DMPl are incubated with 3 ng 32 P-BS1 in the absence (lane 2) or presence (other lanes) of the indicated, unlabeled oligonucleotide competitors. The only complex detected on native gels is indicated.
  • Figure 6B Parallel EMS As are performed as in Figure 6A. using radiolabeled BS 1 or BS2 probes and 600 ng per lane of the indicated competing oiigonucleotides.
  • Figure 6C Assays are performed as in Figure 6A. using a bacterial GST-ETS2 fusion protein in place of Sf9 lysates containing DMPl . Autoradiographic exposure times are 6 hours.
  • Figures 7A-7B are gels showing the binding of radiolabeled BS2 and BS1 oiigonucleotides to proteins in mammalian cells. Lysates of Sf9 cells containing recombinant DMPl (lanes 1), mouse NIH-3T3 fibroblasts (lanes 2-8), or mouse CTLL lymphocytes (lanes 9-15) are incubated with radiolabeled BS2 ( Figure 7 A.) or BS1 ( Figure 7B) probes, either in the absence (lanes 2 and 9) or presence (other lanes) of the indicated competing oiigonucleotides (600 ng). Two distinct BS2-containing complexes (labeled A-complex and B-complex at the right of Figure 7 A.) are detected, only the first of which corresponds in mobility to that formed with recombinant DMPl (lane 1).
  • Figures 8A-8C are gels showing the expression of DMPl in mammalian cells.
  • Figure 8A Lysates of NIH-3T3 cells prepared in RIPA buffer are precipitated with antiserum to DMPl (serum AJ, lane 3) or with nonimmune serum (lane 2), and denatured immunoprecipitates are electrophoretically separated on gels.
  • Lane 1 (taken from the same gel) is loaded with Sf9 lysate containing recombinant DMPl . Proteins transferred to nitrocellulose are detected using a 1 : 1 mixture of antisera AJ and AF at 1/100 dilution.
  • Lane 1 was exposed for various times (18 hours shown) to position the hypo- and hyper phosphorylated forms of recombinant DMPl relative to the protein detected in NIH-3T3 cells. Lanes 2 and 3 exposed for 9 days are cropped from the same film.
  • Figure 8B Lysates from Sf9 cells containing DMPl (lane 1) or from NIH-3T3 cells (lanes 2-7) are incubated with a 32P-labeled BS2 probe plus antiserum AF (lanes 3-7), together with a cognate (lane 4) or irrelevant (lane 5) peptide, or with 600 ng of competing BS2 (lane 6) or M3 (lane 7) oligonucleotide.
  • EMS A performed with a radiolabeled BS2 probe and extracts from NIH-3T3 (lanes 2-6) or CTLL (lanes 7-12) cells. The extracts are either left untreated (none), pre-cleared with nonimmune serum (NI), or immuno-depleted with the indicated antisera to DMPl (AF, AJ, or AH) prior to incubation with the probe. Exposure time is 18 hours.
  • Figures 9A-9C are graphs showing the transactivation of reporter plasmids in 293T cells transfected with recombinant DMPl .
  • Figure 9A Increasing concentrations of reporter plasmids containing a luciferase gene driven by a minimal SV40 promoter with 5' concatamerized BS1 (open circles), BS2 (closed circles), or M3 (closed squares) sequences, or no additions (open triangles) are transfected into 293T cells, and luciferase activity is determined 48 hours later.
  • Figure 9B Increasing concentrations of reporter plasmids containing a luciferase gene driven by a minimal SV40 promoter with 5' concatamerized BS1 (open circles), BS2 (closed circles), or M3 (closed squares) sequences, or no additions (open triangles) are transfected into 293T cells, and luciferase activity is determined 48 hours later.
  • Reporter plasmids (same as Figure 9A, 1 ⁇ g each) are cotransfected with increasing quantities of DMPl expression plasmid, and luciferase activity is measured 48 hours later.
  • Figure 9C The BS2-containing reporter plasmid was cotransfected with the DMPl expression vector (1 ⁇ g) together with the indicated quantities of pRc/RSV expression plasmids containing cyclin D2 and/or CDK4. Background luciferase activity for the BS2 reporter plasmid in the absence of DMPl (see 9B, 0 input) was set to 1.0 arbitrary activation units. The activation relative to this value (i.e.
  • the activation index normalized to 0 input is plotted on the Y-axis.
  • the total input DNA concentrations were adjusted where necessary by addition of parental pRc/RSV plasmid DNA lacking inserts to yield 4 ⁇ g (9A), 3 ⁇ g (9B), and 2 ⁇ g (9C) of each transfection.
  • the error bars indicate standard deviations from the mean.
  • Figure 10 depicts the restriction sites of SEQ ID NO:2 which were employed to generate ten deletion mutants of DMPl used herein; the myb-like site of DMPl (diagonal lines), and the K319E point mutation.
  • Figure 11 Ideogram of chromosome 7 showing the position of clone 11098 at 7.21.
  • the present invention describes a novel amino acid polymer that binds cyclin D2 and can function as a transcription factor by binding specifically to a unique nonamer consensus sequence in DNA thereby activating the transcription of genes which are regulated by the consensus sequence.
  • the present invention includes the amino acid polymer and the corresponding nucleic acids that encode its amino acid sequence.
  • the present invention also includes methods of making, detecting, isolating, and using the amino acid polymer as a transcription factor.
  • Antibodies raised against the amino acid polymer, their use for detection of the amino acid polymer, corresponding antisense nucleic acids and ribozymes are also disclosed.
  • the invention further relates to identification of a DNA-binding site for the cyclin D-associated transcription factor, and to controlling expression of a heterologous gene under control of this binding site and the transcription factor.
  • the present invention is based, in part, on identification of a murine transcription factor termed DMPl , isolated in a yeast two-hybrid screen using cyclin D2 as bait.
  • This novel transcription factor is composed of a central DNA-binding domain containing three atypical myb repeats flanked by highly acidic segments located at its amino- and carboxyterminal ends.
  • Recombinant DMPl specifically binds to oiigonucleotides containing the nonamer consensus sequence CCCG(G/T)ATGT and, when transfected into mammalian cells, activates transcription of a reporter gene driven by a minimal promoter containing concatamerized DMPl binding sites.
  • DMPl mRNA Low levels of DMPl mRNA are normally expressed, albeit ubiquitously, in mouse tissues and cell lines, and are detected in both quiescent and proliferating macrophages and fibroblasts without significant oscillation throughout the cell cycle.
  • DMPl protein is detected in cell lysates by sequential immuno precipitation and immunoblotting, and using GTA core-containing consensus oiigonucleotides as probes.
  • These extracts contained electrophoretic mobility shift assay (EMSA) activity with antigenic and oligonucleotide binding specificities indistinguishable from those of the recombinant DMPl protein.
  • ESA electrophoretic mobility shift assay
  • the present invention provides an amino acid polymer that binds to cyclin D and to a specific DNA sequence.
  • the amino acid polymer has the sequence set forth in SEQ ID NO: 1.
  • the invention further provides an antigenic fragment of the amino acid polymer, which can be used, e.g. , after conjugation with a carrier protein, to generate antibodies to the amino acid polymer.
  • the present invention contemplates the amino acid polymer containing synthetic amino acids, derivitized by acetylation or phosphorylation, or substituted with conservative amino acids that provide the same biochemical properties.
  • amino acid polymer as used herein, is used interchangeably with the term “polypeptide” and denotes a polymer comprising amino acids connected by peptide bonds.
  • the amino acid polymer of this invention is a "cyclin D2 associated transcription factor", or “transcription factor” which is alternatively termed herein DMPl .
  • the monomeric form of DMPl contains about 760 amino acids.
  • about 760 amino acids means between 685 to 835 amino acids, i.e., roughly plus or minus 10% .
  • Murine DMPl has the amino acid sequence set forth in SEQ ID NO: l , as used herein, is a specific form of the amino acid polymer of the present invention.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e. , capable of eliciting an immune response without a carrier.
  • Proteins having a slightly altered amino acid sequence from that described herein and presented in FIGURE 1A are contemplated by the present invention. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits.
  • amino acid residues described herein are preferred to be in the "L” isomeric form and include both naturally occurring amino acids as well as amino acid analogs such as norleucine. However, residues in the "D” isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy 1 group present at the carboxy 1 terminus of a polypeptide.
  • amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy 1-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the amino acid polymer of the present invention may be obtained in several ways including by isolation from animal cells, by synthetic means such as solid-phase peptide synthesis or by isolation from recombinant cells that contain one or more copies of a DNA transcript encoding the amino acid polymers.
