WO1997043415A9 - Cyclin d binding factor, and uses thereof - Google Patents
Cyclin d binding factor, and uses thereofInfo
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- dmpl
- acid polymer
- seq
- sequence
- Prior art date
Links
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Definitions
- 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
The invention discloses a direct interaction between D-type cyclins and a myb-like transcription factor, DMP1, which specifically interacts with cyclin D2. The present invention also provides evidence that D-type cyclins regulate gene expression in an RB-independent manner. Also included is DMP2, the transcription factor composed of a central DNA-binding domain containing three atypical myb repeats flanked by highly acidic segments located at its amino- and carboxyterminal ends. The invention includes amino acid sequences coding for DMP1, and DNA and RNA nucleotide sequences that encode for the amino acid sequences. A use of DMP1 as a transcription factor is disclosed due to its specificity in binding to oligonucleotides containing the nonamer consensus sequence CCCG(G/T)ATGT. In this aspect of the invention, DMP1 when transfected onto mammalian cells, activates the transcription of a reporter gene driven by a minimal promoter containing concatamerized DMP1 binding sites.
Description
CYCLIN D BINDING FACTOR, AND USES THEREOF
RESEARCH SUPPORT
The research leading to the present invention was supported in part by the National Cancer Institute grants CA 20180 and CA 21765. The government may have certain rights in the present invention. Support for this invention was also provided by The Howard Hughes Medical Institute and the AMERICAN LEBANESE SYRIAN ASSOCIATED CHARITIES.
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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. For the cell cycle of eucaryotes, 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. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the Gl 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. During the S phase, 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. During the subsequent M period, 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.
In mammalian cells, a molecular event that temporally coincides with passage through the R point is the phosphorylation of the retinoblastoma protein (RB). In its hypophosphorylated state, RB prevents the cell from exiting the Gl phase by combining with transcription factors such as E2F to actively repress transcription from promoters containing E2F binding sites. However, hyperphosphorylation of RB late in Gl phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes. As many 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. In fibroblasts, 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. However, 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.
Since 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.
Alternatively, 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. However, up until now, no such RB-independent transcription factor has been identified.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.
SUMMARY OF THE INVENTION
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. In one embodiment the cyclin D-associated transcription factor is a substrate of cyclin D2-CDK4 kinase. In another embodiment, the transcription factor consists of about 760 amino acids.
More particularly, 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. (2) 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.
(3) The amino acid polymer binds preferentially to a specific DNA nucleotide sequence.
(4) The amino acid polymer is a substrate of the cyclin D2-CDK4 complex.
(5) The amino acid polymer contains three atypical tandem myb repeats.
(6) D-type cyclins bind less avidly to the amino acid polymer than to retinoblastoma protein, both in vitro and in Sf9 cells. (7) 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. (8) Cyclin D-dependent kinases phosphorylate the amino acid polymer at an atypical recognition sequence. (9) The amino acid polymer binds preferentially to nucleic acids containing the nonamer sequence CCCGTATGT.
(10) Relative to the cyclin D-CDK4 complex, cyclin E-CDK2 complexes phosphorylate the amino acid polymer poorly, if at all.
(I I) 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.
(12) Overexpression of the amino acid polymer can arrest the cell cycle in Gl phase preventing proliferating cells from replicating their chromosomal DNA.
(13) Enforced transient expression of cyclin D2 or D2-CDK4 in mammalian cells negatively regulate the ability of the amino acid polymer to transactivate reporter gene expression.
(14) The amino acid polymer activates transcription more readily in quiescent cells lacking cyclin D expression, than in proliferating cells containing cyclin D.
(15) Enforced expression of cyclin D-CDK4 does not influence the stability of the amino acid polymer.
(16) 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. Although any person having skill in the art would know that many of the above characteristics may be determined in vitro, the present invention includes the same or analogous characteristics that are determined either in situ or in vivo.
In one aspect of the present invention 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. In a more specific embodiment, the binding site has the core trinucleotide sequence GTA. In some embodiments the nucleotide sequence contains a nonamer consensus sequence CCCG(G/T)ATGT. In other embodiments the nucleotide sequences contain multiple concatamers of the nonamer consensus sequence. In preferred embodiments 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. In preferred embodiments the amino acid polymer is mammalian. In the most preferred embodiments, the amino acid polymer is human. In a specific example, the amino acid polymer is obtained from a murine cell and has the sequence of SEQ ID NO: l . In another embodiment, the amino acid polymer is obtained from a human cell and contains the amino acid sequence of SEQ ID NO:24. In a related embodiment the isolated amino acid polymer is obtained from a human cell, is encoded on human chromosome 7 at a position which corresponds to 7q21 , 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.
As shown in the Examples, infra, 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. In one specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO: 16. In another such embodiment 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. In one specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO: 18. In another such embodiment the peptide has an amino acid sequence of SEQ ID NO: 18 having conservative amino acid substitutions. In yet another specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO:20. In still another such embodiment the peptide has an amino acid sequence of SEQ ID NO:20 having conservative amino acid substitutions. In yet another specific embodiment of this type, the peptide has an amino acid sequence consisting of SEQ ID NO: 18 and SEQ ID NO:20. In still another such embodiment the peptide consisting of an amino acid sequence of SEQ ID NO: 18 and SEQ ID NO:20 having conservative amino acid substitutions. 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. In one specific embodiment of this type, the peptide has an amino acid sequence of SEQ ID NO:22. In another such embodiment 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. In this aspect of the invention, DMPl activates the transcription of a heterologous gene including
reporter genes driven by a minimal promoter containing concatamerized DMPl binding sites. If necessary, 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.
In still another aspect of the invention, 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. 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 35S-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 firefly luciferase.
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.
Naturally, in addition to the transcription factor, the present invention provides nucleic acids that contain nucleotide sequences or degenerate variants thereof, which encode the amino acid polymers of the present invention. In this aspect of the 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. In a specific
embodiment, the nucleic acid encodes a full length human DMPl containing the amino acid sequence of SEQ ID NO:24. In a related embodiment the nucleic acid encodes an isolated amino acid polymer which is encoded on human chromosome 7 at a position which corresponds to 7q21, and contains about 760 amino acids, including the 262 amino acids of SEQ ID NO:24.
In addition, 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. In one such embodiment 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. In one specific embodiment of this type, 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. In one such embodiment 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. In one specific embodiment of this type, 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. In one specific embodiment of this type, 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. In one specific embodiment of this type, 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. In one specific embodiment of this type, 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. In a preferred embodiment, the nucleic acids hybridize to the foregoing nucleotide sequences under stringent conditions.
In preferred embodiments 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. In a specific embodiment, 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. In a preferred embodiment of this aspect of the invention the nucleotide probe has a detectable label containing the radioactive element, 32P, and the detecting step includes performance of an electrophoretic mobility shift assay. In another specific embodiment, 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. ,
CCCGTATGT, to a selected gene forming a controllable transcript, and transfecting a mammalian cell with the recombinant gene. In some embodiments of the invention, the endogenous transcription factor of the invention in the mammalian cell will be sufficient to activate selective transcription of the heterologous gene. In other embodiments 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. In these other embodiments, 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.
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.
In a further aspect, 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. In a related embodiment, 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.
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.
In one particular embodiment of this type, the second expression vector encodes an amino acid polymer having the amino acid sequence of SEQ ID NO: l . In another embodiment of this type 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. In still another embodiment, the promoter is an artificial DMPl -responsive promoter. In a preferred embodiment of this type, 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. In another preferred embodiment, the reporter gene is firefly luciferase. In one embodiment, the cell is a mammalian cell, such as a mouse NIH-3T3 fibroblast. In preferred embodiments, 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. Alternatively, 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. In a preferred embodiment 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.
In one such embodiment, 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. In this case 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 . Again 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. Such 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.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
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. Three canonically spaced tryptophan residues (W) within each c-myb repeat are double underlined, and sites corresponding to DNA contacts in repeat-2 are indicated by asterisks. Eleven and six residue "inserts" required for maximal alignment of the two sequences are indicted above repeat-2 and repeat-3. The nucleotide sequence will be deposited in GenBank.
Figure 2 is a gel showing the binding in vitro of D-type cyclins to RB and DMPl fusion proteins. [35S]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 [35S]methionine. Figure 3A. Lysates are divided in half, and proteins in one aliquot are separated directly on denaturing gels. Figure 3B. 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 [35S]methionine (lanes 1-8) or 32P-orthophosphate (lanes 9-12) and half of the [35S]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 [35S]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 32P-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).
Autoradiographic exposure times are 18 hours for Figure 7 A and 6 hours for Figure 7B.
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. Complexes resolved on nondenaturing gels include those previously designated A and B (Figure 7A.) and a supershifted complex designated S in the left margin. Exposure time is 18 hours. Figure 8C. 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. Reporter plasmids (same as Figure 9A, 1 μg 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 μg) 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. For each set of experiments, the total input DNA concentrations were adjusted where necessary by addition of parental pRc/RSV plasmid DNA lacking inserts to yield 4 μg (9A), 3 μg (9B), and 2 μg (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.
DETAILED DESCRIPTION OF THE INVENTION
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. 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. Correspondingly low levels of DMPl protein are 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.
Cyclin D-associated Transcription Factor As noted above, the present invention provides an amino acid polymer that binds to cyclin D and to a specific DNA sequence. In a specific embodiment, 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. Furthermore, as set forth below, 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.
The term "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. As used herein "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 (SEQ ID NO:2), displaying substantially equivalent or altered activity 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.
The 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. NH2 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.
It should be noted that all 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.
In a specific embodiment, 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. , in a biological sample or in a culture after fermentation of recombinant cells, and then treated to "eluted" the protein from the oligonucleotide conjugated to the solid phase support. As one of ordinary skill in the art can readily appreciate, other 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. In another embodiment, the solid phase support may be a useful chromatographic support, such as the carbohydrate polymers SEPHAROSE, SEPHADEX, and agarose. As used herein, a solid phase support is not limited to a specific type of support. Rather, a large number of supports are available and are known to one of ordinary skill in the art. 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. For example, 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
Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California). In synthesis of oiigonucleotides, a 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. , in a column; it can be used in suspension followed by filtration, sedimentation, magnetic association, or centrifugation; by automated sorting (analogous to flow cytometry); or by washing, as in a membrane, well, plastic film, etc.
The term "polypeptide" is used 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. As used herein the term "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 (Nα-amino protected Nα-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 Nα- amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc Nα-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. In addition, the method of the invention can be used with other Nπ-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. Thus, polypeptides of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g., |8-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties. 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, α-helices, β turns, β sheets, γ-turns, and cyclic peptides can be generated.
In one aspect of the invention, the peptides may comprise a special amino acid at the C- terminus which incorporates either a C02H or CONH2 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. In one embodiment, 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.
Secondary structural analysis (e.g. , Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of transcription factor that assume specific secondary structures.
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.
By providing an abundant source of recombinant transcription factor, the present invention enables quantitative structural determination of transcription factor, or domains thereof. In particular, enough material is provided for nuclear magnetic resonance (NMR), infrared (IR), Raman, and ultraviolet (UV), especially circular dichroism (CD), spectroscopic analysis. In particular NMR provides very powerful structural analysis of molecules in solution, which more closely approximates their native environment (Marion et al., 1983, Biochem. Biophys. Res. Comm. 113:967-974; Bar et al. , 1985, J. Magn. Reson. 65:355- 360; Ki ura et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77: 1681-1685). Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, A., 1974, Biochem. Exp. Biol. 11 :7-13).
More preferably, 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).
Genes Encoding The Transcription Factor
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. As used herein, the term "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.
Accordingly, in the practice of the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g. , Sambrook, Fritsch &
Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Therefore, if appearing herein, the following terms shall have the definitions set out below.
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. Preferably, 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. Preferably, 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. The term 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. In discussing the structure of particular double-stranded DNA molecules, 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. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5x SSC, 0.1 % SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5 % SDS. Moderate stringency hybridization conditions correspond to a higher Tm, e.g. , 40% formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest Tm, 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 Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA,
DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al. , supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e. , oiigonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably 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.
In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of 55°C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60°C; in a more preferred embodiment, the Tm is 65°C.
"Homologous recombination" refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific 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. In eukaryotic cells, 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. For purposes of defining the present invention, 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. Within the promoter sequence will be found 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.
As used herein, the term "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).
Accordingly, the term "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). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.
In a specific embodiment, 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.
Similarly, in a particular embodiment, 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). Preferably, 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, whether genomic DNA or cDNA, 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). For example, 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). Preferably, 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.
Further selection can be carried out on the basis of the properties of the gene, e.g. , if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, or partial amino acid sequence of the transcription factor protein as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, 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. For example, 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. In a specific embodiment, 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. Preferably, derivatives are made that have enhanced or increased functional activity relative to native transcription factor.
Due to the degeneracy of nucleotide coding sequences, other DNA 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. Likewise, 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. , as set forth in SEQ ID NO: l, including altered sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a conservative amino acid substitution. These entail "conservative substitutions" as defined herein. These 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. For example, 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.
Particularly preferred substitutions are:
- Lys for Arg and vice versa such that a positive charge may be maintained;
- Glu for Asp and vice versa such that a negative charge may be maintained;
- Ser for Thr such that a free -OH can be maintained; and - Gin for Asn such that a free NH2 can be maintained.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, 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.
The genes encoding 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. For example, 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. In the production of the gene encoding a derivative or analog of transcription factor, care should be taken to ensure that the modified gene remains within the same translational reading frame as the transcription factor gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.
