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• W2060542994 abstract "In animal cells the progression of the cell cycle through G1/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G1/S transition of the cell cycle. Supershift experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2Fcis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells. In animal cells the progression of the cell cycle through G1/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G1/S transition of the cell cycle. Supershift experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2Fcis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells. retinoblastoma rapid amplification of cDNA ends polymerase chain reaction glutathione S-transferase electrophoretic mobility shift assay β-glucuronidase chloramphenicol acetyltransferase ubiquitin-carboxyl extension cauliflower mosaic virus 4-morpholineethanesulfonic acid nuclear localization signal cyclin-dependent kinase Cell division in plants is mainly restricted to meristems and is regulated both temporally and spatially in response to plant growth regulators and environmental signals. Meristematic cell division occurs during the entire plant life, and because plant cells cannot migrate, control of cell proliferation is responsible for the formation of plant organs and structures (1.Meyerowitz E.M. Cell. 1997; 88: 299-308Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Remarkably, however, plant cells can readily undergo multiple cycles of endoreduplication, and additionally most of them are totipotent and can reprogram cell division even after completing their differentiation. These unique features suggest a unique flexibility in the control of the cell cycle in plants. In animal cells a pivotal role in the progression of cell cycle is played by the E2F family of transcription factors, which are key components of a critical checkpoint regulating entry of cells into S phase (2.Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1954) Google Scholar, 3.Helin K. Curr. Opin. Genet. Dev. 1998; 8: 28-35Crossref PubMed Scopus (427) Google Scholar). Progression through the G1 and S phase of the cell cycle ultimately depends on the activation of genes coding for regulatory proteins and for the enzymes involved in nucleotide and DNA synthesis. The expression of many of these genes is largely under the control of the E2F family of transcription factors, which appear to be activated by multiple mitogenic signaling pathways. The E2F proteins bind to specific DNA sequences through a winged helix motif, forming prevalently heterodimers with distantly related partners of the DP (DRTF1 polypeptide) family (4.Zheng N. Fraenkel E. Pabo C.O. Pavletich N.P. Genes Dev. 1999; 13: 666-674Crossref PubMed Scopus (215) Google Scholar). In mammalian cells six distinct E2F proteins associate with two different DP members and bind to similar DNA elements, which are conserved in the promoters of several genes that are activated in late G1 and near the boundary G1/S. E2F gene targets include cell growth regulators such as cyclin A, Cdc2, c-Myc, and proliferating cell nuclear antigen and enzymes such as dihydrofolate reductase, thymidine kinase, ribonucleotide reductase, and DNA polymerase α (5.Leone G. DeGregori J. Yan Z. Jakoi L. Ishida S. Williams R.S. Nevins J.R. Genes Dev. 1998; 12: 2120-2130Crossref PubMed Scopus (307) Google Scholar, 6.Lavia P. Jansen-Dürr P. BioEssays. 1999; 21: 221-230Crossref PubMed Scopus (142) Google Scholar). The importance of the E2F factors for cell cycle progression is further highlighted by the demonstration that transient overexpression of several members of the E2F family is able to induce S phase in quiescent cells in the absence of growth factors (7.Lukas J. Petersen B.O. Holm K. Bartek J. Helin K. Mol. Cell. Biol. 1996; 16: 1047-1057Crossref PubMed Scopus (265) Google Scholar, 8.Müller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar). The activity of the E2F factors is in part regulated by members of the pocket protein family, which includes the product of the retinoblastoma tumor suppressor gene (pRB)1and the related proteins p107 and p130. These proteins possess a highly conserved A/B pocket domain that is the target of viral transforming proteins such as adenovirus E1A (2.Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1954) Google Scholar). The pocket proteins are also the targets of cyclin-dependent kinase activities, and once hypophosphorylated they can bind the activation domain of the various E2F transcription factors, thereby repressing their transcriptional activity. Furthermore, recent results indicate that the pocket proteins can recruit to the E2F complex a transcriptional repressor such as the HDAC1 histone deacetylase (9.Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar, 10.Ferreira R. Magnaghi-Jaulin L. Robin P. Harel-Bellan A. Trouche D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10493-10498Crossref PubMed Scopus (221) Google Scholar). The recruitment of histone deacetylase to promoters containing E2F DNA binding sites is believed to lead to chromatin condensation and to an efficient silencing of transcription. It is therefore now widely believed that the binding of the E2Fs to promoter elements can lead to both repression and activation of transcription, depending on their association with the pocket proteins. Such a concept also provides an explanation for the observation that the E2Fs can act as oncogenes as well as tumor suppressor genes (11.Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 12.Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). The mammalian E2F factors have similar primary structures with a highly conserved DNA-binding domain, found near the N terminus, followed by a DP dimerization domain containing a leucine heptad repeat. Next to the dimerization domain is conserved another region, named marked box, which in human E2Fs is the target of the adenovirus E4 protein (13.Jost C.A. Ginsberg D. Kaelin Jr., W.G. Virology. 1996; 220: 78-90Crossref PubMed Scopus (15) Google Scholar). The C-terminal region contains a transactivation domain that is characterized by the presence of several acidic residues and the presence of a short conserved region involved in the binding to the pocket proteins. E2F-6 lacks the activation domain and the pRB binding region, and it has been shown to function as an inhibitor of E2F-dependent transcriptional activity (14.Cartwright P. Müller H. Wagener C. Holm K. Helin K. Oncogene. 1998; 17: 611-623Crossref PubMed Scopus (163) Google Scholar). E2F-1, E2F-2, and E2F-3, but not the other mammalian E2Fs, possess a conserved domain at the N terminus that can bind to cyclin A/CDK2 (15.Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin Jr., W.G. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (412) Google Scholar). The DP-1 and DP-2 partners of E2F contain DNA binding and dimerization regions related to the E2F proteins but lack activation domains and other conserved regions (3.Helin K. Curr. Opin. Genet. Dev. 1998; 8: 28-35Crossref PubMed Scopus (427) Google Scholar). A plant homologue of the retinoblastoma tumor suppressor gene has been discovered and characterized in maize (16.Grafi G. Burnett R.J. Helentjaris T. Larkins B.A. DeCaprio J.A. Sellers W.R. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 93: 8962-8967Crossref Scopus (202) Google Scholar, 17.Xie Q. Sanz-Burgos A.P. Hannon G.J. Gutierrez C. EMBO J. 1996; 15: 4900-4908Crossref PubMed Scopus (190) Google Scholar, 18.Ach R.A. Durfee T. Miller A.B. Taranto P. Hanley-Bowdoin L. Zambryski P.C. Gruissem W. Mol. Cell. Biol. 1987; 17: 5077-5086Crossref Scopus (197) Google Scholar), suggesting that the transition from G1 to S phase, a key passage during cell cycle, is regulated similarly in animal and plant cells. The existence of pRB proteins that can interact with plant d-type cyclins and viral replication proteins indicated that homologues of the E2F factors might be present in plant cells and could be involved in the regulation of genes responsible for S phase progression. In this paper we describe the isolation and characterization of DcE2F, a E2F-like gene from carrot cells that is expressed in proliferating cells. We demonstrate that the carrot E2F protein is a functional transcription factor that can bind a canonical E2F cis-element in association with a mammalian DP protein and can transactivate through this binding site a reporter gene in both plant and mammalian cells. During the preparation of this manuscript, the isolation of wheat and tobacco E2F homologues was also described (19.Ramirez-Parra E. Xie Q. Boniotti M.B. Gutierrez C. Nucleic Acids Res. 1999; 27: 3527-3533Crossref PubMed Scopus (98) Google Scholar, 20.Sekine M. Ito M. Uemukai K. Maeda Y. Nakagami H. Shinmyo A. FEBS Lett. 1999; 460: 117-122Crossref PubMed Scopus (80) Google Scholar). Taken together with these results, our data demonstrate that the pRB/E2F pathway is conserved in plants, and the isolation of plant E2F provides a new tool to understand how plant growth and development is controlled. Plants of Daucus carota L. cv. Lunga di Amsterdam were grown under normal greenhouse conditions. Carrot cell suspension cultures were maintained as described previously (21.Nielsen E. Rollo F. Parisi B. Cella R. Sala F. Plant Sci. Lett. 1979; 15: 113-125Crossref Scopus (27) Google Scholar). For cellular synchronization, quiescent cells from a carrot culture grown to plateau were washed and incubated for 48 h in fresh Muzashige and Skoog liquid medium lacking hormones and sucrose. The cells were released from starvation by dilution in fresh Muzashige and Skoog medium containing growth regulators and sucrose. At different time points after release, small samples were used for DNA synthesis assay by [3H]thymidine pulse labeling experiments, and the remaining aliquots were collected and immediately frozen in liquid nitrogen before isolation of total RNA. For the isolation of carrot nuclei, cells from an actively dividing suspension culture were washed in protoplast isolation buffer (22.Banks M.S. Evans P.K.A. Plant Sci. Lett. 1976; 7: 409-416Crossref Scopus (72) Google Scholar) and resuspended in approximately 5 volumes of enzyme mixture containing 1% cellulase Onozuka R-10 (Yakult) 0.5% Pectinase (Serva) in protoplast isolation buffer. After incubation for about 6 h at 25 °C, the suspension was centrifuged for 5 min at 200 × g, and the pelleted protoplasts were washed three times with protoplast isolation buffer. After resuspension in approximately 10 volumes of ice-cold resuspension buffer (0.4 m sucrose, 25 mm Tris-HCl, pH 7.6, 10 mm MgCl2, 0.3% Triton X-100, 5 mm β-mercaptoethanol, 0.5 mmphenylmethylsulfonyl fluoride), the protoplasts were disrupted in a Teflon homogenizer (1,000 revolutions/min, 5 strokes for three times). The homogenate was then centrifuged for 5 min at 3,000 ×g, and the pellets were resuspended in two volumes of ice-cold wash buffer (0.4 m sucrose, 50 mmTris-HCl, pH 7.6, 5 mm MgCl2, 20% glycerol, 5 mm β-mercaptoethanol, 0.5 mmphenylmethylsulfonyl fluoride). The nuclei were finally spun down at 2,000 × g for 5 min, and the pellets were stored at −80 °C. Pelleted nuclei were resuspended in ice-cold lysis buffer (25 mm Hepes, pH 7.6, 40 mm KCl, 0.5 mm EDTA, 5 mmMgCl2, 2 mm dithiothreitol, 20% glycerol, 1 mg/ml antipain, 1 mg/ml leupeptin) and were lysed by adding 0.1 volume of cold 4 m (NH4)2SO4. After incubation for 30 min with constant movement at 4 °C, the lysate was centrifuged at 15,000 × g, and the nuclear proteins of the supernatant were precipitated by slowly adding 0.5 volume of cold 4 m(NH4)2SO4 and incubating 60 min at 4 °C with constant movement. The nuclear proteins were then