  • the cyclin D associate transcription factor may be isolated by affinity binding to an oligonucleotide that comprises the DMPl binding site, e.g. , the nonanucleotide CCCGTATGT. This oligonucleotide may be conjugated (covalently associated) to a solid phase support, allowed to bind with DMPl present, e.g.
  • affinity binding partners can be used in addition to an oligonucleotide comprising the DMPl binding site, including anti-DMPl antibodies and cyclin D, particularly cyclin D2.
  • a solid phase support for use in the present invention will be inert to the reaction conditions for binding.
  • a solid phase support for use in the present invention must have reactive groups in order to attach a binding partner, such as an oligonucleotide containing the DMPl binding site, cyclin D, or an antibody to the cyclin D-associated transcription factor, or for attaching a linker or handle which can serve as the initial binding point for any of the foregoing.
  • the solid phase support may be a useful chromatographic support, such as the carbohydrate polymers SEPHAROSE, SEPHADEX, and agarose.
  • a solid phase support is not limited to a specific type of support.
  • Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, magnetic beads, membranes (including but not limited to nitrocellulose, cellulose, nylon, and glass wool filters), plastic and glass dishes or wells, etc.
  • solid phase supports used for peptide or oligonucleotide synthesis can be used, such as polystyrene resin (e.g. , PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE ® resin (obtained from
  • silica based solid phase support may be preferred.
  • Silica based solid phase supports are commercially available (e.g. , from Peninsula Laboratories, Inc.; and Applied Biosystems, Inc.).
  • the solid phase support can be formulated as a chromatography support, e.g.
  • polypeptide in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other the bonds, e.g. , ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • Synthetic polypeptides prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N ⁇ - amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409).
  • Both Fmoc and Boc N ⁇ -amino protected amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to those who practice this art.
  • the method of the invention can be used with other N ⁇ -protecting groups that are familiar to those skilled in this art.
  • Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, IL; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35: 161-214, or using automated synthesizers, such as sold by ABS.
  • polypeptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g.,
  • Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, ⁇ -helices, ⁇ turns, ⁇ sheets, ⁇ -turns, and cyclic peptides can be generated.
  • the peptides may comprise a special amino acid at the C- terminus which incorporates either a C0 2 H or CONH 2 side chain to simulate a free glycine or a glycine-amide group. Another way to consider this special residue would be as a D or L amino acid analog with a side chain consisting of the linker or bond to the bead.
  • the pseudo-free C-terminal residue may be of the D or the L optical configuration; in another embodiment, a racemic mixture of D and L-isomers may be used.
  • the present invention further advantageously provides for determination of the structure of the transcription factor, which can be provided in sufficient quantities by recombinant expression (infra) or by synthesis. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay, etc.
  • the structure of transcription factor of the invention can be analyzed by various methods known in the art. Structural analysis can be performed by identifying sequence similarity with other known proteins. The degree of similarity (or homology) can provide a basis for predicting structure and function of transcription factor, or a domain thereof. In a specific embodiment, sequence comparisons can be performed with sequences found in GenBank, using, for example, the FASTA and FASTP programs (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-48).
  • the protein sequence can be further characterized by a hydrophilicity analysis (e.g. , Hopp and Woods, 1981 , Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the transcription factor protein.
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.
  • the present invention enables quantitative structural determination of transcription factor, or domains thereof.
  • enough material is provided for nuclear magnetic resonance (NMR), infrared (IR), Raman, and ultraviolet (UV), especially circular dichroism (CD), spectroscopic analysis.
  • NMR nuclear magnetic resonance
  • IR infrared
  • UV ultraviolet
  • CD circular dichroism
  • NMR nuclear magnetic resonance
  • IR infrared
  • UV ultraviolet
  • CD circular dichroism
  • co-crystals of transcription factor and a transcription factor-specific ligand, preferably DNA can be studied. Analysis of co-crystals provides detailed information about binding, which in turn allows for rational design of ligand agonists and antagonists. Computer modeling can also be used, especially in connection with NMR or X-ray methods (Fletterick, R. and Zoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the present invention contemplates isolation of a gene encoding a transcription factor of the invention, including a full length, or naturally occurring form of transcription factor, and any antigenic fragments thereof from any animal, particularly mammalian or avian, and more particularly human, source.
  • a gene refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
  • the invention further relates, as set forth below, to preparation of recombinant expression vectors under control of DNA sequences recognized by the transcription factor of the invention.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e. , capable of replication under its own control.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • Heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a "nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxy cytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • Double stranded DNA-DNA, DNA- RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g. , restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i. e. , the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55°
  • 5x SSC 0.1 % SDS, 0.25% milk, and no formamide
  • 30% formamide 5x SSC, 0.5 % SDS
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g. , 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g. , 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al. , supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.
  • a minimum length for a hybridizable nucleic acid is at least about 24 nucleotides; preferably at least about 36 nucleotides; and more preferably the length is at least about 48 nucleotides.
  • standard hybridization conditions refers to a T m of 55°C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65°C.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g. , the immunoglobulin superfamily) and homologous proteins from different species (e.g. , myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667).
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck et al., supra).
  • sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 30% of the amino acids are identical, or greater than about 60% are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.
  • the term "corresponding to” is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
  • a gene encoding transcription factor can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining transcription factor gene are well known in the art, as described above (see, e.g. , Sambrook et al., 1989, supra). Accordingly, any animal cell potentially can serve as the nucleic acid source for the molecular cloning of a transcription factor gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g.
  • a DNA "library” by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • Identification of the specific DNA fragment containing the desired transcription factor gene may be accomplished in a number of ways. For example, if an amount of a portion of a transcription factor gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).
  • a set of oiigonucleotides corresponding to the partial amino acid sequence information obtained for the transcription factor protein can be prepared and used as probes for DNA encoding transcription factor, as was done in a specific example, infra, or as primers for cDNA or mRNA (e.g., in combination with a poly-T primer for RT-PCR).
  • a fragment is selected that is highly unique to transcription factor of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In a specific embodiment, stringency hybridization conditions are used to identify a homologous transcription factor gene.
  • cDNA clones, or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e.g. , has similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for transcription factor.
  • the ability of the transcription factor to bind to a specific DNA sequence e.g. , the sequence
  • CCCG(G/T)ATGT is indicative of its identity as a transcription factor of the invention.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of transcription factor of the invention, that have the same or homologous functional activity as transcription factor, and homologs thereof from other species.
  • the production and use of derivatives and analogs related to transcription factor are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e. , capable of exhibiting one or more functional activities associated with a full-length, wild-type transcription factor of the invention.
  • Transcription factor derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity relative to native transcription factor.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a transcription factor gene may be used in the practice of the present invention. These include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of transcription factor genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the transcription factor derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a transcription factor protein, e.g.
  • amino acid residues within the sequence may be substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • conservative substitutions include substitutions of one or more amino acid residues within the sequence by an amino acid of a similar polarity, which acts as a functional equivalent, may result in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e. , His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces /3-turns in the protein's structure.
  • transcription factor derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned transcription factor gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the transcription factor-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutations enhance the functional activity of the mutated transcription factor gene product.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al., 1978, J. Biol. Chem.
  • a DMPl fusion protein can be expressed.
  • a DMPl fusion protein comprises at least a functionally active portion of a non-DMPl protein joined via a peptide bond to at least a functionally active portion of a DMPl polypeptide.
  • the non- DMPl sequences can be amino- or carboxy-terminal to the DMPl sequences.
  • a recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a functionally active portion of a non-DMPl protein joined in-frame to the DMPl coding sequence, and preferably encodes a cleavage site for a specific protease, e.g.
  • the fusion protein is a GST-DMP1 fusion proteins that bind directly to D-type cyclins in vitro, including radiolabeled D-type cyclins.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oiigonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc. , so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g. , E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • the nucleotide sequence coding for transcription factor, or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence. Such elements are termed herein a "promoter. "
  • the nucleic acid encoding the transcription factor of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding transcription factor and/or its flanking regions.
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. , baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g. , baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant transcription factor protein of the invention may be expressed chromosomal ly, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • the cell into which the recombinant vector comprising the nucleic acid encoding transcription factor is cultured in an appropriate cell culture medium under conditions that provide for expression of transcription factor by the cell.
  • Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • transcription factor protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control transcription factor gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al. , 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the /3-lactamase promoter (Villa- Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al. , 1983, Proc. Natl. Acad. Sci. U.S.A.
  • albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al. , 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al. , 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al. , 1986, Science 234:1372-1378).
  • Vectors are introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • Transgenic Animal Models of DMPl Activity the functional activity of DMPl can be evaluated transgenically.
  • a transgenic mouse (or other animal) model can be used.
  • the dmpl gene can be introduced transgenically using standard techniques, either to provide for over expression of the gene, or to complement animals defective in the gene.