Additionally, 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. Preferably, such 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. 253:6551 ; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44: 177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
In a specific embodiment, 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. , thrombin or Factor Xa, preferably at the DMPl-non-DMPl juncture. In a specific embodiment, 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. A large number of 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. Alternatively, 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. Preferably, 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. For example, 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 2μ plasmid.
Expression of Transcription Factor Polypeptides 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. " Thus, 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. 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, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomal ly, after integration of the coding sequence by recombination. In this regard, 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).
Expression of 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. 78: 1441- 1445), 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. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements
from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al. , 1984, Cell 38:639-646; Ornitz et al. , 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115- 122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al. , 1986, Cell 45:485- 495), 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. 1: 161-171), 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 As noted above, the functional activity of DMPl can be evaluated transgenically. In this respect, 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)].
Alternatively, 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. Alternatively, recombinant techniques can be used to introduce mutations, such as nonsense and amber mutations, or mutations that lead to expression of an inactive protein. In another embodiment, 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 (antisense-sense) 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,
241:593-595 (1988). An important advantage of this approach is that only a small portion of the gene need be expressed for effective inhibition of expression of the entire cognate mRNA. 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.
Expression Vectors Regulated bv the Transcription Factor In addition to expression vectors that provide for expression of the transcription factor of the invention, 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. Preferably, such a vector is a plasmid. More
preferably, the cyclin D transcription factor recognition sequence is genetically engineered into the promoter in the expression vector.
In a specific embodiment, infra, introduction of the DNA recognition sequence for the murine cyclin D transcription factor termed DMPl was inserted in the SV40 minimal promoter and fused to a luciferase reporter gene. These plasmids express less background activity than the SV40 promoter alone.
Accordingly, 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.
In a further embodiment, 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. In one embodiment, these elements are provided on separate vectors, e.g. , as exemplified infra. In another embodiment, these elements are provided in a single expression vector.
Antibodies to the Transcription Factor According to the invention, 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. Acad. Sci. U.S.A. 80:2026-2030), chimeric (Morrison et al., 1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takeda et al. , 1985, Nature 314:452-454), single chain (U.S. Patent 4,946,778), Fab fragments, and an Fab expression library. 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. Alternatively, 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.
For the production of polyclonal antibody, various host animals can be immunized by injection, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, 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). Various 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.
In the production of antibodies, 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. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, 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.
Inhibition of Transcription Factor Expression 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.
Investigators have identified two types of ribozymes, Tetrahymena-type and " hammerhead" -type (Hasselhoff and Gerlach, 1988). 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.
Therapeutic Methods and Gene Therapy 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 addition, 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. In addition to treating diseases or disorders by administration of the cyclin D-associated transcription factor of the invention (DMPl), 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. In a specific embodiment, 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. In a related embodiment, the cyclin D domain of DMPl can be modified so that the cyclins no longer can act as negative effectors of DMPl. In this case a transgene vector for expression of such a modified DMPl of the present invention can be used. In still another embodiment, an inhibitor of the cyclins could be administered to prevent cyclin-DMPl binding.
In the above instances, 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. In yet another embodiment, an analogue of DMPl can be used. Under all of the above circumstances, increased expression of genes under control of DMPl may be necessary to restore appropriate cell cycle and growth characteristics to a transformed cell.
Examples of tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, edulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
On the other hand, 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. Alternatively, 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.
In such cases dysproliferative changes (such as metaplasias and dysplasias) are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung. Thus, 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.
As the present invention provides for detecting the level and activity of DMPl in cells, such as cancer cells or dysproliferative cells, the need to increase or decrease the activity of DMPl in a given cell can be readily determined. In one embodiment, a gene for regulation of DMPl (e.g. , a dmpl gene or an antisense gene) is introduced in vivo in a viral vector. Such vectors 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. 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. Thus, in a specific embodiment, tumors can be specifically targeted. Examples of 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. 90:626-630), and a defective adeno-associated virus vector (Samulski et al., 1987, J. Virol. 61 :3096- 3101 ; Samulski et al., 1989, J. Virol. 63:3822-3828).
Preferably, for in vitro administration, 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. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g. , Wilson, 1995, Nature Medicine). In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.
In another embodiment 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;
Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., 1988, J. Virol. 62: 1120; Temin et al., U.S. Patent No. 5, 124,263;
International Patent Publication No. WO 95/07358, published March 16, 1995, by
Dougherty et al. ; and Kuo et al., 1993, Blood 82:845.
Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.
Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of 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)). The use of 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). The use of 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.
It is also possible to introduce the vector in vivo as a naked DNA plasmid. 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).
In a preferred embodiment of the present invention, 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. In a specific embodiment, a gene therapy vector of the invention comprises the trinucleotide sequence GTA; preferably a vector of the invention comprises the nonanucleotide sequence CCCGTATGT. Thus, 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. In one embodiment, 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. USA, 92: 1023-1027 (19950); Thompson, Thromb. and
Haemostatis, 66: 119-122 (1991)]; and various other well known therapeutic genes such as, but not limited to, .-globin, dystrophin, insulin, erythropoietin, growth hormone, glucocerebrosidase, j3-glucuronidase, α-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, ornithine transcarbamylase, apolipoproteins, and the like. In general, see U.S. Patent No. 5,399,346 to Anderson et al.
In another aspect, 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. Examples of such heterologous genes for immunomodulatory (or immuno-effector) molecules include, but are not limited to, interferon-α, interferon-7, interferon-j3, interferon-ω, interferon-τ, 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.
In a further embodiment, 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. In one embodiment, these elements are provided on separate vectors, e.g. , as exemplified infra. These elements may be provided in a single expression vector.
Detection of Transcription Factor
As suggested earlier, 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.
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. As mentioned earlier, 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 procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. For example, a "competitive" procedure is described in U.S. Patent Nos. 3,654,090 and 3,850,752. A "sandwich" procedure is described in U.S. Patent Nos. RE 31 ,006 and 4,016,043. Still other procedures are known such as the "double antibody," or "DASP" procedure.
The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
A number of 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 3H, l4C, 32P, 35S, 36C1, 51Cr, "Co, 58Co, 59Fe, *Υ, 125I, "'I, and 186Re.
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, β-glucuronidase, β-D-glucosidase, β-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.
Other means for detecting specific binding are well known in the art, including biosensors such as the BIAcore™ 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).
Since DMPl can act as a cell cycle inhibitor when expressed in a tumor cell, a specific peptide domain of DMPl is likely to be responsible for this property. In particular, 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. In a particular embodiment the transactivation domain comprises amino acids 459 to 761 of SEQ ID NO: l or SEQ ID NO: 18. In a related embodiment 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. In a specific embodiment this DNA-binding domain consists of amino acids 87-458 (SEQ ID NO: 16) of SEQ ID NO: l . In particular, 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. For example, natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize DMPl activity.
Knowledge of the primary sequence of DMPl , and the similarity of that sequence with proteins of known function, can provide an initial clue as the inhibitors or antagonists of
the protein. Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g. , using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.
Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al. , Proc. Natl. Acad. Sci. , 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)], very large libraries can be constructed (106-108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method [Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al. J. Immunologic Method 102:259-274 (1987)] and the method of Fodor et al. [Science 251:767-773 (1991)] are examples. Furka et al. [14th International Congress of Biochemistry, Volume 5, Abstract FR-.013 (1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton [U.S. Patent No.
4,631,211, issued December 1986] and Rutter et al. [U.S. Patent No. 5,010, 175, issued April 23, 1991] describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
In another aspect, synthetic libraries [Needels et al., Proc. Natl. Acad. Sci. USA
90: 10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90: 10922-10926 (1993); Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 9428028, each of which is incorporated herein by reference in its entirety], and the like can be used to screen for DMPl ligands, e.g. , agonists or antagonists, according to the present invention.
The screening can be performed with recombinant cells that express the DMPl , or alternatively, using purified protein, e.g. , produced recombinantly, as described above. For example, 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. , Proc. Natl, Acad. Sci. USA, 87:2720-2724 (1990)]; [Wang et al., Proc. Natl. Acad. Sci. USA, 88: 11505-11509 (1991)]; [Cecchini et al., Nucleic Acids Res., 21:5742-5747 (1993)]. 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 , whereas the control cDNA library is obtained from proliferating NIH-3T3 cells that have not been so transfected.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
Various references cited herein by number are listed after the Examples, infra.
MATERIALS AND METHODS Cells and culture conditions Mouse NIH-3T3 fibroblasts and 293T human embryonic kidney cells (18) are maintained in a 10% C02 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). 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.
Isolation of DMPl
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. Of 6 X IO5 colonies screened, 107 grew on SD synthetic medium lacking histidine and express β-galactosidase. Colonies that had been induced to segregate the bait plasmid were mated with yeast strain Y187 containing either pAS2cycD2 or unrelated control plasmids expressing yeast SNF1 or human lamin fused to the GAL4 DNA-binding domain. cDNAs from 36 library-derived plasmids presumed to encode cyclin D2-interacting proteins are sequenced, one of which encodes a cyclin D-binding myb-like protein, here designated DMPl . The nucleotide sequence for the mouse DMPl will be submitted to GenBank.
Because the recovered 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 IO6 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.
In vitro binding and protein kinase assays
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 37CC, and induced with 1 mM isopropyl-/3-D-thiogalactopyranoside (IPTG) for 1 hour. 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). For in vitro binding, 1.5 μg of GST-DMP1 or GST-RB (15) immobilized on glutathione-Sepharose beads are mixed with [35S]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 incoφorated by reference. Proteins are mixed in 0.5 ml of IP Kinase buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol (DTT), 0.1 % Tween-20) containing 10 mg/ml bovine serum albumin (BSA, Cohn Fraction V, Sigma Chemicals, St. Louis MO). After 2 hours at 4°C, 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).
Protein kinase assays are performed using 1.5 μg GST-DMP1 or GST-RB adsorbed to glutathione-Sepharose as substrates. The beads are suspended in a total volume of 25μl 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 [-32P]ATP (6000 Ci/mmol; Amersham), and 2.5-5.0 μl lysate (corresponding to 5 X IO4 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 polyacrylamide gels.
Antisera and immunoblotting
Rabbit antisera to recombinant DMPl are commercially prepared (Rockland, Gilbertsville
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). To detect DMPl in cultured mammalian cells, untreated CTLL cells (4 X 107) or transfected 293T cells (1.5 X 106) are suspended and sonicated in 1 ml of RIPA buffer [50 mM Tris HCl, pH 7.5, containing 150 mM NaCl, 1 % Nonidet P40, 0.5% sodium deoxycholate, and 0.1 % SDS] and clarified by centrifugation. DMPl was precipitated with 10 ul of antiserum AJ, denatured and electrophoretically separated on 9% polyacrylamide gels containing SDS, and transferred to nitrocellulose. The filter is incubated with a 1/100 dilution of AJ and AF antisera, and sites of antibody binding were detected using 125I-protein A (Amersham) as described (12).
Expression of recombinant DMPl in insect cells
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). For preparation of radiolabeled cell lysates, 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 [35S]methionine (1000 Ci/mmol; ICN, Irvine CA) in methionine-free medium or for 4 additional hours with 250 uCi/ml of carrier-free 32P-orthophosphate (9000 Ci/mmol, Amersham) in phosphate-free medium. Cells suspended in 0.25 ml Kinase buffer containing protease and phosphatase inhibitors [2.5 mM EGTA, 0.1 mM phenylmethyl sulfonylfluoride (PMSF), 2% aprotinin, 1 mM β-glycerophosphate, O. lmM Na3V04, and 0.1 mM NaF] are lysed by repeated freezing and thawing and clarified by centrifugation. For detection of DMPl or its complexes with D-type cyclins, 10-20 μl 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 Na3V04, and 0.1 mM NaF. Antiserum AF (10 μl 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. Where indicated, 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.
Selection of DMPl binding consensus oiigonucleotides
Binding site selection and amplification by polymerase chain reaction (PCR) 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. Mixing is performed in 125 μl of Binding buffer (25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM EDTA, 1.5 mM MgCl2, 0.1 % Nonidet P40, 1 mM DTT, 5% glycerol) containing 25 μg
poly (dl-dC) (Boehringer Mannheim) and 25 μg 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 μl 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 32P using the Klenow fragment of DNA polymerase and α-32P-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 μg protein) or Sf9 lysates (corresponding to 5 X IO2 infected cells) containing ~ 4ng recombinant DMPl are mixed with 3 ng of 32P-labeled probe (1 X 105 cpm) in 15 ul Binding buffer containing 2.5 μg of poly(dl-dC) and 2.5 μg BSA and incubated at 4°C for 30 minutes. For competition experiments, the indicated amounts of unlabeled oiigonucleotides are added to the reactions before addition of the labeled probe. In some experiments, 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 μg 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).
Transactivation assay
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. The latter "reporter" plasmid (1 μg) together with increasing amounts of pRc/RSV-DMPl expression plasmid compensated by decreasing quantities of control pRc/RSV DNA (total of both = 2.5 μg) were transfected into 293T cells (1.5 X IO5 cells per 60 mm diameter culture dish) by calcium phosphate precipitation (7). Two days later, cells were harvested, washed three times with PBS, and lysed in 1 ml of 25 mM glycylglycine (Sigma), pH 7.8, 15 mM MgS04, 4 mM EDTA, 1 mM DTT, and 1 % Triton X-100. After clearing by centrifugation, 50 μl aliquots were assayed diluted to 350 μl using 15 mM potassium phosphate buffer, pH 7.8, containing 15 mM MgS04, 4 mM EGTA, 2 mM ATP, 1 mM DTT, and 67 uM luciferin (Sigma). Total light emission was measured for duplicate samples during the initial 20 seconds after luciferin injection with an Optocomp I luminometer (MGM Instruments, Hamden CT).