recovered by centrifugation for 10 min at 4 °C in microcentrifuge and then resuspended in dialysis buffer (same as lysis buffer but without MgCl2) and dialyzed 3 h at 4 °C against the same buffer. The resulting nuclear extracts were divided in small aliquots and stored in liquid nitrogen. Poly(A)+ RNA was isolated from carrot cell culture using oligo(dT) cellulose (Roche Molecular Biochemicals) following a standard batch procedure (23.Bartels D. Thompson R.D. Nucleic Acids Res. 1983; 11: 2961-2978Crossref PubMed Scopus (115) Google Scholar). Total RNA was isolated by the hot phenol method (24.Shirzadegan M. Christie P. Seemann J.R. Nucleic Acids Res. 1991; 19: 6055Crossref PubMed Scopus (182) Google Scholar). For Northern blot analysis, the RNA samples were resolved in formaldehyde gels, transferred to Hybond-N membranes (Amersham Pharmacia Biotech), and hybridized with a DcE2F probe labeled by the random primer method (Amersham Pharmacia Biotech). To verify the level of synchronization, after removal of the DcE2F probe, the filter was subsequently hybridized with a carrot UBI-CEP probe (25.Balestrazzi A. Cella R. Carbonera D. Plant Physiol. 1998; 117: 720Google Scholar). To amplify carrot E2F-like cDNAs, 3′-RACE reactions were performed on carrot poly(A)+ RNA using a degenerate primer of sequenceGCGAATTCMGIMGIATHTAYGA (where I is inosine, M is A/C, H is A/T/C, Y is C/T, and the nucleotides in italics represent the added cloning site) containing all the possible codons for the conserved amino acid sequence RRIYD. Nested PCR reactions were performed with a second primer of sequence GCGAATTCGAYATHACIAAYGT containing all the possible codons for the amino acid sequence DITNV. Reverse transcription and PCR reactions were conducted as described previously (26.Albani D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar). After electrophoretic analysis of the nested reactions, the major PCR fragment, corresponding to a partial DcE2F cDNA, was isolated and subcloned into the plasmid pBluescriptII KS+ (Stratagene). To isolate full-length DcE2F clones, approximately 500,000 plaque-forming units from a carrot cell suspension cDNA library (27.Bernacchia G. Primo A. Giorgetti L. Pitto L. Cella R. Plant J. 1998; 13: 317-329Crossref PubMed Scopus (32) Google Scholar) constructed in λZAPII (Stratagene) were screened with the partial DcE2F probe. Hybridization and washings were performed as previously reported (26.Albani D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar). Two hybridizing plaques were purified and the plasmids containing the DcE2F cDNAs were excised in vivoaccording to the manufacturer's protocol. Sequencing was performed on both strands of the longest cDNA clone (Amersham Pharmacia Biotech sequencing kit). For the construction of the pRSET-DcE2F expression vector the DcE2F cDNA was digested with the enzymes BamHI and HindIII, and the resulting DNA fragment, containing the entire DcE2F coding region preceded by 35 base pairs of the upstream untranslated region, was inserted into the corresponding sites of the polylinker of pRSET-A (Invitrogen). The pRSET-DcE2F plasmid was then introduced intoEscherichia coli BL21(DE3) for the production of a recombinant DcE2F protein carrying a histidine-tagged N-terminal extension of 48 amino acids. The HIS-DcE2F protein was purified under nondenaturing condition by metal affinity chromatography on nickel-nitrilotriacetic acid resin (Qiagen) as previously reported (26.Albani D. Hammond-Kosack M.C.U. Smith C. Conlan S. Colot V. Holdsworth M. Bevan M.W. Plant Cell. 1997; 9: 171-184PubMed Google Scholar). For purification under denaturing conditions, cell lysis and affinity chromatography were performed in phosphate buffer containing 8m urea. Electrophoretic analysis of the eluted proteins revealed a single polypeptide of the expected dimensions indicating a purification to near homogeneity of the recombinant DcE2F