  • Transgenic vectors including viral vectors, or cosmid clones (or phage clones) corresponding to the wild type locus of candidate gene, can be constructed using the isolated dmpl gene, as described below. Cosmids may be introduced into transgenic mice using published procedures [Jaenisch, Science, 240: 1468-1474 (1988)].
  • a transgenic animal model can be prepared in which expression of the dmpl gene is disrupted. Gene expression is disrupted, according to the invention, when no functional protein is expressed.
  • One standard method to evaluate the phenotypic effect of a gene product is to employ knock-out technology to delete the gene.
  • recombinant techniques can be used to introduce mutations, such as nonsense and amber mutations, or mutations that lead to expression of an inactive protein.
  • dmpl genes can be tested by examining their phenotypic effects when expressed in antisense orientation in wild-type animals. In this approach, expression of the wild-type allele is suppressed, which leads to a mutant phenotype.
  • RNA RNA duplex formation prevents normal handling of mRNA, resulting in partial or complete elimination of wild-type gene effect.
  • This technique has been used to inhibit TK synthesis in tissue culture and to produce phenotypes of the Kruppel mutation in Drosophila, and the Shiverer mutation in mice Izant et al.. Cell, 36:1007-1015 (1984); Green et al. , Annu. Rev. Biochem., 55:569-597 (1986); Katsuki et al., Science,
  • the antisense transgene will be placed under control of its own promoter or another promoter expressed in the correct cell type, and placed upstream of the SV40 polyA site. This transgene will be used to make transgenic mice, or by using gene knockout technology.
  • the present invention provides expression vectors for expression of heterologous proteins under control of the transcription factor of the invention.
  • Such vectors include the nonanucleotide consensus sequence recognized by the cyclin D- associated transcription factor operably associated with a heterologous gene or a cassette insertion site for a heterologous gene.
  • a vector is a plasmid.
  • the cyclin D transcription factor recognition sequence is genetically engineered into the promoter in the expression vector.
  • DMPl murine cyclin D transcription factor
  • the present invention provides any of the foregoing expression systems described above in connection with expression of the DMPl transcription activator comprising the specific DNA sequence bound by DMPl operably associated with the gene or cassette insertion site for a gene.
  • the present invention provides for co-expression of the transcription factor (DMPl) and a gene under control of the specific DNA recognition sequence by providing expression vectors comprising both a DMPl coding gene and a gene under control of, inter alia, the DMPl DNA recognition sequence.
  • these elements are provided on separate vectors, e.g. , as exemplified infra. In another embodiment, these elements are provided in a single expression vector.
  • transcription factor polypeptide produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins may be used as an immunogen to generate antibodies that recognize the transcription factor polypeptide.
  • Such antibodies include but are not limited to polyclonal, monoclonal (Kohler and Milstein, 1975, Nature 256:495-497; Kozbor et al., 1983, Immunology Today 4:72; Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; PCT/US90/02545; Cote et al., 1983, Proc. Natl.
  • the anti-transcription factor antibodies of the invention may be cross reactive, e.g. , they may recognize transcription factor from different species. Polyclonal antibodies have greater likelihood of cross reactivity.
  • an antibody of the invention may be specific for a single form of transcription factor, such as murine transcription factor. Preferably, such an antibody is specific for human transcription factor.
  • the transcription factor polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g. , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an transcription factor polypeptide, one may assay generated hybridomas for a product which binds to an transcription factor polypeptide fragment containing such epitope. For selection of an antibody specific to an transcription factor polypeptide from a particular species of animal, one can select on the basis of positive binding with transcription factor polypeptide expressed by or isolated from cells of that species of animal.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the transcription factor polypeptide, e.g. , for Western blotting, imaging transcription factor polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc.
  • the present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the transcription factor at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (see Weintraub, 1990; Marcus-Sekura, 1988, Anal. Biochem. 172:298). In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into organ cells.
  • Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988, supra; Hambor et al., 1988, J. Exp. Med. 168: 1237).
  • Preferably synthetic antisense nucleotides contain phosphoester analogs, such as phosphorothiolates, or thioesters, rather than natural phophoester bonds. Such phosphoester bond analogs are more resistant to degradation, increasing the stability, and therefore the efficacy, of the antisense nucleic acids.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Am. Med. Assoc. 260:3030). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead "-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target MRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • Various diseases or disorders mediated by inappropriate cell cycle activity due to increased or decreased activity of the cyclin D-associated transcription factor of the invention may be addressed by introducing genes that encode either antisense or ribozyme molecules that inhibit expression of the transcription factor (where the disease or disorder is associated with excessive transcription factor activity), or a gene that encodes an agent, such as a cyclin D, that inhibits the transcription factor (where the disease or disorder is associated with decreased transcription factor activity).
  • in vitro or in vivo transfection with one of the foregoing genes may be useful for evaluation of cell cycle activity in an animal model, which in turn may serve for drug discovery and evaluation.
  • the invention contemplates using the DMPl DNA-binding site for regulation of heterologous gene expression under control of DMPl for gene therapy, as set forth below.
  • DMPl can act as a cell cycle inhibitor when expressed in a tumor cell.
  • the present invention is directed to the treatment of tumors and other cancers by modulating the activity of DMPl , e.g. , by enhancing expression of the transcription factor to increase its activity.
  • the cyclin D domain of DMPl can be modified so that the cyclins no longer can act as negative effectors of DMPl.
  • a transgene vector for expression of such a modified DMPl of the present invention can be used.
  • an inhibitor of the cyclins could be administered to prevent cyclin-DMPl binding.
  • control of proliferation of a cancer cell is accomplished by blocking cell proliferation with DMPl, or an active fragment thereof thus, regulating uncontrolled cell proliferation characteristic of cancer cells.
  • DMPl an analogue of DMPl
  • an analogue of DMPl can be used.
  • increased expression of genes under control of DMPl may be necessary to restore appropriate cell cycle and growth characteristics to a transformed cell.
  • agents such as drugs that inhibit the ability of DMPl to bind DNA and/or transactivate its target genes could be administered to stimulate quiescent cells to grow.
  • the invention provides for introducing an antisense nucleotide or a ribozyme specific for dmpl mRNA; providing excess oligonucleotide containing the GTA trinucleotide sequence, and more preferably the CCCGTATGT nonanucleotide sequence to compete for binding of the transcription factor to its corresponding binding sites on gene promoters; or by increasing the level of regulatory activity effected by cyclin D to inhibit DMPl activity.
  • dysproliferative changes such as metaplasias and dysplasias
  • epithelial tissues such as those in the cervix, esophagus, and lung.
  • the present invention provides for treatment of conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed. , W.B. Saunders Co. , Philadelphia, pp. 68-79).
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder. For a review of such disorders, see Fishman et al. , 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia.
  • a gene for regulation of DMPl (e.g. , a dmpl gene or an antisense gene) is introduced in vivo in a viral vector.
  • a viral vector include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • tumors can be specifically targeted.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., 1991, Molec. Cell. Neurosci. 2:320-330), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (1992, J. Clin. Invest.
  • an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g. , adenovirus vector, to avoid immuno- deactivation of the viral vector and transfected cells.
  • immunosuppressive cytokines such as interleukin-12 (IL-12), interferon- ⁇ (IFN- ⁇ ), or anti-CD4 antibody
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g. , Wilson, 1995, Nature Medicine).
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g. , Wilson, 1995, Nature Medicine).
  • the gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33: 153;
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417; see Mackey, et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989, Science 337:387-388).
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et. al., 1988, supra).
  • Targeted peptides e.g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g. , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al. , Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • a gene therapy vector as described above employs a transcription control sequence that comprises the DNA consensus sequence recognized by the transcription factor of the invention, i.e. , a DMPl binding site, operably associated with a therapeutic heterologous gene inserted in the vector. That is, a specific expression vector of the invention can be used in gene therapy.
  • a gene therapy vector of the invention comprises the trinucleotide sequence GTA; preferably a vector of the invention comprises the nonanucleotide sequence CCCGTATGT.
  • the present invention specifically provides for expression of a heterologous gene under control of the cyclin D-associated transcription factor of the invention.
  • Such an expression vector is particularly useful to regulate expression of a therapeutic heterologous gene in conjunction with stages of the cell cycle regulated by the cyclin D- associated transcription factor of the invention.
  • the present invention contemplates constitutive expression of the heterologous gene, even if at low levels, in cells that ubiquitously express the cyclin D-associated transcription factor of the invention.