Fluorescence in situ hybridization for Chromosome Determination. Phytohemagglutinin- stimulated human peripheral blood lymphocytes from a normal donor were used as the source of metaphase chromosomes. Purified DNA from PI clone 11098 was labeled with digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) by nick translation and hybridized overnight at 37°C to fixed metaphase chromosomes in a solution containing sheared human DNA, 50% formamide, 10% dextran sulfate, and 2X SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluorescein- conjugated sheep antibodies to digoxigenin (Boehringer Mannheim, Indianapolis, IN). The 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). 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)].
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.
Staining for the Expression of DMPl and Incoφoration of BrdU in Transfected Cells. 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 incoφoration. 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. To stain for DMPl expression, mouse monoclonal anti-Flag antibodies (12 μg/ml) [Kodak] were incubated with the cells in TBS-Ca+ without FCS for one hour at room temperature. After washing the cells, 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. After washing the cells, streptavidin linked to Texas red [Amersham] was then added at a 1 :500 dilution for 30 minutes at room temperature. To stain for BrdU incoφoration, 1.5N HCl was added to the cells for ten minutes at room temperature to denature the DNA. After washing the cells, sheep anti-BrdU antibodies [Fitzgerald] at a 1 : 12 dilution were then added for one hour at room temperature. After washing the cells, rabbit FITC-conjugated anti-sheep antibodies [Vector] at 1: 100 dilution was then incubated for 30 minutes at room temperature.
Isolation of Clone 11098. 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.
EXAMPLE 1 Isolation and Molecular Features of DMPl
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 105 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. These 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. Among the latter group is 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. heart, brain, spleen, lung, liver, kidney, testis) and mouse cell lines (NIH-3T3 fibroblasts, BAC1.2F5 macrophages, CTLL T cells, and MEL erythroleukemia cells), and it is nonperiodically expressed throughout the cell cycle in synchronized macrophages and fibroblasts (data not shown). Overlapping 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. The cyclin D-binding myb-like protein encoded by this clone is designated DMPl .
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). Authentic myb proteins bind to YAACNG (Y = pyrimidine) consensus sequences in DNA, with usually two or, rarely, only one of the myb repeats being sufficient to confer binding (6, 16,40,41 ,52). Scattered amino acid identities enabled us to align the repeat sequences within mouse c-myb with those of DMPl (Fig IB). In particular, there is an exact conservation of KQCR-W-N in repeat-2 (denoted by asterisks), which in c-myb contacts the DNA-binding site (42). However, the first repeat of DMPl contains a tyrosine substituted for the first tryptophan and leucine for the third. Moreover, 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. These features distinguish the repeats of DMPl from myb proteins and predicted that, if DMPl binds DNA, its consensus binding site would likely differ from the myb recognition sequence.
EXAMPLE 2
Interaction of DMPl with D-type cyclins
Because DMPl interacted with cyclin D2 in yeast, the ability of a glutathione S- transferase (GST)-DMPl fusion protein to bind D-type cyclins in vitro is examined. GST is fused to residues 176- 761 of DMPl (in lieu of GAL4 in the original cDNA clone), and the bacterially synthesized recombinant protein is incubated with [35S]methionine-Iabeled D-type cyclins prepared by transcription and translation in vitro. As a positive control, GST-RB which can specifically bind D-type cyclins in this assay is used (15). Bound cyclins recovered on washed glutathione-Sepharose beads are analyzed by electrophoresis on denaturing gels. Figure 2 (lanes 6 and 10) 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). No labeled proteins bind to GST alone (lanes 4, 8, 12), and neither cyclin A nor cyclin E bind to GST-RB or to GST-DMP1. A cyclin D2 mutant disrupted in an amino-terminal Leu-X-Cys-X-Glu pentapeptide that is required for high efficiency GST-RB binding is not detectably compromised in its interaction with GST-DMP1 (negative data not shown); in
agreement, DMPl bears no homology to RB or to RB-related family members (pl07 and pl30).
We next co-expressed full length DMPl together with D-type cyclins under baculovirus vector control in insect Sf9 cells. After metabolically labeling infected cells with [ 5S]methionine, we precipitated DMPl with an antiserum directed to a peptide representing its nine C-terminal residues. Electrophoretic separation of unfractionated metabolically labeled lysates from infected cells enabled direct autoradiographic visualization and relative quantitation of the recombinant mouse proteins (Fig 3A). Cells infected with a vector containing DMPl cDNA (lane 2) 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.
When DMPl and different D-type cyclins are coexpressed in Sf9 cells (Fig 3A, lanes 3, 5, and 8), anti-DMPl coprecipitate cyclin D2 and D3 (Fig 3B, lanes 6 and 9) and bring down cyclin Dl less efficiently (Fig 3B, lane 4). Antisera to D-type cyclins reciprocally precipitate DMPl (not shown). In analogous experiments using RB in place of DMPl , stronger binding is also observed using cyclin D2 or cyclin D3 versus cyclin Dl suggesting that differences in their binding efficiency may not be physiologic. Using coinfected cells containing approximately equivalent levels of DMPl and cyclin D2 or cyclin D3, only 5-15% of the cyclin is stably bound to DMPl, whereas binding to RB in such experiments is — 1 : 1. Overall, these results are completely consistent with the in vitro binding data obtained with DMPl and RB (Fig 2).
When Sf9 cells producing DMPl are coinfected with baculoviruses encoding both a D-type cyclin and CDK4 (Fig 3A, lanes 4, 6, and 9), complex formation between the
cyclins and DMPl is significantly diminished (Fig 3B, lanes 5, 7, and 10). The latter effect could be due at least in part to competition between CDK4 and DMPl for binding to cyclin. However, coproduction of a cyclin D- binding but catalytically inactive CDK4 mutant (Fig 3A, lane 7) at levels equivalent to those of wild- type CDK4 (Fig 3A, lane 6) is much less effective in preventing an interaction of DMPl with cyclin D2 (Fig 3B, lane 8 versus 7). Therefore, phosphorylation of DMPl by cyclin D-CDK4 complexes (see below) 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.
EXAMPLE 3 DMPl is a Substrate for Cyclin D Dependent Kinases In comparison to many known CDKs, the cyclin D-dependent kinases exhibit an unusual preference for RB over histone HI as an in vitro substrate (33,34,39). To test whether cyclin D-dependent kinases could phosphorylate DMPl, 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. Whereas 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). Under equivalent conditions, 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.
Based on data suggesting that DMPl is post-translationally modified when expressed in Sf9 cells and that coexpression of cyclin D-dependent kinases could reduce its binding to D cyclins (Fig 3), we expressed DMPl in Sf9 cells alone or together with cyclin D2-CDK4 or cyclin D2-CDK6. Infected cells are metabolically labeled with
[35S]methionine, and DMPl is immunoprecipitated from cell lysates and resolved on denaturing gels. Using less radioactive precursor than for the experiments shown in Figure 3, 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). Similar data are obtained when infected cells are labeled with [3 P]orthophosphate instead of [35S]methionine (Fig 4B, lanes 9-12). Additional control experiments performed with the [32P]phosphate-Iabeled proteins confirm that the observed effects of alkaline phosphatase on DMPl mobility are due to removal of phosphate groups and are blocked by 1 mM sodium orthovanadate. Moreover, two dimensional separation of radiolabeled DMPl tryptic phosphopeptides reveal complex fingeφrint patterns, consistent with multiple phosphorylation sites (data not shown). Therefore, both components of the DMPl doublet are phosphoproteins. Its basal phosphorylation can be mediated by endogenous kinases present in insect cells, but co-expression of cyclin D-dependent kinases augments accumulation of the hypeφhosphorylated, more slowly migrating species.
Hypeφhosphorylation 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 hypeφhosphorylation is not as readily induced by cyclin E-CDK2 (Fig 4C, lane 10).
Kinase assays performed with the same lysates (Fig 4D) confirm that the cyclin D-CDK4 complexes are highly active as RB kinases (Fig 4D, lanes 6-8), whereas mutant CDK4 is defective (lane 9). Despite its relative inactivity on DMPl (Fig 4C, lane 10), cyclin E-CDK2 readily phosphorylates both RB (Fig 4D, lane 10) and histone HI (lane 13), but cyclin D2-CDK4 fails to phosphorylate the latter (lane 12). Thus, cyclin D-CDK4 and cyclin E-CDK2 differ in their relative substrate specificities for both histone HI and DMPl .
EXAMPLE 4 Recombinant DMPl Binds to Specific DNA Sequences
To determine whether DMPl would bind specifically to DNA, 30 base-pair random oiigonucleotides flanked by PCR primers are prepared and then incubated with Sf9 cell lysates containing the full length DMPl protein. Oiigonucleotides bound to washed DMPl immunoprecipitates are amplified by PCR, and after six rounds of reprecipitation
and reamplification, the final products are recloned and their sequences determined From 27 sets of sequences, the consensus CCCG(G/T)ATGT is derived (Fig 5A) Repeating the experiment with a histidine-tagged DMPl polypeptide produced in bacteria in place of the baculovirus-coded protein, oiigonucleotides containing GGATG are again isolated, but the preference for the 5' CCC triplet is less pronounced Computer searches indicate that the DMPl oligonucleotide consensus also represents a binding site for the Etsl and Ets2 transcription factors [namely, (G/C)(A/C)GGA(A/T)G(T/C)] All Ets family proteins bind to sequences with a GGA core, with their individual binding specificities determined by adjacent flanking sequences (31,50) Because the selected DMPl binding site included either GGA or, less frequently, GTA in the corresponding position (Fig 5A), two oiigonucleotides are synthesized (designated BS 1 and BS2 in Fig 5B) that differ only in this manner Four mutant oiigonucleotides are prepared (M1-M4 in Fig 5B), at least one of which (Ml) is predicted to bind neither DMPl nor Ets proteins, and another (M3) that, in contradistinction to BS2, should interact with Etsl or Ets2 but not DMPl
Using electrophoretic mobility shift assays (EMSA) performed after mixing a titrated excess (3 ng) of 32P-end labeled BS1 probe with Sf9 lysates producing DMPl ( — 4 ng recombinant protein per reaction), a BS1 -containing protein complex is detected that was competed with an excess of unlabeled BS1 oligonucleotide but not with mutant oiigonucleotides Ml and M2 (Figure 6A) Because Ml is disrupted in three of three completely conserved residues (Fig 5B), its failure to compete is not suφπsmg, but the inability of M2 to compete indicates that CCC sequences 5' of the G(G/T)A core are also important for DMPl binding More subtle mutations withm this region may be tolerated, because high concentrations of M4 competed for BS1 binding to both Ets2 and DMPl in subsequent studies (Fig 5B and see below) DMPl also binds a BS2 probe, and the binding is competed by excess BS2 or BS1 (Fig 6B) In agreement with the site selection frequencies (Fig 5A), binding of 32P-BS1 under equivalent conditions was competed more efficiently by excess unlabeled BS1 than by BS2 (Fig 6B) M3, which is predicted to interact only with Ets proteins, does not compete with BS1 or BS2 probes for binding to DMPl (Fig 6B) In contrast, a bacterially produced GST-Ets2 fusion protein does not bind detectably to a labeled BS2 oligonucleotide (not shown) under conditions where BS1 binding was readily detected (Fig 6C) In agreement, Ets2 binding to BS1 could be competed with excess unlabeled BS1 and M3, but not by BS2 (Fig 6C) Therefore, although both DMPl and Ets2 can each bind to BS1 sequences, their exclusive interactions
with BS2 and M3, respectively, help to distinguish DMPl and Ets binding activities (summarized in Fig 5B).
Under identical EMSA conditions, use of extracts from Sf9 cells coexpressing cyclin D-CDK4 complexes (and containing predominantly hypeφhosphorylated forms of DMPl) do not affect the efficiencies or patterns of DMPl binding to radiolabeled BS1 or BS2 probes. Nor are there apparent differences in the recovery of DMPl -probe complexes between lysates lacking or containing cyclin D. Although as much as 15 % of DMPl molecules form stable complexes with D-type cyclins when the two are coexpressed (Fig 3), both polyvalent and monoclonal antibodies to cyclin D are unable to supershift any of the DMPl -oligonucleotide complexes formed with the same Sf9 extracts, indicating that the interaction of DMPl with cyclin D might inhibit DNA binding.
EXAMPLE 5 DMPl Expression and DNA Binding Activity in Mammalian Cells
Using antisera directed either against a DMPl C-terminal peptide (serum AF, Figs 3 and 4), the GST-DMP1 fusion protein (serum AH, residues 176-761), or its putative DNA-binding domain (serum AJ, residues 221-439), DMPl is not detected in mammalian cells by immunoprecipitation of the protein from metabolically labeled cell lysates. However, sequential immunoprecipitation (with serum AJ) and immunoblotting (with sera AJ plus AH) reveals low levels of DMPl in lysates of proliferating NIH-3T3 fibroblasts (Fig 8 A, lane 3). Most of the protein has a mobility corresponding to that of the hypeφhosphorylated form synthesized in Sf9 cells (lane 1). [The baculovirus-coded protein was separated on the same gel as the immunoprecipitates from NIH-3T3 cells, and their positions were aligned after multiple autoradiographic exposures] .