protein. The bacterial GST-DP1 expression vector was introduced into E. coli XL-1 blue cells, and the recombinant protein was purified by chromatography on glutathione-agarose (Amersham Pharmacia Biotech) according to manufacturer's instructions. The His-DcE2F protein eluted under denaturing conditions was dialyzed and used directly for rabbit immunization. For Western analysis, proteins fractionated by SDS-polyacrylamide gel electrophoresis were transferred to Hybond-C extra membranes (Amersham Pharmacia Biotech) using a semi-dry blotting apparatus (Hoefer Scientific Instruments). The blots were incubated with the anti-DcE2F polyclonal serum, and immunodetection was performed using ECL chemiluminescence detection reagents (Amersham Pharmacia Biotech). For the electrophoretic mobility shift assays (EMSAs) with carrot nuclear extracts or recombinant DcE2F and DP-1 proteins, the cloned double-stranded oligonucleotides were gel purified and labeled by a fill-in reaction with Klenow DNA polymerase in the presence of [α-32P]dATP (Amersham Pharmacia Biotech). The sequences of the probes were 5′-aattcTTTTCCCGCGCTTTTgaatt-3′ for the canonical E2F binding site (EC) and 5′-aattcTTTTCCATCGCTTTTgaatt-3′ for the mutated E2F binding site (EM). In both sequences the lowercase letters represent the EcoRI cloning sites. The DNA binding reactions with carrot nuclear extracts were conducted incubating 1.5–6 μg of nuclear proteins with 50,000 cpm of radiolabeled oligonucleotide probes and 2 μg of sheared salmon sperm DNA in 15 μl of 25 mmHepes, pH 7.5, 100 mm KCl, 1 mmMgCl2, 1 mm EDTA, 5% glycerol, and 10 mm dithiothreitol for 30 min at room temperature. The DNA binding of the recombinant proteins was conducted similarly without adding the salmon sperm DNA. For the competition experiments, increasing amounts of annealed unlabeled oligonucleotides were included in the reactions with the EC probe. The supershift experiments were carried out by preincubating the binding reactions with 1 μl of polyclonal anti-DcE2F serum or preimmune serum for 30 min at room temperature before adding the probe. The protein-DNA complexes were electrophoresed for 3 h at 4 °C on 4% polyacrylamide gels in 0.5× TBE. The EMSAs in mammalian cells were performed essentially as described (28.Helin K. Lees J.A. Vidal M. Dyson N. Harlow E. Fattaey A. Cell. 1992; 70: 337-350Abstract Full Text PDF PubMed Scopus (520) Google Scholar) using a E2F binding probe of sequence 5′-ATTTAAGTTTCGCGCCCTTTCTCAA-3′. Extracts were prepared from U2OS cells transfected with each of the indicated expression plasmids (6 μg/100-mm dish). 10 μg of the extracts were used for Western blotting as shown in Fig. 8 A. To determine the protein composition of the complexes in EMSAs, the following antibodies were used: KH95 (anti E2F-1) (29.Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (415) Google Scholar), TFD10 (anti-DP-1) (8.Müller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar), and 12CA5 (anti-hemagglutinin tag) (30.Field J. Nikawa J. Broek D. MacDonald B. Rodgers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (726) Google Scholar). To generate an expression plasmid for DcE2F, the full open reading frame of DcE2F was amplified by PCR using primers specific for DcE2F and containing BamHI restriction sites in the ends. The PCR product was subsequently cloned intoBamHI-digested pCMVHA (29.Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (415) Google Scholar) to generate pCMVHADcE2F. pCMVE2F-1 and pCMVDP-1 have been described previously (29.Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (415) Google Scholar, 31.Helin K. Harlow E. Fattaey A. Mol. Cell. Biol. 1993; 13: 6501-6508Crossref PubMed Scopus (400) Google Scholar). For the construction of the DcE2F effector plasmid to be used in plant protoplasts, the cDNA was digested with HindIII, blunt-ended with Klenow DNA polymerase, and then digested with BamHI yielding a DNA fragment, containing the entire DcE2F coding region. This fragment was inserted into the BamHI and blunt-ended SacI sites found upstream of the terminator sequence of the nosgene of Agrobacterium tumefaciens cloned as aSacI/EcoRI fragment into pUC19. The DcE2F/NOS gene fusion was then digested with BamHI andEcoRI, and the resulting DNA fragment was inserted downstream of the cauliflower mosaic virus (CaMV) duplicated 35 S promoter in the plasmid pFF19 (32.Timmermans M.C. Maliga P. Vieira J. Messing J. J. Biotechnol. 1990; 14: 333-344Crossref PubMed Scopus (132) Google Scholar) to give rise to the p35 S-DcE2F construct. The p35 S-DP1 effector was obtained by cloning into pFF19 aBamHI/SalI-digested DNA fragment from pBSK-DP1 (29.Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (415) Google Scholar). For the construction of the chimeric 6XE2F-minimal 35 S promoter-GUS reporter construct, a DNA fragment containing the six E2F binding sites was isolated from the pGL3TATAbasic-6XE2F vector (8.Müller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar) after digestion with AspI, blunt ending with Klenow DNA polymerase and digestion with XhoI. This fragment was then cloned into HindIII (filled in with Klenow DNA polymerase) and SalI sites of pBI221.9 to give rise to the pBI221-E2F reporter construct. The transactivation experiments in plant cells were conducted with protoplasts isolated from carrot somatic embryos at the heart torpedo stages of development. The embryos were incubated with 1% cellulase and 0.2% pectinase in protoplast isolation buffer (27.2 mg/liter KH2PO4, 101 mg/liter KNO3, 1.4 g/liter CaCl2, 246 mg/liter MgSO4, 0.16 mg/liter KI, 0.025 mg/liter CuSO4, 10 mm MES, and 0.7 m sorbitol, pH 5.5). The protoplasts were pelleted and washed twice in the same buffer without enzymes and then resuspended at a density of 7.5 × 106 protoplasts ml−1 in 10 mm Hepes, 130 mm KCl, 10 mm NaCl, 4 mm CaCl2, 0.2m mannitol, pH 7.2. For electroporation, aliquots of 0.4 ml of the protoplast suspension were placed in the electroporation cuvettes (ECU-104, Equibio) and then mixed with 10 μg of each test plasmid, 5 μg of CaMV 35 S-CAT plasmid (as internal control) and sonicated calf thymus DNA to a total of 50 μg of DNA. After incubation on ice for 10 min, the electrical pulse was delivered by the Electroporator II (Invitrogen) charged to 330 V electric potential, 500 microfarad capacitance, and 500 Ω resistance. After 10 min on ice, the protoplasts were diluted with 2.1 ml of culture medium (Gamborg's B5 medium supplemented with 300 mg/liter CaCl2-H2O, 825 mg/liter NH4NO3, 100 mg/liter sodium pyruvate, 200 mg/liter malic acid, 200 mg/liter citric acid, 300 mg/liter casamino acids, 200 mg/liter yeast extract, 20 g/liter saccarose, 76 g/liter mannitol, 0.1 mg/liter 2,4-dichlorophenoxyacetic acid, 0.2 mg/liter 6BAP, 10−6m α-naphtaleneacetic acid, 5 × 10−7m zeatin riboside, pH 5.6) and incubated 40 h in the dark at 25 °C. GUS activity was measured as described by Gallie et al. (33.Gallie D.R. Lucas W.J. Walbot V. Plant Cell. 1989; 1: 301-311Crossref PubMed Scopus (161) Google Scholar). CAT assays was performed using the CAT detection kit (Roche Molecular Biochemicals) as described by the manufacturer. Transactivation assays in mammalian cells were performed as described previously (8.Müller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar) using pGL3TATAbasic-6xE2F as reporter construct and pCMVβ-gal (CLONTECH) to normalize for transfection efficiency. For a 60-mm tissue culture dish, 30 ng of the indicated expression plasmid was transfected in combination with 1 μg of pGL3TATAbasic-6xE2F and 500 ng of pCMVβ-gal. Cells were harvested 36 h after addition of the DNA-calcium phosphate coprecipitate. Following the discovery of plant homologues of the retinoblastoma tumor suppressor protein, a yeast two-hybrid assay has shown that the maize pRB protein can bind to human