  • Various therapeutic heterologous genes can be inserted in a gene therapy vector of the invention under the control of, inter alia, the DMPl binding site, such as but not limited to adenosine deaminase (ADA) to treat severe combined immunodeficiency (SCID); marker genes or lymphokine genes into tumor infiltrating (TIL) T cells (Kasis et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:473; Culver et al., 1991, ibid. 88:3155); genes for clotting factors such as Factor VIII and Factor IX for treating hemophilia [Dwarki et al. Proc. Natl. Acad. Sci.
  • ADA adenosine deaminase
  • SCID severe combined immunodeficiency
  • marker genes or lymphokine genes into tumor infiltrating (TIL) T cells Kasis et al., 1990, Proc. Nat
  • the present invention provides for regulated expression of the heterologous gene in concert with expression of proteins under control of the cyclin D- associated transcription factor upon commitment to DNA synthesis. Concerted control of such heterologous genes may be particularly useful in the context of treatment for proliferative disorders, such as tumors and cancers, when the heterologous gene encodes a targeting marker or immunomodulatory cytokine that enhances targeting of the tumor cell by host immune system mechanisms.
  • heterologous genes for immunomodulatory (or immuno-effector) molecules include, but are not limited to, interferon- ⁇ , interferon-7, interferon-j3, interferon- ⁇ , interferon- ⁇ , tumor necrosis factor- ex, tumor necrosis factor-/., inter leukin-2, interleukin-7, interleukin-12, interleukin-15, B7- 1 T cell costimulatory molecule, B7-2 T cell costimulatory molecule, immune cell adhesion molecule (ICAM) -I T cell costimulatory molecule, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • IAM immune cell adhesion molecule
  • the present invention provides for coexpression of the transcription factor (DMPl) and a therapeutic heterologous gene under control of the specific DNA recognition sequence by providing a gene therapy expression vector comprising both a DMPl coding gene and a gene under control of, inter alia, the DMPl DNA recognition sequence.
  • DMPl transcription factor
  • these elements are provided on separate vectors, e.g. , as exemplified infra. These elements may be provided in a single expression vector.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of a binding partner of the transcription factor, such as an anti- amino acid polymer antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb, or oligonucleotide containing the specific sequence.
  • a binding partner of the transcription factor such as an anti- amino acid polymer antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb, or oligonucleotide containing the specific sequence.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present amino acid polymer.
  • the amino acid polymer can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular transcription activation activity in suspect target cells.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the amino acid polymer or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, l4 C, 32 P, 35 S, 36 C1, 51 Cr, "Co, 58 Co, 59 Fe, * ⁇ , 125 I, "'I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • biosensors such as the BIAcoreTM system (Pharmacia Biosensor AB, Uppsala, Sweden), or optical immunosensor systems. These systems can be grouped into four major categories: reflection techniques; surface plasmon resonance; fiber optic techniques, and integrated optic devices. Reflection techniques include ellipsometry, multiple integral reflection spectroscopy, and fluorescent capillary fill devices. Fiber-optic techniques include evanescent field fluorescence, optical fiber capillary tube, and fiber optic fluorescence sensors. Integrated optic devices include planer evanescent field fluorescence, input grading coupler immunosensor, Mach-Zehnder interferometer, Hartman interferometer and difference interferometer sensors.
  • Holographic detection of binding reactions is accomplished detecting the presence of a holographic image that is generated at a predetermined image location when one reactant of a binding pair binds to an immobilized second reactant of the binding pair (see U.S. Patent No. 5,352,582, issued October 4, 1994 to Lichtenwalter et al.).
  • Examples of optical immunosensors are described in general in a review article by G.A. Robins (Advances in Biosensors), Vol. 1 , pp. 229- 256, 1991. More specific description of these devices are found for example in U.S. Patents 4,810,658; 4,978,503; 5,186,897; R.A. Brady et al. (Phil. Trans. R. Soc. Land. B 316, 143-160, 1987) and G.A. Robinson et al. (in Sensors and Actuators, Elsevier, 1992).
  • the transactivation domain of a DMPl (or an expression vector containing a nucleic acid encoding the same) can be administered to stimulate the expression of the genes under control of DMPl -responsive promoters that aid in the prevention of cell proliferation.
  • the transactivation domain comprises amino acids 459 to 761 of SEQ ID NO: l or SEQ ID NO: 18.
  • the transactivation domain comprises amino acids 1-86 (SEQ ID NO:20) and 459 to 761 (SEQ ID NO: 18) of SEQ ID NO: l.
  • DMPl also contains a specific DNA-binding domain that by itself is incapable of transactivating genes controlled by DMPl -responsive promoters.
  • this DNA-binding domain consists of amino acids 87-458 (SEQ ID NO: 16) of SEQ ID NO: l .
  • the DNA-binding domain of a DMPl (or an expression vector containing a nucleic acid encoding the same) can be administered to inhibit the expression of the genes under control of DMPl -responsive promoters by competing with endogenous DMPl and thereby aid in cell proliferation.
  • DMPl, the DMPl-binding domain, and/or the transactivation domain of DMPl also can be used to identify DMPl target genes that are responsible for the regulation of cell growth.
  • Drug Assays Identification and isolation of a gene encoding an DMPl of the present invention provides for expression of DMPl in quantities greater than can be isolated from natural sources, or in indicator cells that are specially engineered to indicate the activity of DMPl expressed after transfection or transformation of the cells. Accordingly, in addition to rational design of agonists and antagonists, including drugs, based on the structure of DMPl polypeptide, the present invention contemplates an alternative method for identifying specific ligands and/or effectors of DMPl using various screening assays known in the art.
  • Any screening technique known in the art can be used to screen for DMPl agonists or antagonists.
  • the present invention contemplates screens for small molecule effectors, ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize activates DMPl in vivo.
  • natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize DMPl activity.
  • the screening can be performed with recombinant cells that express the DMPl , or alternatively, using purified protein, e.g. , produced recombinantly, as described above.
  • purified protein e.g. , produced recombinantly
  • the ability of labelled or unlabelled DMPl, the DNA-binding domain of DMPl, the cyclin D binding domain of DMPl , and/or the transactivation domain of DMPl, all of which have been defined herein can be used to screen libraries, as described in the foregoing references.
  • Genes that are under the control of a DMPl -responsive promoter can be identified through the use of the subtractive library method enhanced by the polymerase chain reaction (PCR), which allows performance of multiple cycles of hybridization using small amounts of starting material [Wieland et al.
  • PCR polymerase chain reaction
  • Two cDNA libraries can be prepared from NIH-3T3 fibroblast cells, for example.
  • One cDNA library is obtained from cells transfected with an expression vector encoding DMPl
  • the control cDNA library is obtained from proliferating NIH-3T3 cells that have not been so transfected.
  • Mouse NIH-3T3 fibroblasts and 293T human embryonic kidney cells (18) are maintained in a 10% C0 2 sterile incubator at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 units/ml penicillin and streptomycin (GIBCO/BRL Gaithersburg MD).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • 2 mM glutamine 2 mM glutamine
  • 100 units/ml penicillin and streptomycin GIC/BRL Gaithersburg MD
  • Mouse CTLL T lymphocytes are grown in RPMI 1640 medium using the same supplements plus 100 units/ml recombinant mouse interleukin-2 (a generous gift of Dr. Peter Ralph, formerly of Cetus Corp, now Chiron).
  • Spodoptera frugiperda Sf9 cells are maintained at 27°C in Grace's medium containing 10% FBS, yeastolate, lactalbumin hydrolysate, and gentimycin (all from GIBCO/BRL) in 100 ml spinner bottles.
  • a yeast two hybrid system (5, 14) as employed previously (20) was used to isolate cDNAs encoding cyclin D2 binding proteins.
  • a BamHI-HindlH cDNA fragment encoding mouse cyclin D2 (35,36) is subcloned into plasmid pAS2 in frame with the yeast GAL4 DNA-binding domain to generate the pAS2cycD2 bait plasmid.
  • Yeast strain Y190 whose HIS3 and LacZ genes are induced by GAL4, is transformed with pAScycD2 and then with a pACT library (Clonetech, Palo Alto CA) containing cDNAs prepared from mouse T-lymphoma cells fused 3' to the GAL4 transcription activation domain.
  • DMPl cDNA (2.6 kb 3' of GAL4) is shorter than the single mRNA species detected in mouse tissues by Northern blotting analysis
  • plaque lifts representing 4 X IO 6 phages from a mouse C19 erythroleukemia cell cDNA library (5' stretch gtlO, Clonetech) are screened with a radiolabeled DMPl probe, and two cDNAs containing additional 5' sequences are isolated. These contain 200 and 373 bp segments overlapping those at the 5' end of the probe plus — 800 bp of novel 5' sequences. The latter sequences are fused within the region of overlap to those in the 2.6 kb DMPl cDNA to generate a putative full-length cDNA of 3.4 kb.