Using the non-Ets-interacting 32P-labeled BS2 probe to screen for DNA binding activity in mammalian cells by EMSA, complexes with mobility indistinguishable from that generated with the recombinant protein in Sf9 lysates (Fig 7A, lane 1, complex A) are detected with lysates from NIH-3T3 fibroblasts (lanes 2-8) and CTLL T cells (lanes 9-15). A faster migrating complex which lacks DMPl is also seen (complex B, see below). As predicted, A-complexes containing bound 32P-BS2 are competed by both unlabeled BS1 (lanes 3, 10) and BS2 (lanes 4, 11), but not by the M3 Ets- specific recognition sequence (lanes 7, 14). Using the same lysates, more total binding activity is detected with a BS 1 probe (Fig 7B;
compare autoradiographic exposure times for panels A and B), the vast majority of which is competed by M3 (lanes 7, 14) but not by BS2 (lanes 4, 11). Therefore, the EMSAs performed with 32P-BS1 primarily detect Ets-type DNA binding activity, whereas that performed with 32P-BS2 scores an activity indistinguishable from that of bona fide DMPl .
To confirm that DMPl activity is responsible for the A-complexes observed in EMSAs done with the BS2 probe, antiserum to the DMPl C-terminus (AF) is added to the binding reactions (Fig 8B). This generates a "supershifted" complex of slower mobility (labeled S, lane 3) which is eliminated by competition with the cognate DMPl peptide (PI, lane 4) but not with an unrelated control peptide (P2, lane 5). Formation of the A and S complexes is blocked by competition with the unlabeled BS2 oligonucleotide but not with M3 , whereas B complexes remain and must therefore contain a protein(s) other than DMPl or Etsl Ets2. Consistent with these findings, preincubation of NIH-3T3 or CTLL extracts with any of three different antisera to DMPl (AF, AJ, or AH) but not with nonimmune serum (NI) eliminates the formation of A, but not B, complexes in EMSAs (Fig 8C). Therefore, the BS2-containing A-complex formed with extracts of mammalian cells contained authentic DMPl .
EXAMPLE 6 DMPl Can Activate Transcription
To determine if DMPl has the capacity to activate transcription, 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). When the cDNA encoding DMPl is cloned into a pRc/RSV mammalian expression plasmid and cotransfected with limiting amounts (1 μg) of the BS2-driven reporter plasmid into 293T cells, significant transactivation of luciferase activity at levels — 20-fold that seen with the BS2 reporter plasmid alone are observed (Fig 9B). A 7-fold activation of the BS1 -driven reporter in response to DMPl (Fig 9B) is of even greater absolute magnitude but is initiated from a 4-5 fold higher basal level (Figs 9 A and 9B). In contrast, using promoters lacking BS2
sites or containing Ets-specific M3 sites, transactivation by DMPl is not observed. Gross overexpression of DMPl in these experiments is documented by immunoprecipitation and immunoblotting, and the majority of the ectopically produced protein is localized to the cell nucleus (data not shown).
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. However, in the absence of ectopically expressed DMPl, "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. Similarly, when the GGA-containing consensus oligonucleotide probe is used for EMSA, competition studies indicate that Ets family members predominate in complexes resolved from lysates of NIH-3T3 and CTLL cells.
Complexes formed with the GTA-containing BS2 probe could be depleted or supershifted with antisera to DMPl and are not competed by unlabeled Ets-binding M3 oligonucleotide (Fig. 8), whereas those formed with the GGA-containing BS1 probe are resistant to these treatments (negative data not shown). Particularly in cases such as these where total Ets binding activity greatly exceeds that of DMPl , the use of oligonucleotide probes containing the GTA core is essential for unambiguously demonstrating endogenous DMPl DNA binding activity by EMSA.
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 hypeφhosphorylated in cells co-expressing catalytically active, but not mutant, cyclin D-CDK4 complexes. Immune complexes containing cyclin D-CDK4 can also hypeφhosphorylate DMPl in vitro. However, 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.
The observed interactions of 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 . Third, 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. However, even though there are fourteen 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. Conversely, cyclin E-CDK2 complexes phosphorylated DMPl poorly, if at all, and no physical interactions between DMPl and cyclin E or cyclin A are detected. Finally, phosphorylation of RB by cyclin D-CDK4 complexes cancels its ability to bind D-type cyclins, so that in coinfected Sf9 cells, stable ternary complexes could only be generated between RB, D-type cyclin, and catalytically inactive CDK4 subunits.
However, 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. In agreement, 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 hypeφhosphorylation of DMPl can decrease its ability to bind cyclin D, the role of cyclin D binding is not solely to trigger CDK4-mediated phosphorylation.
Together, these findings provide evidence that 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. However, 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.
EXAMPLE 7
Functional Analysis of DMPl Domains:
Introduction The ability of 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 32P 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. Notably, 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 32P-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 32P labeled bands were blocked by the addition of the cold BS2 probe.
Transfection TYPE Amino Acids of 32P-labeled Product SEQ ID NO:l Band
None None None No
DMPl wildtype 1-761 Yes
Ml EcoNI 1-661 Yes
M2 StuI 1-520 Yes
M3 Ncol 1-458 Yes
M4 BstBI 1-380 1 No
M5 Xbal and 87-761 Yes BstEII
M6 Bstll and 1-86; 170-761 No
Eco47-3
M7 Eco47-3 and 1-169; 238-761 No Sad
M8 Sad and 1-237; 381-761 No BstBI
M9 5' deletion 234-761 No
M10 BstEII and 87-458 Yes Ncol
Mi l K319E 1-318;E;320-761 Yes, but a very faint band
Next, the series of DMPl deletion mutants and the K319E point mutant were expressed in mammalian cells and in insect Sf9 cells (see Figure 10, Table 1) to determine the DMPl gene transactivation domain. Using a reporter gene (luciferase) programmed by an artificial DMPl -responsive promoter, sequences at the DMPl carboxy lterminus, namely amino acids 459 to 761 of SEQ ID NO: l were shown to be necessary for gene transactivation (Table 2). Elimination of these sequences did not effect DNA binding in an EMSA assay (Table 1) but resulted in a dramatic reduction of reporter gene transcription (Table 2). The extreme N-terminal sequence of DMPl can also contribute to transactivation (amino acids 1-86 of SEQ ID NO: l).
Table 2
The results of transfecting NIH-3T3 fibroblasts (10 ml cultures) with expression vectors (3μg/10ml) encoding murine wild-type DMPl , corresponding deletion mutations, or a point mutation of DMPl (K319E). The effects were measured either by determining the expression of luciferase by a luciferase reporter plasmid under the control of a DMP1- responsive promoter (pGL2BS2, 8μg/10ml), or as the percent of cells that incoφorate BrdU. Transactivation of luciferase reporter plasmids was normalized by arbitrarily setting the amount of luciferase activity determined in presence of an expression vector without an insert, to 1.0. Transfection efficiencies were normalized by the levels of secreted endocrine alkaline phosphatase assays. The cells were treated as described in Examples 7 and 8. The transfection products are as defined in Figure 10, Table 1.
Transfection Product Luciferase Activity %CeIIs BrdU Positive
None 1.0 80
DMPl 8.4 12
Ml 6.7 12
M2 3.6 20
M3 2.3 24
M4 1.0 52
M5 6.2 32
M6 1.0 48
M7 0.9 36
M8 1.1 54
M9 1.0 50
M10 1.1 56
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. In addition, 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.
EXAMPLE 8: DMPl Arrests Cell Cycle Progression in Gl Phase:
Introduction: Expression of high concentrations of the transcription factor, DMPl , in NIH-3T3 fibroblasts is shown to arrest the cell cycle in Gl phase, and to prevent the proliferating cells from replicating their chromosomal DNA. The effect is dependent upon the ability of DMPl to bind to cellular DNA, which indicates that genes negatively regulated by DMPl play an important role in cell cycle progression. The coexpression of the growth promoting Gl cyclins Dl , D2 or E can override the ability of DMPl to induce Gl arrest.
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 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 incoφoration and DMPl expression as described in the Materials and Methods.
Immunofluorescence showed that cells expressing DMPl did not incoφorate BrdU. Thus the nuclei of these cells were stained red, which indicates DMPl has been expressed, or alternatively, green, which indicates BrdU incoφoration has occurred (see MATERIALS and METHODS).
In contrast, cells expressing a DMPl point mutant in place of DMPl did not arrest cells in Gl . The DMPl point mutant, K319E, binds to DNA with a diminished affinity, if at all (Table 1). The cells expressing K319E DMPl also incoφorated BrdU thereby generating dual-labeled nuclei (red + green = yellow).
Furthermore, in nonstarved cells which were incubated in 10% FCS, 90% of the cells incorporated BrdU in the absence of DMPl transfection, whereas only 30% of the cells incoφorated BrdU when the cells were transfected with expression vectors containing DMPl. In the serum starved cells, 80% of the cells incoφorated BrdU in the absence of DMPl expression, whereas only 15% of the cells incoφorated BrdU. Co-transfection of cells with expression vectors containing DMPl and cyclins D2, or E hindered the ability of DMPl to induce cell cycle arrest, thereby overriding the inhibition of BrdU incoφoration due to DMPl (Table 3). Thus, DMPl blocks BrdU incoφoration less efficiently in the presence of 10% FCS than in serum starved cells.
The series of DMPl deletion mutants and the K319E point mutant were found to also effect the percentage of cells that incoφorated BrdU, though generally to a lesser extent than wildtype DMPl (Tables 2 and 3). Notably, however, Ml had an equivalent effect on BrdU incoφoration as the wildtype DMPl.
Table 3
The results of transfecting NIH-3T3 fibroblasts with expression vectors encoding murine wild-type DMPl, DMPl deletion mutants, or the point mutation K319E, on the percentage of cells that incoφorate BrdU. Starved (0.1 % FCS) or nonstarved (10% FCS) cells were labeled for 22 hours. M6, M8, and Mil are defined in Figure 10, Table 1.
Transfection Additive %Cells BrdU Positive Product in 0.1% FCS in 10% FCS
None None 80 90
DMPl None 15 30
DMPl cyclin D2 57 80
DMPl cyclin E 56 82
M6 None 47 44
M8 None 54 54
Mi l None 47 54
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 .
Table 4
Effect of potential antagonists and agonists on the DMPl transactivation of the expression of luciferase by a luciferase reporter plasmid under the control of a DMPl -responsive promoter. Transactivation of luciferase reporter plasmids was normalized by arbitrarily setting the amount of luciferase activity to determined in presence of an expression vector without an insert to 1.0.
DMPl Additive Co-additive Luciferase Activity
No None None 1.0
Yes None None 8.4
Yes cyclin Dl None 1.5
Yes cyclin D2 None 1.6
Yes cyclin D3 None 1.6
Yes cyclin A None 5.5
Yes cyclin E None 1.4
Yes cyclin H None 5.5
Yes CDK2 None 7.6
Yes CDK4 None 6.7
Yes P16 None 9.2
Yes P19 None 8.0
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.
EXAMPLE 10 Chromosomal Localization Of PI Clone 11098 Bv Fluorescence in situ Hybridization: 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 7q, a position which corresponds to 7q21. (Figure 11).
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12. Downing, J. R., C. W. Rettenmier, and C. J. Sherr. 1988. Ligand-induced tyrosine kinase activity of the colony stimulating factor- 1 receptor in a murine macrophage cell line. Mol Cell Biol 8: 1795-1799.
13. Downing, J. R., S. A. Shurtleff, and C. J. Sherr. 1991. Peptide antisera to human colony- stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase. Mol Cell Biol 1 1 :2489-2495.
14. Durfee, T., K. Becherer, P.-L. Chen, S.-H. Yeh, Y. Yang, A. Kilburn, W.-H. Lee, and S. J. Elledge. 1993. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes & Devel. 7:555-569. 15. Ewen, M. E., H. K. Sluss, C. J. Sherr, H. Matsushime, J. Kato, and D. M.
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16. Gabrielson, O. S. , A. Sentenac, and P. Fromageot. 1991. Specific DNA binding by c-myb: evidence for a double helix-turn-helix-related motif. Science 253:1140-1143. 17. Gonda, T. J., N. M. Gough, A. R. Dunn, and J. de Blaquiere. 1985. Nucleotide sequence of cDNA clones of the murine myb proto-oncogene. EMBO J 4:2003-2008.
18. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol
36:59-72. 19. Guan, K., C. W. Jenkins, Y. Li, M. A. Nichols, X. Wu, C. L. O'Keefe, A. G.
Matera, and Y. Xiong. 1994. Growth suppression by pi 8, a pl6INK4/MTSl_ and pl4INK4/MTS2_related CDK6 inhibitor, correlates with wild-type pRb function. Genes
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20. Hirai, H., M. F. Roussel, J. Kato, R. A. Ashmun, and C. J. Sherr. 1995. Novel INK4 proteins, pl9 and pl8, are specific inhibitors of cyclin D-dependent kinases CDK4 and CDK6. Mol Cell Biol. 15:2672-2681.
21. Inaba, T., L. H. Shapiro, T. Funabiki, A. E. Sinclair, B. G. Jones, R. A. Ashmun, and A. T. Look. 1994. DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein. Mol Biol Cell 14:3403-3413.
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24. Klempnauer, K. H., T. J. Gonda, and J. M. Bishop. 1982. Nucleotide sequence of the retro viral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced onco gene. Cell 31:453-463.
25. Klempnauer, K. H. and A. E. Sippel. 1987. The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain. EMBO J 6:2719-2725.
26. Koh, J., G. H. Enders, B. D. Dynlacht, and E. Harlow. 1995. Tumour-derived pl6 alleles encoding proteins defective in cell cycle inhibition. Nature 375:506-510.
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29. Lukas, J., H. Muller, J. Bartkova, D. Spitkovsky, A. A. Kjerulff, P. Jansen-Durr, M. Strauss, and J. Bartek. 1994. DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin Dl function in Gl . J Cell Biol 125:625-638.