andDrosophila E2F transcription factors (34.Huntley R. Healy S. Freeman D. Lavender P. de Jager S. Greenwood J. Makker J. Walker E. Jackman M. Xie Q. Bannister A.J. Kouzarides T. Gutierrez C. Doonan J.H. Murray J.A. Plant Mol. Biol. 1998; 37: 155-169Crossref PubMed Scopus (128) Google Scholar). A transcription interference assay has further shown that maize pRB can inhibit E2F-dependent transcriptional activation in animal cells (34.Huntley R. Healy S. Freeman D. Lavender P. de Jager S. Greenwood J. Makker J. Walker E. Jackman M. Xie Q. Bannister A.J. Kouzarides T. Gutierrez C. Doonan J.H. Murray J.A. Plant Mol. Biol. 1998; 37: 155-169Crossref PubMed Scopus (128) Google Scholar). These remarkable properties of maize pRB strongly indicated that plant homologues of the animal E2F family of transcription factors were likely to exist and to be involved in the regulation of genes responsible for the progression of the cell cycle through the G1/S transition. To investigate the presumed presence of E2F-like factors in plant cells, DNA binding assays using carrot nuclear extracts were performed (Fig. 1). Animal E2F/DP complexes as well as cellular E2F DNA binding activities have been shown to specifically recognize a consensus sequence TTT(C/G)(G/C)CG(C/G), and mutations of the internal invariable CG doublet have been shown to greatly reduce E2F binding efficiency (4.Zheng N. Fraenkel E. Pabo C.O. Pavletich N.P. Genes Dev. 1999; 13: 666-674Crossref PubMed Scopus (215) Google Scholar). Accordingly, EMSAs on carrot nuclear extracts were performed with a canonical E2F binding probe of sequence 5′-TTTTCCCGCGCTTTT-3′ and with a mutated probe of sequence 5′-TTTTCCATCGCTTTT-3′. As shown in Fig. 1, carrot nuclear extracts contain DNA binding activities, which, as expected for E2F complexes, recognize the canonical sequence but are unable to bind to the mutated probe. Furthermore, as also shown in Fig.1, competition experiments with excess of unlabeled probes confirmed the binding specificity for the canonical sequence and demonstrated that E2F-like DNA binding activities exist in carrot cells. Although E2F-like DNA binding activities were detected in carrot nuclear extracts, it remained uncertain whether they could correspond to a real functional homologue of the E2F transcription factors or could simply represent unrelated nuclear factors that are able to discriminate between the two EMSA probes. To verify the molecular nature of these DNA binding activities, we proceeded to the isolation of a carrot homologue of animal E2Fs. The indication that all the E2F binding activities in animal cells are heterodimers, formed by E2F and DP partners, precluded the possibility to isolate E2F homologues by Southwestern screening procedures. All the animal E2F and DP factors, however, possess highly conserved DNA-binding domains that are found near the N termini of the proteins. In view of the perfect identity of a portion of this domain in the mammalian E2Fs and in a E2F homologue of Drosophila melanogaster (35.Ohtani K. Nevins J.R. Mol. Cell. Biol. 1994; 14: 1603-1612Crossref PubMed Scopus (111) Google Scholar, 36.Dynlacht B.D. Brook A. Dembski M. Yenush L. Dyson N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6359-6363Crossref PubMed Scopus (129) Google Scholar), we proceeded to the isolation of a plant E2F homologue using PCR techniques. A pair of degenerate primers encoding the conserved amino acid sequences RRIYD and DITNV were used in 3′-RACE reactions performed with poly(A)+ RNA isolated from actively dividing carrot cells. Using the same strategy we also attempted the isolation of a DP homologue with a pair of degenerate primers encoding the amino ac" @default.
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• W2060542994 title "DcE2F, a Functional Plant E2F-like Transcriptional Activator from Daucus carota" @default.
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