  • a BglH fragment encoding amino acids 176-761 of DMPl (Figure 1) is subcloned into the BamHI site of the pGEX-3X plasmid (Pharmacia, Uppsala Sweden), and overnight cultures of transformed bacteria are diluted 10-fold with fresh medium, cultured for 2-4 more hours at 37 C C, and induced with 1 mM isopropyl-/3-D-thiogalactopyranoside (IPTG) for 1 hour.
  • IPTG isopropyl-/3-D-thiogalactopyranoside
  • Induced bacteria are lysed by sonication in phosphate-buffered saline (PBS) containing 1 % Triton X-100, and recombinant glutathione-S-transferase (GST)-DMPl protein is purified by absorption and elution from glutathione-Sepharose beads as described(35).
  • PBS phosphate-buffered saline
  • GST glutathione-S-transferase
  • GST-DMP1 or GST-RB (15) immobilized on glutathione-Sepharose beads are mixed with [ 35 S]methionine-labeled mouse D-type cyclins, prepared by transcription (Stratagene Transcription System, La Jolla CA) and translation (rabbit reticulocyte system from Promega, Madison WI) in vitro, as per the manufacturer's instructions, hereby inco ⁇ orated by reference.
  • IP Kinase buffer 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.1 % Tween-20
  • BSA bovine serum albumin
  • the beads are collected by centrifugation, washed 4 times with IP Kinase buffer, and the bound proteins are denatured and analyzed by electrophoresis on 11 % polyacrylamide gels containing sodium dodecyl sulfate (SDS) (1).
  • SDS sodium dodecyl sulfate
  • Protein kinase assays are performed using 1.5 ⁇ g GST-DMP1 or GST-RB adsorbed to glutathione-Sepharose as substrates.
  • the beads are suspended in a total volume of 25 ⁇ l Kinase buffer (50 mM HEPES, pH 7.5, 10 mM Mg-Cl, 1 mM DTT) containing lmM EGTA, 10 mM /3-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF, 20 uM ATP, 1 uCi [- 32 P]ATP (6000 Ci/mmol; Amersham), and 2.5-5.0 ⁇ l lysate (corresponding to 5 X IO 4 cell equivalents) from Sf9 cells coinfected with the indicated cyclins and CDKs. After incubation for 20 minutes at 30°C (with linear incorporation kinetics), the total proteins in the reaction are denatured and, following centrifugation of the beads, separated on denaturing poly
  • PA using hexahistidine (His)-tagged fusion proteins produced in bacteria (32) and containing fused DMPl residues 221-439 (serum AJ to myb-repeat domain) or residues 176-761 (serum AH).
  • Antiserum AF is raised against a synthetic peptide representing the nine C-terminal DMPl residues conjugated to keyhole limpet hemocyanin as described (13).
  • All antisera specifically precipitate multiple phosphorylated forms of the full-length DMPl protein from Sf9 lysates infected with a DMPl -producing baculovirus vector and do not crossreact with mammalian cyclins (D-types, E, A, or B) or CDKs (2, 4, and 6).
  • D-types, E, A, or B mammalian cyclins
  • CDKs 2, 4, and 6
  • BamHI linkers are added to an Xbal-EcoRV cDNA fragment containing the entire DMPl coding sequence, and the fragment is inserted into the BamHI site of the pAcYMl baculovirus vector (37). Production of virus and infection of Spodoptera frugiperda (Sf9) cells are performed as previously described (23).
  • cells infected with the indicated recombinant viruses encoding DMPl , CDKs, and/or cyclins are metabolically labeled 40 hours post-infection for 8 additional hours with 50 uCi/ml of [ 35 S]methionine (1000 Ci/mmol; ICN, Irvine CA) in methionine-free medium or for 4 additional hours with 250 uCi/ml of carrier-free 32 P-orthophosphate (9000 Ci/mmol, Amersham) in phosphate-free medium.
  • 10-20 ⁇ l lysate is diluted to 0.5 ml in EBC buffer (50 mM Tris Hcl, pH 8.0, 120 mM NaCl, 0.5 % Nonidet P-40, 1 mM EDTA, and 1 mM DTT) containing 2% aprotinin, 1 mM .-glycerophosphate, 0.1 mM Na 3 V0 4 , and 0.1 mM NaF.
  • EBC buffer 50 mM Tris Hcl, pH 8.0, 120 mM NaCl, 0.5 % Nonidet P-40, 1 mM EDTA, and 1 mM DTT
  • Antiserum AF (10 ⁇ l adsorbed to protein A-Sepharose beads) directed to the DMPl C-terminus was added, beads are recovered after incubation for 4 hours at 4°C, and adsorbed proteins are denatured and resolved on denaturing gels.
  • metabolically labeled Sf9 lysates are treated with calf intestinal phosphatase after immune precipitation(23). Determination of cyclin dependent kinase activities in the cell extracts is performed using soluble GST-RB or histone HI (Boehringer Mannheim, Indianapolis IN) as substrates.
  • Binding site selection and amplification by polymerase chain reaction is performed as described (21).
  • Single-stranded oiigonucleotides containing 30 random bases interposed between fixed forward (5'-CGCGGATCCTGCAGCTCGAG-3') and reverse (5'- TGCTCTAGAAGCTTGTCGAC-3') primers are prepared, and then double-stranded oiigonucleotides are generated using them as templates with the forward and reverse primers.
  • the double-stranded oiigonucleotides are mixed with recombinant DMPl protein immunoprecipitated from Sf9 cells and immobilized to protein A beads.
  • Binding buffer 25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM EDTA, 1.5 mM MgCl 2 , 0.1 % Nonidet P40, 1 mM DTT, 5% glycerol
  • 25 ⁇ g poly (dl-dC) (Boehringer Mannheim) and 25 ⁇ g BSA, followed by incubation with gentle rotation for 30 minutes at 4°C. Beads are collected by centrifugation, washed 3 times with Binding buffer, and suspended in 50 ⁇ l distilled water.
  • Bound oligomers eluted into the supernatant by boiling are reamplified by PCR using the same primers. After 6 rounds of binding and amplification, recovered oiigonucleotides are subcloned into the BamHI to Hindlll sites of pSK bluescript plasmids (Stratagene, La Jolla CA) and their sequences are determined using a Sequenase version 2.0 kit (U.S. Biochemicals, Cleveland OH).
  • Electrophoretic mobility shift assay (EMSA)
  • Double-stranded oiigonucleotides containing potential DMPl binding sites (BS1 and BS2) and mutated versions (M1-M4) (Figure 5B) are end-labeled with 32 P using the Klenow fragment of DNA polymerase and ⁇ - 32 P-dATP (6000 Ci/mmol; Dupont NEN) (8).
  • Nuclear extracts from mouse NIH-3T3 or CTLL cells are prepared with buffer containing 0.4 M NaCl (2).
  • Mammalian cell extracts (15 ⁇ g protein) or Sf9 lysates (corresponding to 5 X IO 2 infected cells) containing ⁇ 4ng recombinant DMPl are mixed with 3 ng of 32 P-labeled probe (1 X 10 5 cpm) in 15 ul Binding buffer containing 2.5 ⁇ g of poly(dl-dC) and 2.5 ⁇ g BSA and incubated at 4°C for 30 minutes.
  • the indicated amounts of unlabeled oiigonucleotides are added to the reactions before addition of the labeled probe.
  • a bacterially produced GST-Ets2 fusion protein containing the complete Ets2 DNA-binding domain (10) is used in place of Sf9 extracts containing recombinant DMPl .
  • Protein-DNA complexes are separated on nondenaturing 4% polyacrylamide gels as described (8). Where indicated, antiserum to DMPl together with 2.5 ⁇ g salmon testis DNA (Sigma; used to reduce nonspecific DNA binding activity caused by serum addition) is preincubated with extracts for 30 minutes at 4°C prior to initiation of binding reactions. Immune complexes are either removed by adsorption to protein A-Sepharose beads (immunodepletion experiments) or are allowed to remain ("supershift" experiments).
  • An Xbal-EcoRV fragment containing the entire DMPl coding sequence is subcloned by blunt end ligation into a Spel-Xbal fragment of the Rc/RSV vector (Invitrogen, La Jolla CA) to enable DMPl expression in mammalian cells.
  • 6X concatamerized BS1 , 8X concatamerized, BS2, or 7X concatamerized M3 oiigonucleotides ( Figure 5B) are inserted into the Xhol-Smal sites of pGL2 (Promega) 5' to a minimal simian virus 40 (SV40) early promoter driving firefly luciferase gene expression.