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44. Quelle, D. E., R. A. Ashmun, S. A. Shurtleff, J. Kato, D. Bar-Sagi, M. F. Roussel, and C. J. Sherr. 1993. Overexpression of mouse D-type cyclins accelerates Gl phase in rodent fibroblasts. Genes & Devel 7: 1559-1571.
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The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.
Various publications in addition to the immediately foregoing are cited herein, the disclosures of which are incoφorated by reference in their entireties.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Hirai, Hiroshi Sherr, Charles
(ii) TITLE OF INVENTION: CYCLIN-D BINDING FACTOR, AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 27
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: David A. Jackson, Esq.
(B) STREET: 411 Hackensack Ave, Continental Plaza, 4th
Floor
(C) CITY: Hackensack
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07601
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jackson Esq., David A.
(B) REGISTRATION NUMBER: 26,742
(C) REFERENCE/DOCKET NUMBER: 1340-1-002 N
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-487-5800
(B) TELEFAX: 201-343-1684
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 761 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(v) FRAGMENT TYPE:
( i) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
Met Ser Thr Val Glu Glu Asp Ser Asp Thr Val Thr Val Glu Thr Val 1 5 10 15
Asn Ser Val Thr Phe Thr Gin Asp Thr Asp Gly Asn Leu lie Leu His 20 25 30
Cys Pro Gin Asn Asp Pro Asp Glu Val Asp Ser Glu Asp Ser Thr Glu 35 40 45
Pro Pro His Lys Arg Leu Cys Leu Ser Ser Glu Asp Asp Gin Ser lie 50 55 60
Asp Asp Ala Thr Pro Cys lie Ser Val Val Ala Leu Pro Leu Ser Glu 65 70 75 80
Asn Asp Gin Ser Phe Glu Val Thr Met Thr Ala Thr Thr Glu Val Ala 85 90 95
Asp Asp Glu Leu Ser Glu Gly Thr Val Thr Gin lie Gin lie Leu Gin 100 105 110
Asn Asp Gin Leu Asp Glu lie Ser Pro Leu Gly Thr Glu Glu Val Ser 115 120 125
Ala Val Ser Gin Ala Trp Phe Thr Thr Lys Glu Asp Lys Asp Ser Leu 130 135 140
Thr Asn Lys Gly His Lys Trp Lys Gin Gly Met Trp Ser Lys Glu Glu 145 150 155 160 lie Asp lie Leu Met Asn Asn lie Glu Arg Tyr Leu Lys Ala Arg Gly 165 170 175 lie Lys Asp Ala Thr Glu lie lie Phe Glu Met Ser Lys Asp Glu Arg 180 185 190
Lys Asp Phe Tyr Arg Thr lie Ala Trp Gly Leu Asn Arg Pro Leu Phe 195 200 205
Ala Val Tyr Arg Arg Val Leu Arg Met Tyr Asp Asp Arg Asn His Val 210 215 220
Gly Lys Tyr Thr Pro Glu Glu lie Glu Lys Leu Lys Glu Leu Arg lie 225 230 235 240
Lys His Gly Asn Asp Trp Ala Thr lie Gly Ala Ala Leu Gly Arg Ser 245 250 255
Ala Ser Ser Val Lys Asp Arg Cys Arg Leu Met Lys Asp Thr Cys Asn 260 265 270
Thr Gly Lys Trp Thr Glu Glu Glu Glu Lys Arg Leu Ala Glu Val Val 275 280 285
His Glu Leu Thr Ser Thr Glu Pro Gly Asp lie Val Thr Gin Gly Val 290 295 300
Ser Trp Ala Ala Val Ala Glu Arg Val Gly Thr Arg Ser Glu Lys Gin 305 310 315 320
Cys Arg Ser Lys Trp Leu Asn Tyr Leu Asn Trp Lys Gin Ser Gly Gly 325 330 335
Thr Glu Trp Thr Lys Glu Asp Glu lie Asn Leu lie Leu Arg lie Ala 340 345 350
Glu Leu Asp Val Ala Asp Glu Asn Asp lie Asn Trp Asp Leu Leu Ala 355 360 365
Glu Gly Trp Ser Ser Val Arg Ser Pro Gin Trp Leu Arg Ser Lys Trp 370 375 380
Trp Thr He Lys Arg Gin He Ala Asn His Lys Asp Val Ser Phe Pro 385 390 395 400
Val Leu He Lys Gly Leu Lys Gin Leu His Glu Asn Gin Lys Asn Asn 405 410 415
Pro Val Leu Leu Glu Asn Lys Ser Gly Ser Gly Val Pro Asn Ser Asn 420 425 430
Cys Asn Ser Ser Val Gin His Val Gin He Arg Val Ala Arg Leu Glu 435 440 445
Asp Asn Thr Ala He Ser Pro Ser Pro Met Ala Ala Leu Gin He Pro 450 455 460
Val Gin He Thr His Val Ser Ser Thr Asp Ser Pro Ala Ala Ser Ala 465 470 475 480
Asp Ser Glu Thr He Thr Leu Asn Ser Gly Thr Leu Gin Thr Phe Glu 485 490 495
He Leu Pro Ser Phe Pro Leu Gin Pro Thr Gly Thr Pro Gly Thr Tyr 500 505 510
Leu Leu Gin Thr Ser Ser Ser Gin Gly Leu Pro Leu Thr Leu Thr Thr 515 520 525
Asn Pro Thr Leu Thr Leu Ala Ala Ala Ala Pro Ala Ser Pro Glu Gin 530 535 540
He He Val His Ala Leu Ser Pro Glu His Leu Leu Asn Thr Ser Asp 545 550 555 560
Asn Val Thr Val Gin Cys His Thr Pro Arg Val He He Gin Thr Val 565 570 575
Ala Thr Glu Asp He Thr Ser Ser Leu Ser Gin Glu Glu Leu Thr Val 580 585 590
Asp Ser Asp Leu His Ser Ser Asp Phe Pro Glu Pro Pro Asp Ala Leu 595 600 605
Glu Ala Asp Thr Phe Pro Asp Glu He Pro Arg Pro Lys Met Thr He 610 615 620
Gin Pro Ser Phe Asn Asn Ala His Val Ser Lys Phe Ser Asp Gin Asn 625 630 635 640
Ser Thr Glu Leu Met Asn Ser Val Met Val Arg Thr Glu Glu Glu He 645 650 655
Ala Asp Thr Asp Leu Lys Gin Glu Glu Pro Pro Ser Asp Leu Ala Ser 660 665 670
Ala Tyr Val Thr Glu Asp Leu Glu Ser Pro Thr He Val His Gin Val 675 680 685
His Gin Thr He Asp Asp Glu Thr He Leu He Val Pro Ser Pro His 690 695 700
Gly Phe He Gin Ala Ser Asp Val He Asp Thr Glu Ser Val Leu Pro 705 710 715 720
Leu Thr Thr Leu Thr Asp Pro He Phe Gin His His Gin Glu Ala Ser 725 730 735
Asn He He Gly Ser Ser Leu Gly Ser Pro Val Ser Glu Asp Ser Lys 740 745 750
Asp Val Glu Asp Leu Val Asn Cys His 755 760
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2903 base pairs
(B) TYPE: nucleic acid <C) STRANDEDNESS: double (D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus rausculus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 248..2533
(D) OTHER INFORMATION: /codon_start= 248 /product= "DMP-1"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GAATCCGGCT CGCTCACCCC AGCTGCAGCC ACTCTCTCCC GCGGCTGCTT CCTCCATCCT 60
GGTATTTTTT GGAGCCTCCA TCCTGGTTCT TCCAAAGTGC CCGGACCCAA AACAGGAAAG 120
GATCACAGAT GCACAAGCAT GGAGGAGAAG CAGTCTGGTT AACGTGAGTG ATGCTGCTGG 180
CCGAAGCACA GAGGTGGGAT TCTATGGGAA GGCCTGTAGC TAATCCACCT GTGGTCTAGA 240
TTTGAGTATG AGCACAGTTG AAGAGGATTC TGACACAGTA ACAGTAGAAA CTGTGAACTC 300
TGTGACGTTT ACTCAGGACA CGGACGGGAA TCTCATTCTT CATTGCCCTC AGAATGACCC 360
TGATGAAGTA GACTCAGAAG ACAGTACTGA ACCTCCACAT AAGAGGCTTT GTTTGTCCTC 420
TGAGGATGAT CAAAGCATTG ATGACGCTAC GCCATGCATA TCAGTCGTGG CACTCCCACT 480
TTCAGAAAAT GATCAGAGCT TTGAGGTGAC CATGACGGCA ACTACAGAGG TGGCAGATGA 540
TGAACTTTCT GAGGGAACTG TGACACAAAT TCAGATTTTA CAGAATGATC AACTAGATGA 600
AATATCTCCA TTGGGTACTG AGGAAGTCTC AGCAGTTAGC CAAGCGTGGT TTACAACTAA 660
AGAAGATAAG GATTCTCTCA CTAACAAAGG ACATAAATGG AAGCAGGGGA TGTGGTCCAA 720
GGAAGAAATT GATATTTTAA TGAACAACAT CGAGCGCTAT CTGAAGGCTC GCGGAATAAA 780
AGATGCTACA GAAATCATCT TTGAGATGTC AAAAGACGAA AGAAAAGATT TCTACAGGAC 840
TATAGCGTGG GGGCTGAACC GGCCTTTGTT TGCAGTTTAT AGAAGAGTGC TGCGCATGTA 900
TGATGACAGG AACCATGTGG GAAAATACAC TCCTGAAGAG ATCGAGAAGC TCAAGGAGCT 960
CCGGATAAAA CACGGCAATG ACTGGGCAAC AATAGGGGCG GCCCTAGGAA GAAGCGCCTC 1020
TTCTGTCAAA GACCGCTGCC GGCTGATGAA GGATACCTGC AACACAGGGA AATGGACAGA 1080
AGAAGAAGAA AAGAGACTTG CAGAGGTAGT TCATGAATTA ACAAGCACGG AGCCAGGTGA 1140
CATCGTCACA CAGGGTGTGT CTTGGGCAGC TGTAGCTGAA AGAGTGGGTA CCCGCTCAGA 1200
AAAGCAATGC CGTTCTAAAT GGCTCAACTA CCTGAACTGG AAGCAGAGTG GGGGTACTGA 1260
ATGGACCAAG GAAGATGAAA TCAATCTCAT CCTAAGGATA GCTGAGCTTG ATGTGGCCGA 1320
TGAAAATGAC ATAAACTGGG ATCTTTTAGC TGAAGGATGG AGCAGTGTCC GTTCACCACA 1380
GTGGCTTCGA AGTAAATGGT GGACCATCAA AAGGCAAATT GCAAACCATA AGGATGTTTC 1440
ATTTCCTGTC CTAATAAAAG GTCTTAAACA GTTACATGAG AACCAAAAAA ACAACCCAGT 1500
GCTTTTGGAG AATAAATCAG GATCTGGAGT TCCAAACAGT AATTGCAATT CCAGTGTACA 1560
GCATGTTCAG ATCAGAGTCG CCCGCTTGGA AGATAATACA GCCATCTCTC CAAGCCCCAT 1620
GGCAGCGTTG CAGATTCCAG TCCAGATCAC CCACGTCTCT TCAACAGACT CCCCTGCTGC 1680
TTCTGCCGAC TCAGAAACAA TCACACTAAA CAGTGGAACA CTACAAACAT TTGAGATTCT 1740
TCCATCTTTT CCATTACAGC CCACTGGTAC TCCAGGCACC TACCTTCTTC AAACAAGCTC 1800
AAGTCAAGGC CTTCCCCTAA CACTGACCAC AAATCCCACA CTAACCCTGG CAGCTGCTGC 1860
TCCTGCTTCT CCTGAACAGA TCATTGTTCA TGCTTTATCC CCAGAACATT TGTTGAACAC 1920
AAGCGATAAT GTCACGGTAC AATGTCACAC ACCAAGAGTC ATCATTCAGA CGGTAGCTAC 1980
AGAGGACATC ACTTCTTCAT TATCCCAAGA GGAACTGACA GTTGATAGTG ATCTTCATTC 2040
ATCTGATTTC CCTGAGCCTC CAGATGCACT AGAAGCAGAC ACTTTCCCAG ACGAAATTCC 2100
TCGGCCTAAG ATGACTATAC AACCATCATT TAATAATGCT CATGTATCTA AATTCAGCGA 2160
CCAAAATAGC ACAGAACTGA TGAACAGTGT CATGGTCAGA ACAGAGGAAG AAATTGCCGA 2220
CACTGACCTT AAGCAGGAAG AACCGCCGTC TGACTTAGCC AGTGCTTATG TTACTGAGGA 2280
TTTAGAGTCT CCCACCATAG TGCACCAAGT TCATCAGACA ATTGATGATG AAACAATACT 2340
TATCGTTCCT TCACCTCATG GCTTTATCCA GGCATCTGAT GTTATAGATA CTGAATCTGT 2400
CTTGCCTTTG ACAACACTAA CAGATCCAAT ATTCCAGCAT CATCAGGAAG CATCAAATAT 2460
AATTGGATCA TCTTTGGGCA GTCCTGTTTC TGAAGACTCA AAGGATGTTG AGGACTTAGT 2520
AAACTGTCAC TAGATTATTA GAAACAGGTA CTTCAAGAAG CCACATTGTG ACTACATTGT 2580
CTCCAAAGAA AGGAGCCATC CCAGGAGTTG TGGTTTGCCA TTCCTCTGGC TTGTACTTAG 2640
CTGCCATGCT TAAGCCATGC ACATTGTTGC TGCTGTTACT TTTACCTCCT TCTCAGTAGA 2700
TCATCTAGGG TCCAATTTTA TAACAGTTGT TATGATGGAG GATAGGAAGT GTGAATTGCC 2760
CAGACTTGTT AGGTTTTATG TCAAGAGGGA GTTGCAGTCA CTGCAGCTAC TTATCATCAC 2820
CAGAGCTTAA CTACTCTGGT TTAAATATAA GTAGTAATAG TGATCTCTGC AGTTAGACAC 2880
ACAGCTCTCG TCCAGACTCA AGC 2903
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2903 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
GAAUCCGGCU CGCUCACCCC AGCUGCAGCC ACUCUCUCCC GCGGCUGCUU CCUCCAUCCU 60
GGUAUUUUUU GGAGCCUCCA UCCUGGUUCU UCCAAAGUGC CCGGACCCAA AACAGGAAAG 120
GAUCACAGAU GCACAAGCAU GGAGGAGAAG CAGUCUGGUU AACGUGAGUG AUGCUGCUGG 180
CCGAAGCACA GAGGUGGGAU UCUAUGGGAA GGCCUGUAGC UAAUCCACCU GUGGUCUAGA 240
UUUGAGUAUG AGCACAGUUG AAGAGGAUUC UGACACAGUA ACAGUAGAAA CUGUGAACUC 300
UGUGACGUUU ACUCAGGACA CGGACGGGAA UCUCAUUCUU CAUUGCCCUC AGAAUGACCC 360
UGAUGAAGUA GACUCAGAAG ACAGUACUGA ACCUCCACAU AAGAGGCUUU GUUUGUCCUC 420
UGAGGAUGAU CAAAGCAUUG AUGACGCUAC GCCAUGCAUA UCAGUCGUGG CACUCCCACU 480
UUCAGAAAAU GAUCAGAGCU UUGAGGUGAC CAUGACGGCA ACUACAGAGG UGGCAGAUGA 540
UGAACUUUCU GAGGGAACUG UGACACAAAU UCAGAUUUUA CAGAAUGAUC AACUAGAUGA 600
AAUAUCUCCA UUGGGUACUG AGGAAGUCUC AGCAGXJUAGC CAAGCGUGGU UUACAACUAA 660
AGAAGAUAAG GAUUCUCUCA CUAACAAAGG ACAUAAAUGG AAGCAGGGGA UGUGGUCCAA 720
GGAAGAAAUU GAUAUUUUAA UGAACAACAU CGAGCGCUAU CUGAAGGCUC GCGGAAUAAA 780
AGAUGCUACA GAAAUCAUCU UUGAGAUGUC AAAAGACGAA AGAAAAGAUU UCUACAGGAC 840
UAUAGCGUGG GGGCUGAACC GGCCUUUGUU UGCAGUUUAU AGAAGAGUGC UGCGCAUGUA 900
UGAUGACAGG AACCAUGUGG GAAAAUACAC UCCUGAAGAG AUCGAGAAGC UCAAGGAGCU 960
CCGGAUAAAA CACGGCAAUG ACUGGGCAAC AAUAGGGGCG GCCCUAGGAA GAAGCGCCUC 1020
UUCUGUCAAA GACCGCUGCC GGCUGAUGAA GGAUACCUGC AACACAGGGA AAUGGACAGA 1080
AGAAGAAGAA AAGAGACUUG CAGAGGUAGU UCAUGAAUUA ACAAGCACGG AGCCAGGUGA 1140
CAUCGUCACA CAGGGUGUGU CUUGGGCAGC UGUAGCUGAA AGAGUGGGUA CCCGCUCAGA 1200
AAAGCAAUGC CGUUCUAAAU GGCUCAACUA CCUGAACUGG AAGCAGAGUG GGGGUACUGA 1260
AUGGACCAAG GAAGAUGAAA UCAAUCUCAU CCUAAGGAUA GCUGAGCUUG AUGUGGCCGA 1320
UGAAAAUGAC AUAAACUGGG AUCUUUUAGC UGAAGGAUGG AGCAGUGUCC GUUCACCACA 1380
GUGGCXJUCGA AGUAAAUGGU GGACCAUCAA AAGGCAAAXJU GCAAACCAUA AGGAUGXJUUC 1440
AUUUCCUGUC CUAAUAAAAG GUCXJUAAACA GXJUACAUGAG AACCAAAAAA ACAACCCAGU 1500
GCUUXJUGGAG AAUAAAUCAG GAUCUGGAGU UCCAAACAGU AAUUGCAAUU CCAGUGUACA 1560
GCAUGXJUCAG AUCAGAGUCG CCCGCUUGGA AGAUAAUACA GCCAUCUCUC CAAGCCCCAU 1620
GGCAGCGUUG CAGAXJUCCAG UCCAGAUCAC CCACGUCUCU UCAACAGACU CCCCUGCUGC 1680
XJUCUGCCGAC UCAGAAACAA UCACACUAAA CAGUGGAACA CUACAAACAU UUGAGAUUCU 1740
UCCAUCUUUU CCAUUACAGC CCACUGGUAC UCCAGGCACC UACCUUCXJUC AAACAAGCUC 1800
AAGUCAAGGC CUUCCCCUAA CACUGACCAC AAAUCCCACA CUAACCCUGG CAGCUGCUGC 1860
UCCUGCUUCU CCUGAACAGA UCAUUGUUCA UGCUUUAUCC CCAGAACAUU UGUUGAACAC 1920
AAGCGAUAAU GUCACGGUAC AAUGUCACAC ACCAAGAGUC AUCAXJUCAGA CGGUAGCUAC I960
AGAGGACAUC ACUUCUUCAU UAUCCCAAGA GGAACUGACA GUUGAUAGUG AUCUUCAUUC 2040
AUCUGAUUUC CCUGAGCCUC CAGAUGCACU AGAAGCAGAC ACUXJUCCCAG ACGAAAXJUCC 2100
UCGGCCUAAG AUGACUAUAC AACCAUCAUU UAAUAAUGCU CAUGUAUCUA AAUUCAGCGA 2160
CCAAAAUAGC ACAGAACUGA UGAACAGUGU CAUGGUCAGA ACAGAGGAAG AAAUUGCCGA 2220
CACUGACCUU AAGCAGGAAG AACCGCCGUC UGACUUAGCC AGUGCUUAUG UUACUGAGGA 2280
UUUAGAGUCU CCCACCAUAG UGCACCAAGU UCAUCAGACA AUUGAUGAUG AAACAAUACU 2340
UAUCGUUCCU UCACCUCAUG GCUUUAUCCA GGCAUCUGAU GUUAUAGAUA CUGAAUCUGU 2400
CUUGCCUUUG ACAACACUAA CAGAUCCAAU AUUCCAGCAU CAUCAGGAAG CAUCAAAUAU 2460
AAUUGGAUCA UCUUUGGGCA GUCCUGUUUC UGAAGACUCA AAGGAUGUUG AGGACUUAGU 2520
AAACUGUCAC UAGAUUAUUA GAAACAGGUA CUUCAAGAAG CCACAUUGUG ACUACAUUGU 2580
CUCCAAAGAA AGGAGCCAUC CCAGGAGXJUG UGGUUUGCCA UUCCUCUGGC UUGUACUUAG 2640
CUGCCAUGCU UAAGCCAUGC ACAUUGXJUGC UGCUGUUACU UUUACCUCCU UCUCAGUAGA 2700
UCAUCUAGGG UCCAAUUUUA UAACAGUUGU UAUGAUGGAG GAUAGGAAGU GUGAAUUGCC 2760
CAGACUUGXJU AGGXJXJJUAUG UCAAGAGGGA GUUGCAGUCA CUGCAGCUAC UUAUCAUCAC 2820
CAGAGCUUAA CUACUCUGGU UUAAAUAUAA GUAGUAAUAG UGAUCUCUGC AGUUAGACAC 2880
ACAGCUCUCG UCCAGACUCA AGC 2903 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 156 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp Glu Lys Leu Lys Lys 1 5 10 15
Leu Val Glu Gin Asn Gly Thr Asp Asp Trp Lys Val lie Ala Asn Tyr 20 25 30
Leu Pro Asn Arg Thr Asp Val Gin Cys Gin His Arg Trp Gin Lys Val 35 40 45
Leu Asn Pro Glu Leu lie Lys Gly Pro Trp Thr Lys Glu Glu Asp Gin 50 55 60
Arg Val lie Lys Leu Val Gin Lys Tyr Gly Pro Lys Arg Trp Ser Val 65 70 75 80 lie Ala Lys His Leu Lys Gly Arg lie Gly Lys Gin Cys Arg Glu Arg 85 90 95
Trp His Asn His Leu Asn Pro Glu Val Lys Lys Thr Ser Trp Thr Glu 100 105 110
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
Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys Val 145 150 155
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECXJLE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : CGCGGATCCT GCAGCTCGAG 20
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECXJLE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer" (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : TGCTCTAGAA GCTTGTCGAC 20
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
Ser Pro Xaa Glx 1
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Lys Gin Cys Arg Xaa Xaa Trp Xaa Asn 1 5
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
Leu Xaa Cys Xaa Glu 1 5
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor BS1"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AATTGACCCG GATGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO : 11 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor BS2"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AATTGACCCG TATGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO : 12 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor Ml"
(iii) HYPOTHETICAL: NO
(xi) SEQXJENCE DESCRIPTION: SEQ ID NO : 12 : AATTGACCCT GCGGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor M2"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AATTGATTTG GATGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor M3"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AATTGACCCG GAAGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "probe/competitor M4"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: AATTGACCAG GATGTAGGTA CGC 23
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Val Thr Met Thr Ala Thr Thr Glu Val Ala Asp Asp Glu Leu Ser Glu 1 5 10 15
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
Phe Thr Thr Lys Glu Asp Lys Asp Ser Leu Thr Asn Lys Gly His Lys 50 55 60
Trp Lys Gin Gly Met Trp Ser Lys Glu Glu lie Asp lie Leu Met Asn 65 70 75 80
Asn lie Glu Arg Tyr Leu Lys Ala Arg Gly lie Lys Asp Ala Thr Glu 85 90 95 lie lie Phe Glu Met Ser Lys Asp Glu Arg Lys Asp Phe Tyr Arg Thr 100 105 110 lie Ala Trp Gly Leu Asn Arg Pro Leu Phe Ala Val Tyr Arg Arg Val 115 120 125
Leu Arg Met Tyr Asp Asp Arg Asn His Val Gly Lys Tyr Thr Pro Glu 130 135 140
Glu lie Glu Lys Leu Lys Glu Leu Arg lie Lys His Gly Asn Asp Trp 145 150 155 160
Ala Thr lie Gly Ala Ala Leu Gly Arg Ser Ala Ser Ser Val Lys Asp 165 170 175
Arg Cys Arg Leu Met Lys Asp Thr Cys Asn Thr Gly Lys Trp Thr Glu 180 185 190
Glu Glu Glu Lys Arg Leu Ala Glu Val Val His Glu Leu Thr Ser Thr 195 200 205