  • SV40 minimal simian virus 40
  • chromosomes were then counterstained with 4,6-diamidino-2-phenylindole (DAPI) and analyzed. Definitive chromosomal assignment was confirmed by cohybridization of clone 11098 with a biotinalyted chromosome 7 centromere-specific probe (D7Zl)(Oncor Inc. , Gaitherburg, M.D.). Specific probe signals were detected by incubating the hybridized slides in fluorescein-conjugated sheep antibodies to digoxigenin and Texas red avidin (Vector Laboratories, Burlington CA).
  • D7Zl biotinalyted chromosome 7 centromere-specific probe
  • Chromosome band assignment was made based on the relative position of the fluorescence signal relative to landmarks on the chromosome such as centromere, telomeres, and heterochromatic euchromatic boundaries [Franke, Cytogenet Cell Genet 65:206-219 (1994)].
  • EST T90434 Human C-Terminal Fragment.
  • EST T90434 was purchased from Genome Systems Inc., St Louis, Missouri. The EST was selected on the basis of the homology of 289 nucleotides sequenced with that of SEQ ID NO:2; 78.4% identity was reported. Upon resequencing the EST, it was found that some of the 289 basepairs had been incorrectly assigned.
  • BrdU is added to NIH-3T3 cells after the experimental treatment and the cells were incubated for twenty-two hours in DMEM plus 10% fetal calf serum (FCS). The cells were then stained for DMPl expression and/or BrdU inco ⁇ oration. The nucleic acids encoding the wildtype DMPl and the deletion and point mutants had been constructed so as to express the corresponding proteins with Flag-tags.
  • mouse monoclonal anti-Flag antibodies (12 ⁇ g/ml) [Kodak] were incubated with the cells in TBS-Ca + without FCS for one hour at room temperature.
  • horse anti-mouse biotinylated antibodies at a 1 :500 dilution were added to the cells in TBS plus 5% FCS and incubated for 30 minutes at room temperature.
  • streptavidin linked to Texas red was then added at a 1 :500 dilution for 30 minutes at room temperature.
  • 1.5N HCl was added to the cells for ten minutes at room temperature to denature the DNA.
  • sheep anti-BrdU antibodies [Fitzgerald] at a 1 : 12 dilution were then added for one hour at room temperature.
  • rabbit FITC-conjugated anti-sheep antibodies [Vector] at 1: 100 dilution was then incubated for 30 minutes at room temperature.
  • a genomic probe for DMPl was prepared by PCR with a primer having a nucleotide sequence of a portion of the C-terminal fragment of human DMPl (obtained by sequencing EST T90434) and human genomic DNA. The probe was then used to obtain Clone 11098 from a PI human genomic DNA library.
  • a yeast two-hybrid screen is used to isolate cDNAs encoding proteins able to interact with cyclin D2. Plasmids containing cDNAs prepared from the RNA of mouse T lymphoma cells and fused 3' to the GAL4 activation domain are transfected into yeast cells containing a "bait plasmid" encoding the GAL4 DNA-binding domain fused in frame with full length mouse cyclin D2 coding sequences. From 6 X 10 5 transformants, 36 plasmids are isolated which, when segregated and mated with yeast containing the cyclin D2 bait plasmid or with control strains expressing unrelated GAL4 fusion proteins, coded for proteins that interacted specifically with D-type cyclins.
  • cDNAs specify several previously identified cyclin D-interacting proteins (i.e. known CDKs and CDK inhibitors) as well as novel polypeptides unrelated to those in searchable data bases.
  • CDKs and CDK inhibitors cyclin D-interacting proteins
  • novel polypeptides unrelated to those in searchable data bases.
  • CDKs and CDK inhibitors cyclin D-interacting proteins
  • novel polypeptides unrelated to those in searchable data bases.
  • a single clone encoding a protein containing three tandem "myb repeats" characteristic of the myb family of transcription factors (17,24,45).
  • Northern blot analysis reveals that a single — 3.8 kb mRNA related to the cloned sequences is present ubiquitously in adult mouse tissues (i.e.
  • cDNAs containing 0.8 kb of additional 5' sequences are isolated from a mouse erythroleukemia (MEL) cell library, enabling the reconstruction of a 3.4 kb cDNA which approximates the length of the mRNA detected by Northern blotting.
  • MEL mouse erythroleukemia
  • the DMPl cDNA contains a long open reading frame that encodes a protein of 761 amino acids with a mass of 84,589 daltons ( Figure 1A), but its apparent molecular weight, based on its electrophoretic mobility on denaturing polyacrylamide gels, is significantly larger (see below).
  • the initiation codon is the most 5' AUG in the nucleotide sequence and is preceded by 247 nucleotides that contain termination codons in all three reading frames.
  • DMPl contains three myb repeats (residues 224-392, underlined in Fig 1A), connoting its role as a transcription factor (6,25,52).
  • the clone recovered in the two-hybrid screen lacked the 5' untranslated region together with sequences encoding amino acids 1-175, which are replaced by the GAL4 activation domain. Both the amino terminal (residues 4-169) and carboxylterminal (residues 579-756) ends of the full length DMPl protein are highly acidic. Fourteen SP and TP doublets are distributed throughout the protein, but none represent canonical proline-directed phosphorylation sites for cycl in-dependent kinases (SPXK/R). A typical nuclear localization signal is not identified.
  • Imperfect tandem myb repeats were first identified in the v-myb gene product of avian myeloblastosis virus and in its cellular proto-oncogene coded c-myb homologs (Fig IB).
  • the prototypic repeat sequence contains three regularly spaced tryptophan residues separated by 18-19 amino acids, with the third tryptophan of a repeat separated by 12 amino acids from the first tryptophan of the next (3, 17,25,45,49).
  • Degenerate repeats that contain tyrosine in place of the third tryptophan or isoleucine in place of the first have been identified in other "myb-like" proteins (49).
  • Scattered amino acid identities enabled us to align the repeat sequences within mouse c-myb with those of DMPl (Fig IB).
  • KQCR-W-N in repeat-2 denoted by asterisks
  • the first repeat of DMPl contains a tyrosine substituted for the first tryptophan and leucine for the third.
  • the second and third repeats which in myb are each required for DNA binding, contain 11 and 6 residue insertions between the first and second tryptophans.
  • GST glutathione S- transferase
  • Bound cyclins recovered on washed glutathione-Sepharose beads are analyzed by electrophoresis on denaturing gels.
  • Figure 2 shows that cyclins D2 and D3 interact strongly with GST-RB in vitro ( - 20% of the total input protein is bound; see legend), whereas, as seen previously (15), cyclin Dl binds much less avidly (lane 2).
  • GST-DMP1 is less efficient than GST-RB in binding cyclins D2 and D3 ( ⁇ 4-fold less binding), and under these conditions, an interaction with Dl is not detected (lanes 3, 7, 11).
  • Cells infected with a vector containing DMPl cDNA produce a family of - 125 kDa proteins (brackets, right margin), as well as smaller species of — 78 and - 54 kDa (arrows, right margin), which are not synthesized in cells infected with a wild-type baculovirus (lane 1).
  • the proteins in the 125 kDa range represented phosphorylated forms of DMPl (see below) which are specifically precipitated with three different DMPl antisera (Fig 3B, lane 3, and see below) but not with nonimmune serum (lane 2).
  • the 78 and 54 kDa species may represent C-terminally truncated DMPl products arising from premature termination or proteolysis, because they were not precipitated with the antiserum to the DMPl C-terminus (Fig 3B). Apart from their phosphorylation, the full-length DMPl proteins had apparent molecular masses significantly larger than that predicted from the cDNA sequence.
  • phosphorylation of DMPl by cyclin D-CDK4 complexes might also inhibit DMPl from binding to D-type cyclins.
  • the fact that catalytically inactive CDK4 subunits do not enter into stable ternary complexes with cyclin D2-DMP1 (Fig 3B, lane 8) also indicates that DMPl-bound cyclin D2 molecules are prevented from interacting as efficiently as unbound cyclin D2 with its catalytic partners.
  • DMPl is a Substrate for Cyclin D Dependent Kinases
  • the cyclin D-dependent kinases exhibit an unusual preference for RB over histone HI as an in vitro substrate (33,34,39).
  • equivalent quantities of GST-DMP1 and GST-RB fusion proteins are compared for their ability to be phosphorylated in vitro by Sf9 lysates containing cyclin D-CDK4.
  • lysates of Sf9 cells infected with control baculoviruses do not efficiently phosphorylate either fusion protein (Fig 4A, lanes 1 and 5)
  • lysates containing active cyclin D-CDK4 complexes phosphorylate both (Fig 4A, lanes 2-4 and 6-8).
  • GST-RB is always a preferred substrate (lanes 6-8), and different preparations of cyclin D3-CDK4 are routinely more active than D2- or Dl- containing holoenzymes in phosphorylating DMPl (lanes 2-4).