Glu Pro Gly Asp lie Val Thr Gin Gly Val Ser Trp Ala Ala Val Ala 210 215 220
Glu Arg Val Gly Thr Arg Ser Glu Lys Gin Cys Arg Ser Lys Trp Leu 225 230 235 240
Asn Tyr Leu Asn Trp Lys Gin Ser Gly Gly Thr Glu Trp Thr Lys Glu 245 250 255
Asp Glu lie Asn Leu lie Leu Arg lie Ala Glu Leu Asp Val Ala Asp 260 265 270
Glu Asn Asp lie Asn Trp Asp Leu Leu Ala Glu Gly Trp Ser Ser Val 275 280 285
Arg Ser Pro Gin Trp Leu Arg Ser Lys Trp Trp Thr lie Lys Arg Gin 290 295 300 lie Ala Asn His Lys Asp Val Ser Phe Pro Val Leu lie Lys Gly Leu 305 310 315 320
Lys Gin Leu His Glu Asn Gin Lys Asn Asn Pro Val Leu Leu Glu Asn 325 330 335
Lys Ser Gly Ser Gly Val Pro Asn Ser Asn Cys Asn Ser Ser Val Gin 340 345 350
His Val Gin lie Arg Val Ala Arg Leu Glu Asp Asn Thr Ala lie Ser 355 360 365
Pro Ser Pro Met 370
(2) INFORMATION FOR SEQ ID NO: 17:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1116 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY : 1inear
(ll) MOLECXJLE TYPE: cDNA
(ill) HYPOTHETICAL: NO
(vi ) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
( l) SEQXJENCE DESCRIPTION: SEQ ID NO: 17-
GTGACCATGA CGGCAACTAC AGAGGTGGCA GATGATGAAC TTTCTGAGGG AACTGTGACA 60
CAAATTCAGA TTTTACAGAA TGATCAACTA GATGAAATAT CTCCATTGGG TACTGAGGAA 120
GTCTCAGCAG TTAGCCAAGC GTGGTTTACA ACTAAAGAAG ATAAGGATTC TCTCACTAAC 180
AAAGGACATA AATGGAAGCA GGGGATGTGG TCCAAGGAAG AAATTGATAT TTTAATGAAC 240
AACATCGAGC GCTATCTGAA GGCTCGCGGA ATAAAAGATG CTACAGAAAT CATCTTTGAG 300
ATGTCAAAAG ACGAAAGAAA AGATTTCTAC AGGACTATAG CGTGGGGGCT GAACCGGCCT 360
TTGTTTGCAG TTTATAGAAG AGTGCTGCGC ATGTATGATG ACAGGAACCA TGTGGGAAAA 420
TACACTCCTG AAGAGATCGA GAAGCTCAAG GAGCTCCGGA TAAAACACGG CAATGACTGG 480
GCAACAATAG GGGCGGCCCT AGGAAGAAGC GCCTCTTCTG TCAAAGACCG CTGCCGGCTG 540
ATGAAGGATA CCTGCAACAC AGGGAAATGG ACAGAAGAAG AAGAAAAGAG ACTTGCAGAG 600
GTAGTTCATG AATTAACAAG CACGGAGCCA GGTGACATCG TCACACAGGG TGTGTCTTGG 660
GCAGCTGTAG CTGAAAGAGT GGGTACCCGC TCAGAAAAGC AATGCCGTTC TAAATGGCTC 720
AACTACCTGA ACTGGAAGCA GAGTGGGGGT ACTGAATGGA CCAAGGAAGA TGAAATCAAT 780
CTCATCCTAA GGATAGCTGA GCTTGATGTG GCCGATGAAA ATGACATAAA CTGGGATCTT 840
TTAGCTGAAG GATGGAGCAG TGTCCGTTCA CCACAGTGGC TTCGAAGTAA ATGGTGGACC 900
ATCAAAAGGC AAATTGCAAA CCATAAGGAT GTTTCATTTC CTGTCCTAAT AAAAGGTCTT 960
AAACAGTTAC ATGAGAACCA AAAAAACAAC CCAGTGCTTT TGGAGAATAA ATCAGGATCT 1020
GGAGTTCCAA ACAGTAATTG CAATTCCAGT GTACAGCATG TTCAGATCAG AGTCGCCCGC 1080
TTGGAAGATA ATACAGCCAT CTCTCCAAGC CCCATG 1116 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 303 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: C-terminal
(vi ) ORIGINAL SOXJRCE :
(A) ORGANISM: Mus musculus
(xi) SEQXJENCE DESCRIPTION: SEQ ID NO:18:
Ala Ala Leu Gin He Pro Val Gin He Thr His Val Ser Ser Thr Asp 1 5 10 15
Ser Pro Ala Ala Ser Ala Asp Ser Glu Thr He Thr Leu Asn Ser Gly 20 25 30
Thr Leu Gin Thr Phe Glu He Leu Pro Ser Phe Pro Leu Gin Pro Thr 35 40 45
Gly Thr Pro Gly Thr Tyr Leu Leu Gin Thr Ser Ser Ser Gin Gly Leu 50 55 60
Pro Leu Thr Leu Thr Thr Asn Pro Thr Leu Thr Leu Ala Ala Ala Ala 65 70 75 80
Pro Ala Ser Pro Glu Gin He He Val His Ala Leu Ser Pro Glu His 85 90 95
Leu Leu Asn Thr Ser Asp Asn Val Thr Val Gin Cys His Thr Pro Arg 100 105 110
Val He He Gin Thr Val Ala Thr Glu Asp He Thr Ser Ser Leu Ser 115 120 125
Gin Glu Glu Leu Thr Val Asp Ser Asp Leu His Ser Ser Asp Phe Pro 130 135 140
Glu Pro Pro Asp Ala Leu Glu Ala Asp Thr Phe Pro Asp Glu He Pro 145 150 155 160
Arg Pro Lys Met Thr He Gin Pro Ser Phe Asn Asn Ala His Val Ser
165 170 175
Lys Phe Ser Asp Gin Asn Ser Thr Glu Leu Met Asn Ser Val Met Val 180 185 190
Arg Thr Glu Glu Glu He Ala Asp Thr Asp Leu Lys Gin Glu Glu Pro 195 200 205
Pro Ser Asp Leu Ala Ser Ala Tyr Val Thr Glu Asp Leu Glu Ser Pro 210 215 220
Thr He Val His Gin Val His Gin Thr He Asp Asp Glu Thr He Leu 225 230 235 240
He Val Pro Ser Pro His Gly Phe He Gin Ala Ser Asp Val He Asp 245 250 255
Thr Glu Ser Val Leu Pro Leu Thr Thr Leu Thr Asp Pro He Phe Gin 260 265 270
His His Gin Glu Ala Ser Asn He He Gly Ser Ser Leu Gly Ser Pro 275 280 285
Val Ser Glu Asp Ser Lys Asp Val Glu Asp Leu Val Asn Cys His 290 295 300
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 909 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECXJLE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi ) ORIGINAL SOXJRCE :
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GCAGCGTTGC AGATTCCAGT CCAGATCACC CACGTCTCTT CAACAGACTC CCCTGCTGCT 60
TCTGCCGACT CAGAAACAAT CACACTAAAC AGTGGAACAC TACAAACATT TGAGATTCTT 120
CCATCTTTTC CATTACAGCC CACTGGTACT CCAGGCACCT ACCTTCTTCA AACAAGCTCA 180
AGTCAAGGCC TTCCCCTAAC ACTGACCACA AATCCCACAC TAACCCTGGC AGCTGCTGCT 240
CCTGCTTCTC CTGAACAGAT CATTGTTCAT GCTTTATCCC CAGAACATTT GTTGAACACA 300
AGCGATAATG TCACGGTACA ATGTCACACA CCAAGAGTCA TCATTCAGAC GGTAGCTACA 360
GAGGACATCA CTTCTTCATT ATCCCAAGAG GAACTGACAG TTGATAGTGA TCTTCATTCA 420
TCTGATTTCC CTGAGCCTCC AGATGCACTA GAAGCAGACA CTTTCCCAGA CGAAATTCCT 480
CGGCCTAAGA TGACTATACA ACCATCATTT AATAATGCTC ATGTATCTAA ATTCAGCGAC 540
CAAAATAGCA CAGAACTGAT GAACAGTGTC ATGGTCAGAA CAGAGGAAGA AATTGCCGAC 600
ACTGACCTTA AGCAGGAAGA ACCGCCGTCT GACTTAGCCA GTGCTTATGT TACTGAGGAT 660
TTAGAGTCTC CCACCATAGT GCACCAAGTT CATCAGACAA TTGATGATGA AACAATACTT 720
ATCGTTCCTT CACCTCATGG CTTTATCCAG GCATCTGATG TTATAGATAC TGAATCTGTC 780
TTGCCTTTGA CAACACTAAC AGATCCAATA TTCCAGCATC ATCAGGAAGC ATCAAATATA 840
ATTGGATCAT CTTTGGGCAG TCCTGTTTCT GAAGACTCAA AGGATGTTGA GGACTTAGTA 900
AACTGTCAC 909 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Ser Thr Val Glu Glu Asp Ser Asp Thr Val Thr Val Glu Thr Val 1 5 10 15
Asn Ser Val Thr Phe Thr Gin Asp Thr Asp Gly Asn Leu He Leu His 20 25 30
Cys Pro Gin Asn Asp Pro Asp Glu Val Asp Ser Glu Asp Ser Thr Glu 35 40 45
Pro Pro His Lys Arg Leu Cys Leu Ser Ser Glu Asp Asp Gin Ser He 50 55 60
Asp Asp Ala Thr Pro Cys He Ser Val Val Ala Leu Pro Leu Ser Glu 65 70 75 80
Asn Asp Gin Ser Phe Glu 85
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 258 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus
(xi) SEQXJENCE DESCRIPTION: SEQ ID NO: 21:
ATGAGCACAG TTGAAGAGGA TTCTGACACA GTAACAGTAG AAACTGTGAA CTCTGTGACG 60
TTTACTCAGG ACACGGACGG GAATCTCATT CTTCATTGCC CTCAGAATGA CCCTGATGAA 120
GTAGACTCAG AAGACAGTAC TGAACCTCCA CATAAGAGGC TTTGTTTGTC CTCTGAGGAT 180
GATCAAAGCA TTGATGACGC TACGCCATGC ATATCAGTCG TGGCACTCCC ACTTTCAGAA 240
AATGATCAGA GCTTTGAG 258 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQXJENCE CHARACTERISTICS:
(A) LENGTH: 223 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECXJLE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(vi ) ORIGINAL SOXJRCE :
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Ser Thr Val Glu Glu Asp Ser Asp Thr Val Thr Val Glu Thr Val 1 5 10 15
Asn Ser Val Thr Phe Thr Gin Asp Thr Asp Gly Asn Leu He Leu His 20 25 30
Cys Pro Gin Asn Asp Pro Asp Glu Val Asp Ser Glu Asp Ser Thr Glu 35 40 45
Pro Pro His Lys Arg Leu Cys Leu Ser Ser Glu Asp Asp Gin Ser He
50 55 60
Asp Asp Ala Thr Pro Cys He Ser Val Val Ala Leu Pro Leu Ser Glu 65 70 75 80
Asn Asp Gin Ser Phe Glu Val Thr Met Thr Ala Thr Thr Glu Val Ala 85 90 95
Asp Asp Glu Leu Ser Glu Gly Thr Val Thr Gin He Gin He Leu Gin 100 105 110
Asn Asp Gin Leu Asp Glu He Ser Pro Leu Gly Thr Glu Glu Val Ser 115 120 125
Ala Val Ser Gin Ala Trp Phe Thr Thr Lys Glu Asp Lys Asp Ser Leu 130 135 140
Thr Asn Lys Gly His Lys Trp Lys Gin Gly Met Trp Ser Lys Glu Glu 145 150 155 160
He Asp He Leu Met Asn Asn He Glu Arg Tyr Leu Lys Ala Arg Gly 165 170 175
He Lys Asp Ala Thr Glu He He Phe Glu Met Ser Lys Asp Glu Arg 180 185 190
Lys Asp Phe Tyr Arg Thr He Ala Trp Gly Leu Asn Arg Pro Leu Phe 195 200 205
Ala Val Tyr Arg Arg Val Leu Arg Met Tyr Asp Asp Arg Asn His 210 215 220
(2) INFORMATION FOR SEQ ID NO: 23
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 669 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY, linear
( l l ) MOLECXJLE TYPE - cDNA
( l i l ) HYPOTHETI CAL NO
(vi ) ORIGINAL SOXJRCE .
(A) ORGANISM: Mus musculus
(xi) SEQXJENCE DESCRIPTION: SEQ ID NO: 23:
ATGAGCACAG TTGAAGAGGA TTCTGACACA GTAACAGTAG AAACTGTGAA CTCTGTGACG 60
TTTACTCAGG ACACGGACGG GAATCTCATT CTTCATTGCC CTCAGAATGA CCCTGATGAA 120
GTAGACTCAG AAGACAGTAC TGAACCTCCA CATAAGAGGC TTTGTTTGTC CTCTGAGGAT 180
GATCAAAGCA TTGATGACGC TACGCCATGC ATATCAGTCG TGGCACTCCC ACTTTCAGAA 240
AATGATCAGA GCTTTGAGGT GACCATGACG GCAACTACAG AGGTGGCAGA TGATGAACTT 300
TCTGAGGGAA CTGTGACACA AATTCAGATT TTACAGAATG ATCAACTAGA TGAAATATCT 360
CCATTGGGTA CTGAGGAAGT CTCAGCAGTT AGCCAAGCGT GGTTTACAAC TAAAGAAGAT 420
AAGGATTCTC TCACTAACAA AGGACATAAA TGGAAGCAGG GGATGTGGTC CAAGGAAGAA 480
ATTGATATTT TAATGAACAA CATCGAGCGC TATCTGAAGG CTCGCGGAAT AAAAGATGCT 540
ACAGAAATCA TCTTTGAGAT GTCAAAAGAC GAAAGAAAAG ATTTCTACAG GACTATAGCG 600
TGGGGGCTGA ACCGGCCTTT GTTTGCAGTT TATAGAAGAG TGCTGCGCAT GTATGATGAC 660
AGGAACCAT 669 (2) INFORMATION FOR SEQ ID NO: 24-
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 262 ammo acids
(B) TYPE: amino ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY- linear
(ii) MOLECULE TYPE peptide (ill) HYPOTHETICAL. NO
( v) FRAGMENT TYPE : C- termmal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 24 :
Ser Phe His Leu Gin Pro Thr Gly Thr Pro Gly Thr Tyr Leu Leu Gin 1 5 10 15
Thr Ser Ser Ser Gin Gly Leu Pro Leu Thr Leu Thr Ala Ser Pro Thr 20 25 30
Val Thr Leu Thr Ala Ala Ala Pro Ala Ser Pro Glu Gin He He Val 35 40 45
His Ala Leu Ser Pro Glu His Leu Leu Asn Thr Ser Asp Asn Val Thr 50 55 60
Val Gin Cys His Thr Pro Arg Val He He Gin Thr Val Ala Thr Glu 65 70 75 80
Asp He Thr Ser Ser He Ser Gin Ala Glu Leu Thr Val Asp Ser Asp 85 90 95
He Gin Ser Ser Asp Phe Pro Glu Pro Pro Asp Ala Leu Glu Ala Asp 100 105 110
Thr Phe Pro Asp Glu He His His Pro Lys Met Thr Val Glu Pro Ser 115 120 125
Phe Asn Asp Ala His Val Ser Lys Phe Ser Asp Gin Asn Ser Thr Glu 130 135 140
Leu Met Asn Ser Val Met Val Arg Thr Glu Glu Glu He Ser Asp Thr 145 150 155 160
Asp Leu Lys Gin Glu Glu Ser Pro Ser Asp Leu Ala Ser Ala Tyr Val 165 170 175
Thr Glu Gly Leu Glu Ser Pro Thr He Glu Glu Gin Val Asp Gin Thr 180 185 190
He Asp Asp Glu Thr He Leu He Val Pro Ser Pro His Gly Phe He 195 200 205
Gin Ala Ser Asp Val He Asp Thr Glu Ser Val Leu Pro Leu Thr Thr 210 215 220
Leu Thr Asp Pro He Leu Gin His His Gin Glu Glu Ser Asn He He 225 230 235 240
Gly Ser Ser Leu Gly Ser Pro Val Ser Glu Asp Ser Lys Asp Val Glu 245 250 255
Asp Leu Val Asn Cys His 260
(2)' INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 800 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CCTCTTTCCA TCTACAGCCC ACTGGCACTC CAGGCACCTA CCTACTTCAA ACAAGCTCAA 60
GCCAAGGCCT TCCCCTAACT CTGACTGCTA GTCCCACAGT AACCCTGACA GCTGCTGCTC 120
CTGCTTCTCC TGAACAGATT ATTGTTCATG CTTTATCCCC AGAACATTTG TTGAACACAA 180
GTGATAATGT TACAGTGCAG TGTCACACAC CAAGAGTCAT CATTCAGACT GTTGCCACAG 240
AGGACATCAC TTCTTCCATA TCCCAAGCAG AACTGACCGT CGATAGTGAT ATTCAGTCAT 300
CTGATTTTCC TGAGCCTCCA GACGCCCTAG AAGCAGACAC TTTCCCAGAT GAAATTCATC 360
ACCCTAAGAT GACTGTGGAG CCATCATTTA ATGATGCTCA TGTATCCAAA TTCAGTGACC 420
AAAATAGCAC AGAACTGATG AATAGTGTTA TGGTCAGAAC AGAAGAAGAA ATCTCTGACA 480
CCGACCTTAA ACAAGAGGAA TCACCCTCTG ATTTAGCCAG TGCTTATGTT ACTGAGGGTT 540
TAGAGTCTCC CACTATAGAA GAACAAGTTG ATCAAACAAT TGATGATGAA ACAATACTTA 600
TCGTTCCTTC ACCACATGGC TTTATCCAGG CATCTGATGT TATAGATACT GAATCTGTCT 660
TGCCTTTGAC AACACTAACA GATCCCATAC TCCAACATCA TCAGGAAGAA TCAAATATCA 720
TTGGATCATC CTTGGGCAGT CCTGTTTCAG AAGATTCAAA GGATGTCGAA GATTTGGTAA 780
ACTGTCATTA GAATAATTCT 800 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 800 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CCUCUXJUCCA UCUACAGCCC ACUGGCACUC CAGGCACCUA CCUACUUCAA ACAAGCUCAA 60
GCCAAGGCCU UCCCCUAACU CUGACUGCUA GUCCCACAGU AACCCUGACA GCUGCUGCUC 120
CUGCUUCUCC UGAACAGAUU AUUGUUCAUG CJUUAUCCCC AGAACAUUUG XJUGAACACAA 180
GUGAUAAUGU UACAGUGCAG UGUCACACAC CAAGAGUCAU CAUUCAGACU GUUGCCACAG 240
AGGACAUCAC XJUCXJUCCAUA UCCCAAGCAG AACUGACCGU CGAUAGUGAU AXJUCAGUCAU 300
CUGAUXJUUCC UGAGCCUCCA GACGCCCUAG AAGCAGACAC XJXJUCCCAGAU GAAAUUCAUC 360
ACCCUAAGAU GACUGUGGAG CCAUCAXJUUA AUGAUGCUCA UGUAUCCAAA UUCAGUGACC 420
AAAAUAGCAC AGAACUGAUG AAUAGUGXJUA UGGUCAGAAC AGAAGAAGAA AUCUCUGACA 480
CCGACCJUAA ACAAGAGGAA UCACCCUCUG AUXJUAGCCAG UGCUUAUGUU ACUGAGGGUU 540
UAGAGUCUCC CACUAUAGAA GAACAAGUUG AUCAAACAAU UGAUGAUGAA ACAAUACUUA 600
UCGUUCCUUC ACCACAUGGC UUUAUCCAGG CAUCUGAUGU UAUAGAUACU GAAUCUGUCU 660
UGCCXJUUGAC AACACUAACA GAUCCCAUAC UCCAACAUCA UCAGGAAGAA UCAAAUAUCA 720
UUGGAUCAUC CJUGGGCAGU CCUGUUUCAG AAGAUUCAAA GGAUGUCGAA GAUUUGGUAA 780
ACUGUCAUUA GAAUAAUUCU 800
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 850 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CCTCTTTCCA TCTACAGCCC ACTGGCACTC CAGGCACCTA CCTACTTCAA ACAAGCTCAA 60
GCCAAGGCCT TCCCCTAACT CTGACTGCTA GTCCCACAGT AACCCTGACA GCTGCTGCTC 120
CTGCTTCTCC TGAACAGATT ATTGTTCATG CTTTATCCCC AGAACATTTG TTGAACACAA 180
GTGATAATGT TACAGTGCAG TGTCACACAC CAAGAGTCAT CATTCAGACT GTTGCCACAG 240
AGGACATCAC TTCTTCCATA TCCCAAGCAG AACTGACCGT CGATAGTGAT ATTCAGTCAT 300
CTGATTTTCC TGAGCCTCCA GACGCCCTAG AAGCAGACAC TTTCCCAGAT GAAATTCATC 360
ACCCTAAGAT GACTGTGGAG CCATCATTTA ATGATGCTCA TGTATCCAAA TTCAGTGACC 420
AAAATAGCAC AGAACTGATG AATAGTGTTA TGGTCAGAAC AGAAGAAGAA ATCTCTGACA 480
CCGACCTTAA ACAAGAGGAA TCACCCTCTG ATTTAGCCAG TGCTTATGTT ACTGAGGGTT 540
TAGAGTCTCC CACTATAGAA GAACAAGTTG ATCAAACAAT TGATGATGAA ACAATACTTA 600
TCGTTCCTTC ACCACATGGC TTTATCCAGG CATCTGATGT TATAGATACT GAATCTGTCT 660
TGCCTTTGAC AACACTAACA GATCCCATAC TCCAACATCA TCAGGAAGAA TCAAATATCA 720
TTGGATCATC CTTGGGCAGT CCTGTTTCAG AAGATTCAAA GGATGTCGAA GATTTGGTAA 780
ACTGTCATTA GAATAATTCT TAGAAATAGG CAGTTCAAGC AAAGAAGGCA CACTGTTAAT 840
TACAACCTCT 850
Claims
1. An isolated amino acid polymer that: (a) has a binding affinity for a D-type cyclin, in vitro; (b) has a binding affinity for a specific DNA nucleotide sequence; and (c) is a transcription factor involved in the activation of genes that prevent cell proliferation.
2. The amino acid polymer of Claim 1 which contains about 760 amino acids.
3. The amino acid polymer of Claim 1 wherein said specific DNA nucleotide sequence comprises a nonamer consensus sequence CCCG(G/T)ATGT.
4. The amino acid polymer of Claim 3 wherein the nonamer consensus sequence is CCCGTATGT.
5. The amino acid polymer of Claim 4 which is a mammalian protein.
6. The amino acid polymer of Claim 5 wherein the mammalian protein is a human protein that is encoded on human chromosome 7 at a position which corresponds to 7q21; and contains about 760 amino acids, including the 262 amino acids of SEQ ID NO: 24.
7. The amino acid polymer of Claim 5 which is a murine protein.
8. The amino acid polymer of Claim 1 selected from the group consisting of a protein having the amino acid sequence of SEQ ID NO: l ; and the amino acid sequence of SEQ ID NO: l with conservative substitutions.
9. An isolated fragment of an amino acid polymer, wherein said amino acid polymer: (a) has a binding affinity for a D-type cyclin, in vitro; (b) has a binding affinity for a specific DNA nucleotide sequence; and (c) is a transcription factor involved in the activation of genes that prevent cell proliferation; and wherein said fragment is selected from the group consisting of a DNA-binding domain of the amino acid polymer; a cyclin binding domain of the amino acid polymer; and a transactivation domain of the amino acid polymer.
10. The fragment of Claim 9 which is the DNA-binding domain of the amino acid polymer having the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 16 with conservative substitutions.
11. The fragment of Claim 9 which is the cyclin binding domain of the amino acid polymer having the amino acid sequence of SEQ ID NO:22, or SEQ ID NO:22 with conservative substitutions.
12. The fragment of Claim 9 which is the transactivation domain of the amino acid polymer having the amino acid sequence of SEQ ID NO: 18, or SEQ ID NO: 18 with conservative substitutions.
13. An antibody to the amino acid polymer of Claim 1.
14. An isolated nucleic acid that encodes the amino acid polymer of Claim 1.
15. The nucleic acid of Claim 14 wherein the amino acid polymer has the amino acid sequence SEQ ID NO: l , or SEQ ID NO: l with conservative substitutions.
16. The nucleic acid of Claim 14 wherein the amino acid polymer is a human protein that is encoded on human chromosome 7 at a position which corresponds to 7q21; and contains about 760 amino acids, including the 262 amino acids of SEQ ID NO:24.
17. The nucleic acid of Claim 16 wherein the nucleic acid comprises the coding region of the nucleotide sequence of SEQ ID NO: 25.
18. An isolated nucleic acid that encodes the fragment of Claim 9.
19. The nucleic acid of Claim 18 which has a nucleotide sequence selected from the group consisting of SEQ ID NO: 17; SEQ ID NO: 19; and SEQ ID NO:23.
20. An expression vector comprising the nucleic acid of Claim 14 operatively linked to an expression control sequence, wherein the nucleic acid is a DNA.
21. An isolated nucleic acid having a nucleotide sequence selected from the group consisting of (a) a DNA sequence of SEQ ID NO:2; (b) an RNA sequence corresponding to SEQ ID NO:3; and (c) a nucleotide sequence that hybridizes to any of the foregoing nucleotide sequences under stringent hybridization conditions.
22. A method for detecting the presence or activity of an amino acid polymer characterized by: (i) a binding affinity for a D-type cyclin, in vitro; and (ii) a binding affinity for a specific DNA nucleotide sequence; comprising the steps of: (a) contacting a biological sample from a mammal with a oligonucleotide probe under conditions that allow binding of the oligonucleotide probe to the amino acid polymer to occur, wherein the nucleotide probe contains the core sequence GTA, and wherein the presence or activity of the amino acid polymer is suspected in the biological sample; and (b) detecting whether said binding has occurred between the amino acid polymer and the nucleotide probe; wherein the detection of said binding indicates the presence or activity of the amino acid polymer in the biological sample.
23. A method of isolating an amino acid polymer comprising (a) contacting a biological sample from a mammal with an oligonucleotide linked to a solid phase support under conditions that allow binding of the oligonucleotide to the amino acid polymer to occur, whereby an amino acid polymer-oligonucleotide-solid phase support binding complex is formed, wherein the oligonucleotide contains the sequence CCCGTATGT, and wherein the presence of the amino acid polymer is either known or suspected in the biological sample; (b) washing the amino acid polymer-oligonucleotide-solid phase support binding complex, wherein an impurity is removed and whereby the amino acid polymer becomes a purified amino acid polymer; and (c) disrupting the amino acid polymer-oligonucleotide-solid phase support binding complex, and thereby separating the amino acid polymer from the oligonucleotide linked to the solid phase support, whereby the amino acid polymer is isolated.
24. An isolated amino acid polymer obtained by the method of Claim 23.
25. An expression vector having a transcription control sequence comprising a nonamer sequence CCCGTATGT operably associated with a recombinant gene or a cassette insertion site for a recombinant gene.
26. A method for activating transcription of a recombinant gene in a mammalian cell comprising transfecting the mammalian cell with the expression vector of Claim 25, which expression vector comprises the recombinant gene.
27. The method of Claim 26 further comprising transfecting the mammalian cell with an expression vector that provides for the expression of an amino acid polymer selected from the group consisting of a protein having the amino acid sequence of SEQ ID NO: l; and SEQ ID NO: l with conservative substitutions.
28. A transgenic animal comprising the expression vector of Claim 20 homologously recombined in a chromosome.
29. A transgenic animal in which the gene encoding the amino acid polymer of Claim 1 has been disrupted so as to be unable to express a functional transcription factor.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU31323/97A AU3132397A (en) | 1996-05-16 | 1997-05-16 | Cyclin d binding factor, and uses thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US64883796A | 1996-05-16 | 1996-05-16 | |
US1781596P | 1996-05-16 | 1996-05-16 | |
US08/648,837 | 1996-05-16 | ||
US60/017,815 | 1996-05-16 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1997043415A1 WO1997043415A1 (en) | 1997-11-20 |
WO1997043415A9 true WO1997043415A9 (en) | 1998-04-02 |
Family
ID=26690353
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/008480 WO1997043415A1 (en) | 1996-05-16 | 1997-05-16 | Cyclin d binding factor, and uses thereof |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU3132397A (en) |
WO (1) | WO1997043415A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6303772B1 (en) * | 1996-05-16 | 2001-10-16 | St. Jude Children's Research Hospital | Cyclin D binding factor, and uses thereof |
WO2012013249A1 (en) * | 2010-07-30 | 2012-02-02 | Université de Liège | Dentin matrix protein 1 (dmp1) for use in pharmaceutical compositions |
-
1997
- 1997-05-16 WO PCT/US1997/008480 patent/WO1997043415A1/en active Application Filing
- 1997-05-16 AU AU31323/97A patent/AU3132397A/en not_active Abandoned
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