  • Similar results are obtained when immunoprecipitated cyclin D-CDK4 or D-CDK6 complexes are used in lieu of Sf9 extracts as sources of enzyme.
  • DMPl is immunoprecipitated from cell lysates and resolved on denaturing gels.
  • DMPl is more easily resolved into two major species (Fig 4B, lane 2).
  • No protein is precipitated from cells infected with a control baculovirus (lane 1).
  • Coinfection of cells producing DMPl with cyclin D2-CDK4 or cyclin D2-CDK6 results in conversion of the faster migrating DMPl species to the slower mobility form (lanes 3, 4), whereas treatment of DMPl immunoprecipitates with alkaline phosphatase converts both species to a single, more rapidly migrating band (lanes 7, 8).
  • Hype ⁇ hosphorylation of DMPl is not observed following infection of the cells with vectors producing D-type cyclin regulatory subunits alone (Fig 4C, lanes 3-5). The process depends on a functional catalytic subunit (lanes 6-8 versus 3-5), and it is unaffected by a catalytically inactive CDK4 mutant (lane 9). Perhaps surprisingly, DMPl hype ⁇ hosphorylation is not as readily induced by cyclin E-CDK2 (Fig 4C, lane 10).
  • cyclin D-CDK4 and cyclin E-CDK2 differ in their relative substrate specificities for both histone HI and DMPl .
  • DMPl is not detected in mammalian cells by immunoprecipitation of the protein from metabolically labeled cell lysates.
  • sequential immunoprecipitation with serum AJ
  • immunoblotting with sera AJ plus AH
  • Fig 8 A lane 3
  • tandem BS1 , BS2, or M3 consensus sites are inserted 5' to an SV40 minimal promoter and these control elements are fused to a luciferase reporter gene.
  • Reporter plasmids containing either BS1 or M3 binding sites are themselves highly active in a dose-dependent fashion when transfected into 293T kidney cells, likely due to expression of endogenous Ets factors, but the reporter plasmid containing BS2 sites generates even less "background" activity than one containing only a minimal SV40 promoter (Fig 9A).
  • Ets family transcription factors including Etsl and Ets2 can also bind to and activate transcription from those DMPl consensus recognition sites that contain a GGA core.
  • Promoter-reporter plasmids containing consensus binding sites with either a central GGA or GTA trinucleotide could each respond to overexpressed, recombinant DMPl in transactivation assays.
  • background levels of reporter gene activity are significantly higher using the Ets-responsive promoters implying that endogenous Ets activity greatly exceeds that of endogenous DMPl in the cells tested.
  • competition studies indicate that Ets family members predominate in complexes resolved from lysates of NIH-3T3 and CTLL cells.
  • DMPl not only specifically interacts with cyclin D2 when overexpressed in yeast cells, but translated, radiolabeled D-type cyclins bind directly to GST-DMP1 fusion proteins in vitro, and complexes between full-length DMPl and D-type cyclins readily form in intact Sf9 insect cells engineered to co-express both proteins under baculovirus vector control.
  • DMPl undergoes basal phosphorylation when synthesized in Sf9 cells and is further hype ⁇ hosphorylated in cells co-expressing catalytically active, but not mutant, cyclin D-CDK4 complexes.
  • Immune complexes containing cyclin D-CDK4 can also hype ⁇ hosphorylate DMPl in vitro.
  • other kinases also contribute to DMPl phosphorylation in insect cells, given the accumulation of multiply phosphorylated forms of the protein even in cells not engineered to co-express recombinant cyclin-CDK complexes.
  • DMPl and D-type cyclins show some analogy with those previously observed with RB. However, there are many important differences. First, side by side comparisons indicate that D-type cyclins bind less avidly to DMPl than to RB, both in vitro and in Sf9 cells. Second, the efficiency of RB binding to D-type cyclins is influenced by a Leu-X-Cys-X-Glu pentapeptide sequence that D-type cyclins share with certain RB-binding oncoproteins, whereas a cyclin D2 mutant containing substitutions in this region remained able to interact with DMPl .
  • RB is phosphorylated to a much higher stoichiometry than DMPl by cyclin D-CDK4 complexes.
  • CDK4-mediated phosphorylation of RB in vitro or in Sf9 cells can occur at multiple canonical CDK sites.
  • Ser-Pro and Thr-Pro doublets distributed throughout the DMPl protein, none of these represents a typical CDK consensus sequence, suggesting that cyclin D-dependent kinases phosphorylate atypical recognition sequences in this protein.
  • catalytically inactive CDK4 could not enter into stable ternary complexes with DMPl and cyclin D. This again indicates that cyclin D contacts DMPl and RB via different residues (see above), and raises the possibility that DMPl and CDK4 interact with overlapping binding sites on cyclin D, being able to compete with one another for cyclin D binding.
  • introduction of catalytically inactive CDK4 into cells expressing both cyclin D2 and DMPl modestly reduce the extent of D2 binding to DMPl, although to a far lesser extent than wild-type CDK4. Therefore, although hype ⁇ hosphorylation of DMPl can decrease its ability to bind cyclin D, the role of cyclin D binding is not solely to trigger CDK4-mediated phosphorylation.
  • cyclin D influences gene expression via its binding and/or phosphorylation of DMPl .
  • Enforced transient expression of cyclin D2 or D2-CDK4 in mammalian cells negatively regulates the ability of DMPl to transactivate reporter gene expression although the mechanistic basis remains unresolved.
  • This effect of cyclin D is observed with or without addition of exogenous catalytic subunits, but endogenous CDK4 activity can already be significantly activated via cyclin D overexpression alone, while even higher levels of CDK4 activity are likely to be toxic.
  • Enforced expression of cyclin D-CDK4 neither influences the stability of overexpressed DMPl nor its ability to preferentially localize to the nucleus of transfected mammalian cells.
  • Coexpression of cyclin D or cyclin D-CDK4 together with DMPl in Sf9 cells also had no apparent effect on the ability of DMPl to form EMSA complexes with consensus oligonucleotide probes.
  • the majority of DMPl molecules in such extracts do not contain stably bound cyclin, and their extent and sites of phosphorylation are unknown.
  • Oligonucleotide-bound proteins from such extracts or from mammalian cells could be supershifted in EMSAs performed with antisera to DMPl , but polyvalent antisera or monoclonal antibodies to D cyclins are without detectable effect on their electrophoretic mobility, indicating that cyclin D binding and/or cyclin D-CDK4 mediated phosphorylation interferes with the ability of DMPl to bind to DNA. Direct effects on transactivation potential are similarly plausible. In the case where cyclin D regulates DMPl activity in vivo, DMPl functions better in quiescent cells lacking cyclin D expression than in proliferating cells. These observations underscore a role for D-type cyclins in the control of gene expression in an RB-independent fashion.
  • DMPl to act as a transcription factor correlates with its ability to regulate cell growth. Both reporter gene activity and growth arrest depend upon the ability of DMPl to bind to specific DNA sequences and to activate transcription when so bound. Cyclin D overrides the ability of DMPl to regulate transcription of its target genes and to induce growth arrest. This indicates that specific peptide domains of DMPl can act as antagonists of target gene activation or cyclin D mediated regulation. A series of experiments are described which define three specific functional domains of DMPl .
  • results A series of deletion mutants and a point mutant of DMPl, K319E, (in which the lysine at position 319 of SEQ ID NO: l is replaced by a glutamic acid) were prepared and used to determine the DNA-binding domain of DMPl by electrophoretic mobility shift assay (EMSA) using a 32 P labeled BS2 probe.
  • the DNA-binding domain of DMPl was mapped to a central region containing the three MYB repeats plus adjacent flanking sequences: a BstEII to Ncol fragment encoding amino acids 87-458 of SEQ ID NO: l (Table 1). This region alone was necessary and sufficient for DNA binding.
  • the K319E point mutation which converts a positive charge to a negative charge in the middle of the
  • DNA-binding domain has a markedly diminished affinity (i.e. , about 2% of the wildtype) for the DNA probe.
  • Table 1 An EMSA assay with the 32 P-BS2 Probe for transfection lysates of NIH-3T3 fibroblasts having expression vectors encoding murine wild-type DMPl, corresponding deletion mutations, or a point mutation of DMPl (K319E).
  • the EMSA assay was performed as described above, with and without a 100-fold excess of cold BS2 probe. All 32 P labeled bands were blocked by the addition of the cold BS2 probe.
  • Mi l 1.1 48 The series of DMPl deletion mutants and the K319E point mutant were then used to determine the cyclin D binding domain of DMPl .
  • Expression vectors encoding murine wild-type DMPl , the corresponding deletion mutations, or K319E i.e. , wildtype DMPl and Ml-Ml l , defined in Figure 10, Table 1 were cotransfected with an expression vector encoding cyclin Dl into SF9 cells. Wildtype DMPl and Ml-Ml l were expressed containing Flag-tags. SF9 lysates were immunoprecipitated with an antibody raised against the Flag-tag.
  • the immunoprecipitates were resolved individually by gel electrophoresis, and then Western blotted with an antibody raised against cyclin Dl .
  • All of the samples, except M9 contained a band that corresponded to cyclin Dl , indicating that the cyclin Dl was bound to all of the immunoprecipitated DMPl mutants except M9. Therefore, the cyclin Dl binding domain is missing in the M9 deletion mutant.
  • the M5 sample was particularly faint, indicating that a portion of the cyclin Dl binding domain also may be missing in this deletion mutant of DMPl . Therefore, deletion of the N-terminal domain of DMPl (i.e. , amino acids 1-223) abrogates its ability to interact with D-type cyclins, and thus, the region of DMPl from residues 1-223 contains a specific cyclin D interaction motif required for D-type cyclin-DMPl association.
  • Results NIH-3T3 cells were placed on cover slides and transfected with the expression vectors (pFLEX-DMPl or the corresponding vector containing the deletion or point mutants of mouse DMPl plus or minus cyclin D or E) for fourteen hours. The cells were then washed twice and DMEM plus 10% FCS was added and the cells incubated for eight hours. Half of the cells were starved by washing twice with 0.1 % FCS, and then incubated for twenty-four hours in 0.1 % FCS in DMEM. The remaining cells were not starved but were incubated for twenty-four hours in DMEM plus 10% FCS without washing. BrdU was added to both groups of cells and the cells were incubated for twenty-two hours in DMEM plus 10% FCS.
  • the expression vectors pFLEX-DMPl or the corresponding vector containing the deletion or point mutants of mouse DMPl plus or minus cyclin D or E
  • the cells were then restimulated to enter the cell cycle synchronously with DMEM plus 10% FCS. At the same time, 5'-Bromo-2' Deoxyuridine (BrdU) was added to the medium. The cells were fixed 22 hours later in methanol acetone (1 : 1) and stained for BrdU inco ⁇ oration and DMPl expression as described in the Materials and Methods.
  • PrdU 5'-Bromo-2' Deoxyuridine
  • Coexpression of a D-type cyclin with DMPl overrides the ability of DMPl to transactivate a luciferase gene under the control of an artificial DMPl -responsive promoter (Table 4), as well as the ability of DMPl to inhibit cell growth.
  • Coexpression of CDK2, CDK4, or the specific CDK inhibitors, (i.e. , INK4 proteins P16 or P19) with DMPl had little to no effect on the stimulation of luciferase activity due to DMPl .
  • EXAMPLE 9 Nucleotide Sequence Of The C-Terminal Portion Of Human DMPl .
  • a human DMPl fragment contained by EST T90434 was obtained and then fully sequenced.
  • the EST had 2013 basepairs of which 789 were determined to contain coding sequence.
  • the 789 basepairs of coding sequence contains a stop codon (TAG) at its 3' end, and encodes the carboxylterminal third of human DMPl.
  • the remaining 1224 basepairs correspond to the 3' untranslated region.
  • the amino acid sequence of the human fragment of DMPl and the corresponding murine protein have 95.4% similarity and 91.6% identify over this 262 amino acid sequence.
  • Clone 11098 contains a genomic fragment of human DMPl .
  • Chromosomal assignment of clone 11098 gene was made by fluorescence in situ hybridization. The only fluorescence signals identified were located on the long arm of a group C chromosome resembling chromosome 7 on the basis of DAPI banding. The chromosomal assignment was confirmed by cohybridizing clone 11098 with a chromosomes 7 centromere-specific probe (D7Z1). Band assignment was made by determining that clone 11098 is located 30% of the distance from the centromere to the telomere of chromosome arm 7 q , a position which corresponds to 7 q 21. ( Figure 11).
  • Cyclin Dl is a nuclear protein required for cell cycle progression in Gl. Genes & Devel 7:812-821. 5. Bartel, P. L., C.-T. Chien, R. Sternglanz, and S. Fields. 1993. Using the two hybrid system to detect protein-protein interactions, p. 153-179. In (ed.D. A. Hartley) In: Cellular interactions in development: a practical approach. Oxford University Press, Oxford UK. 6. Biedenkapp, H., U. Borgmeyer, A. E. Sippel, and K. H. Klempnauer. 1988.
  • Viral myb oncogene encodes a sequence-specific DNA binding activity. Nature 335:835-837. 7. Chen, C. and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752.
  • Cyclin Dl is dispensable for Gl control in retinoblastoma gene-deficient cells, independent of CDK4 activity. Mol Cell Biol 15:2600-2611.
  • BAS1 has a myb motif and activates HIS4 transcription only in combination with BAS2. Science 246:931-935. 50. Wasylyk, B., S. L. Hahn, and A. Giovane. 1993. The ets family of transcription factors. Eur J Biochem 211:7-18.
  • ORGANISM Mus musculus
  • Lys Asp Phe Tyr Arg Thr lie Ala Trp Gly Leu Asn Arg Pro Leu Phe 195 200 205
  • ORGANISM Mus rausculus
  • ORGANISM Mus musculus
  • GAAUCCGGCU CGCUCACCCC AGCUGCAGCC ACUCUCUCCC GCGGCUGCUU CCUCCAUCCU 60
  • AAAGCAAUGC CGUUCUAAAU GGCUCAACUA CCUGAACUGG AAGCAGAGUG GGGGUACUGA 1260
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ORGANISM Mus musculus
  • Glu Glu Asp Arg lie lie Tyr Gin Ala His Lys Arg Leu Gly Asn Arg 115 120 125
  • Trp Ala Glu lie Ala Lys Leu Leu Pro Gly Arg Thr Asp Asn Ala lie 130 135 140
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • FRAGMENT TYPE internal
  • ORGANISM Mus musculus
  • Gly Thr Val Thr Gin lie Gin lie Leu Gin Asn Asp Gin Leu Asp Glu 20 25 30 lie Ser Pro Leu Gly Thr Glu Glu Val Ser Ala Val Ser Gin Ala Trp 35 40 45
  • Glu lie Glu Lys Leu Lys Glu Leu Arg lie Lys His Gly Asn Asp Trp 145 150 155 160
  • ORGANISM Mus musculus
  • CTCATCCTAA GGATAGCTGA GCTTGATGTG GCCGATGAAA ATGACATAAA CTGGGATCTT 840
  • AAACAGTTAC ATGAGAACCA
  • AAAAAACAAC CCAGTGCTTT TGGAGAATAA ATCAGGATCT 1020
  • ORGANISM Mus musculus
  • ORGANISM Mus musculus
  • CAAAATAGCA CAGAACTGAT GAACAGTGTC ATGGTCAGAA CAGAGGAAGA AATTGCCGAC 600
  • AACTGTCAC 909 (2) INFORMATION FOR SEQ ID NO: 20:
  • ORGANISM Mus musculus
  • ORGANISM Mus musculus
  • ORGANISM Mus musculus

Abstract

L'invention décrit une interaction directe entre des cyclines de type D et un nouveau facteur de transcription de type myb, le DMP1, ayant une interaction spécifique avec la cycline D2. La présente invention démontre en outre que des cyclines de type D régulent l'expression génique indépendamment de la protéine rétinoblastome. L'invention décrit aussi le facteur de transcription DMP2 qui est composé d'un domaine central de liaison ADN contenant trois répétitions de myb atypiques flanquées de segments à forte acidité situés aux extrémités de ses terminaisons amino et carboxy. L'invention inclut un codage de séquences d'aminoacides pour DMP1 et des séquences nucléotidiques d'ADN et d'ARN qui codent pour les séquences d'aminoacides. L'invention décrit une utilisation de DMP1 comme facteur de transcription du fait de son aptitude spécifique à se lier à des oligonucléotides contenant la séquence consensus CCCG(G/T)ATGT de nonamère. Dans cet aspect de l'invention, DMP1, lorsqu'il est transfecté dans des cellules mammifères, active la transcription d'un gène marqueur sous l'effet d'un promoteur minimal contenant des sites de liaison de DMP1 en concatamères.
PCT/US1997/008480 1996-05-16 1997-05-16 Facteur de liaison de la cycline de type d et emplois dudit produit WO1997043415A1 (fr)

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US6303772B1 (en) 1996-05-16 2001-10-16 St. Jude Children's Research Hospital Cyclin D binding factor, and uses thereof
WO2012013249A1 (fr) * 2010-07-30 2012-02-02 Université de Liège Protéine 1 de la matrice dentinaire (dmp1) pour l'utilisation dans des compositions pharmaceutiques

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