FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1998354003> ?p ?o ?g. }

• W1998354003 endingPage "24656" @default.
• W1998354003 startingPage "24649" @default.
• W1998354003 abstract "Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the α2-macroglobulin and Aα-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the α2-macroglobulin and Aα-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. interleukin-1 serum amyloid A SAA3 enhancer factor distal response element CCAAT/enhancer-binding protein electrophoretic mobility shift assay base pair(s) polyacrylamide gel electrophoresis signal transducer and activator of transcription protein: LC, liquid chromatography mass spectrometry collision-induced dissociation Wilm's tumor 1. The defense processes initiated in most vertebrates after infection or tissue injury are termed the acute-phase response (1Kusher I. Ann. N. Y. Acad. Sci. 1982; 389: 39-48Crossref PubMed Scopus (1167) Google Scholar). One characteristic of this response is changes in the circulating plasma protein profile, reflecting the synthesis and secretion of proteins involved in immune function and wound repair (2Koj A. Structure and Function of Plasma Proteins. Plenum Publishing Corp., London1974: 73-125Crossref Google Scholar). After tissue injury or infection, macrophages and monocytes near the damaged site detect the infectious agent or damaged cells and respond with a first wave of synthesis of cytokines, mainly of interleukin-1 (IL-1)1 and tumor necrosis factor. These first-wave cytokines trigger the surrounding cell types, such as fibroblasts and blood vessel endothelial cells, to respond with an amplified second wave of cytokine synthesis, which includes a large amount of IL-6. A significant amount of these cytokines is transported in the blood stream and triggers the acute-phase response in target tissues such as the liver. The liver is one of the major targets for these proinflammatory cytokines because it has the largest number of cells with cytokine receptors as well as a high density of receptors per cell (3Taga T. Kishimoto T. Cellular and Molecular Mechanisms of Inflammation. Academic Press, Inc., New York1990: 219-243Crossref Google Scholar, 4Castell J.V. Geiger T. Gross V. Andus T. Walter E. Hirano T. Kishimoto T. Heinrich P.C. Eur. J. Biochem. 1988; 177: 357-361Crossref PubMed Scopus (267) Google Scholar, 5Geisterfer M. Richards C. Baumann M. Fey G. Gwynne D. Gauldie J. Cytokine. 1993; 5: 1-7Crossref PubMed Scopus (59) Google Scholar). The liver responds to the cytokine stimulation by a burst of synthesis of acute-phase plasma proteins. The magnitude of the changes in the relative plasma concentrations of these proteins ranges from less than 2-fold to several hundredfold after acute inflammation. Elevated expression of acute-phase genes is regulated primarily at the transcriptional level. Analyses of many acute-phase gene promoters have revealed two general types of regulatory cis-acting elements in the transcriptional induction by cytokines: the binding sites for constitutive factors such as C/EBPα, hepatocyte nuclear factor 1, and hepatocyte nuclear factor 3 and the binding sites for inducible transcription factors such as C/EBPβ, C/EBPδ, NFκB, and signal transducer and activator of transcription proteins (STATs). In most cases, full transcriptional activation of these acute-phase gene promoters requires the combined action of a constitutive factor and an inducible transcription factor or factors. For example, induction of β-fibrinogen by IL-6 requires the cooperative interaction of three transcription factors: the constitutively expressed transcription factor hepatocyte nuclear factor 1, the IL-6-inducible C/EBPβ protein, and an unidentified IL-6-responsive factor (6Dalmon J. Laurent M. Gourtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar). In the promoter of the C-reactive protein gene, the binding site for members of the C/EBP family and hepatocyte nuclear factor 1 are required for full promoter activity after cytokine induction (7Majello B. Arcone R. Toniatti C. Giliberto G. EMBO J. 1990; 9: 457-465Crossref PubMed Scopus (145) Google Scholar, 8Toniatti C. Demartis A. Monaci P. Nicosia A. Giliberto G. EMBO J. 1990; 9: 4467-4475Crossref PubMed Scopus (97) Google Scholar). The serum amyloid A (SAA) gene family belongs to one of the major acute-phase proteins. In mice, there are four SAA genes (SAA1, SAA2, SAA3, and SAA5) and a pseudogene (9de Beer M.C. Beach C.M. Shedlofsky S.I. de Beer F.C. Biochem. J. 1991; 280: 45-49Crossref PubMed Scopus (43) Google Scholar, 10Lowell C.A. Stearman R.S. Morrow J.W. J. Biol. Chem. 1986; 261: 8453-8461Abstract Full Text PDF PubMed Google Scholar, 11Stearman R.S. Lowell C.A. Peltzman C.G. Morrow J.F. Nucleic Acids Res. 1986; 14: 797-809Crossref PubMed Scopus (35) Google Scholar). The SAA plasma concentration rises from 0.5 μg/ml to more than 1000 μg/ml 24 h after injection of bacterial lipopolysaccharide (12Hoffman J.S. Benditt E.P. J. Biol. Chem. 1982; 257: 10510-10517Abstract Full Text PDF PubMed Google Scholar). SAA circulates as an apolipoprotein of high density lipoprotein particles, and at the peak of inflammation, it constitutes up to 20% of the total protein in the high density lipoprotein particles (12Hoffman J.S. Benditt E.P. J. Biol. Chem. 1982; 257: 10510-10517Abstract Full Text PDF PubMed Google Scholar). SAA has been suggested to play a role in reverse cholesterol transport of high density lipoprotein by affecting the activity of the enzyme lecithin-cholesterol acyltransferase, which converts cholesterol to cholesterol esters (13Steinmetz A. Hocke G. Saile R. Puchois P. Fruchart J.C. Biochim. Biophys. Acta. 1989; 1006: 173-178Crossref PubMed Scopus (112) Google Scholar). However, continuous overproduction of SAA associated with chronic inflammation often results in secondary amyloidosis, an incurable and frequently fatal disorder (14Yakar S. Livneh A. Kaplan B. Pras M. Semin. Arthritis Rheum. 1995; 24: 255-261Crossref PubMed Scopus (24) Google Scholar). The large increase in the hepatic synthesis of SAA is primarily a consequence of dramatically increased transcription of SAA genes (10Lowell C.A. Stearman R.S. Morrow J.W. J. Biol. Chem. 1986; 261: 8453-8461Abstract Full Text PDF PubMed Google Scholar, 15Morrow J.F. Stearman R.S. Peltzman C.G. Potter D.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4718-4722Crossref PubMed Scopus (125) Google Scholar). Thus, transcriptional induction of SAA genes is an excellent model system for studying differential gene expression in response to a specific stimulus. To dissect the molecular mechanisms of SAA gene regulation, we have studied the promoters of the rat SAA1 (16Li X. Liao W.S.-L. J. Biol. Chem. 1991; 266: 15192-15201Abstract Full Text PDF PubMed Google Scholar, 17Li X. Liao W.S.-L. Nucleic Acids Res. 1992; 20: 4765-4772Crossref PubMed Scopus (65) Google Scholar) and mouse SAA3 genes (18Li X. Huang J.H. Rienhoff H.Y. Liao W.S.-L. Mol. Cell. Biol. 1990; 10: 6624-6631Crossref PubMed Scopus (41) Google Scholar, 19Huang J.H. Rienhoff H.Y. Liao W.S.-L. Mol. Cell. Biol. 1990; 10: 3619-3625Crossref PubMed Scopus (28) Google Scholar, 20Huang J.H. Liao W.S.-L. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). Our studies of the rat SAA1 promoter have shown the functional importance and cooperative interaction between NFκB and C/EBP proteins in cytokine-induced expression. Our studies of the mouse SAA3 promoter demonstrated that a 350-bp promoter fragment was necessary and sufficient to confer cytokine responsiveness. Two elements were identified in this 350-bp promoter fragment: a proximal response element, which contains two adjacent C/EBP binding sequences that enhances SAA3 gene expression in liver-derived cells, and a distal response element (DRE), which confers responsiveness to cytokine induction and has properties of an inducible transcription enhancer (19Huang J.H. Rienhoff H.Y. Liao W.S.-L. Mol. Cell. Biol. 1990; 10: 3619-3625Crossref PubMed Scopus (28) Google Scholar). We demonstrated that DRE consists of three functionally distinct elements: the A element, a weak binding site for C/EBP family proteins; the B element, which also interacts with C/EBP family proteins but with a much higher affinity; and the C element that interacts with a constitutive nuclear factor, which was named SAA3 enhancer factor (SEF). Deletions and site-specific mutation studies revealed that all three elements are required for maximum promoter activity. Deletions and mutations of the C element drastically reduce both basal and inducible activities of SAA3 promoter. Furthermore, although the C element does not interact with C/EBP directly and mutation of this element does not alter C/EBP binding to elements A and B, mutation of the C element nevertheless dramatically reduces the transactivation of the SAA3 promoter by C/EBPδ (20Huang J.H. Liao W.S.-L. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). Taken together, these functional studies clearly demonstrated that SEF is a critical component in the regulation of SAA3 promoter activity. To further our understanding of SAA3 gene regulation, we purified and characterized SEF from HeLa nuclear extracts and provide some evidence that SEF may play a broad role in regulating other gene promoters. HeLa cells were grown in suspension in Spinner's minimum essential medium supplemented with 5% (v/v) bovine calf serum (Hyclone). The cells were maintained by daily dilution with fresh complete medium to 4.5 × 105 cells/ml and were grown to a density of 9 × 105 cells/ml before harvesting. Nuclear extracts were prepared as described previously (21Dignam J.B. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar). The cell pellet from 12 liters of cells was resuspended with 5 volumes of hypotonic buffer (10 mm HEPES, pH 7.6, 1.5 mm MgCl2, 10 mm KCl, 1 mm benzamidine, and freshly added 0.2 mm phenylmethylsulfonyl fluoride and 14 mmβ-mercaptoethanol). After incubation on ice for 20 min, the cells were lysed with a glass Dounce homogenizer with 20 up-and-down strokes. Nuclei were pelleted at 3400 × g for 15 min and resuspended in 3.5 volumes of high salt buffer (20 mmHEPES, pH 7.9, 25% glycerol, 1.5 mm MgCl2, 0.75 m KCl, 1 mm EDTA, 1 mmbenzamidine, 0.2 mm phenylmethylsulfonyl fluoride, and 14 mm β-mercaptoethanol). The nuclear proteins were extracted by gently mixing at 4 °C for 10 min. After centrifugation at 15,000 × g for 30 min, the supernatant (designated crude nuclear extract) was applied immediately onto ion-exchange chromatography columns for SEF purification. The double-stranded synthetic oligonucleotides 5′-CACATTTCTGGAAATGCCTAGAT-3′, which correspond to the mouse SAA3 promoter sequence between nucleotides −169 and −147 that contain the SEF-binding site, were annealed, oligomerized with T4 DNA ligase, and cloned into the Sma I site of pGEM-7Zf(+) (Promega Corp.). A clone containing eight SEF-binding sites was recovered and verified by DNA sequencing. An Eco RI-Bam HI fragment harboring eight SEF-binding sites was purified from an agarose gel and end-labeled at the Eco RI site by using biotin-14-dATP (Life Technologies, Inc.) and the Klenow fragment of DNA polymerase I. The magnetic DNA affinity beads were prepared as described (22Gabrielsen O.S. Huet J. Methods Enzymol. 1993; 218: 508-525Crossref PubMed Scopus (44) Google Scholar). The biotinylated DNA fragment was then incubated with prewashed, streptavidin-coated magnetic beads (Dynal) in TE buffer (10 mm Tris-HCl, pH 8.0, and 1 mm EDTA) containing 1 m NaCl and placed in a roller at room temperature for 30 min. The amount of DNA on the beads was approximately 20 pmol/mg of magnetic bead. After binding, the DNA-conjugated beads were stored at 4 °C in TE buffer containing 0.1m NaCl. Crude HeLa nuclear extracts were diluted to 25 mm NaCl with Buffer A (25 mmTris-HCl, pH 7.3, 10% glycerol, 1 mm benzamidine, 1 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, and 14 mm β-mercaptoethanol and applied to a 160-ml DEAE-Sephacel column at a flow rate of 3 ml/min. After loading, the column was washed extensively with Buffer A, and SEF activity was subsequently eluted with 0.2 m NaCl in Buffer A. The DEAE eluates were loaded directly onto a 50-ml heparin-agarose column at a flow rate of 1 ml/min. After washing with 0.2 m NaCl in Buffer A, the bound SEF activity was eluted with 0.5 m NaCl in Buffer A. The eluate from the heparin-agarose column was then diluted to 0.25 m NaCl with Buffer A before being applied to a phenyl-Sepharose column (2.5 × 10 cm). The phenyl-Sepharose column was washed sequentially with Buffer A containing 0.25m NaCl and Buffer A containing 0.25 m NaCl and 30% ethylene glycol before the SEF activity was eluted with Buffer A containing 65% ethylene glycol. The eluate from phenyl-Sepharose column was first dialyzed in TEG buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 10% glycerol, 0.05% Nonidet P-40, 1 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride, and 14 mm β-mercaptoethanol) for 2 h and then dialyzed in TEG buffer containing 0.1 m NaCl for an additional 2 h. The dialyzed sample was mixed directly with the DNA affinity beads. The amount of beads and poly(dI-dC) used in the incubation depended on the amount of protein in the phenyl-Sepharose eluate. In general, approximately 100 μg of protein was incubated with 1.5 mg of DNA affinity beads and 50 μg of poly(dI-dC). This mixture was incubated in a roller at 4 °C for 30 min before being subjected to magnetic separation. After the magnetic separation, the SEF-bound magnetic beads were washed twice by resuspension in TEG buffer containing 0.1 m NaCl. SEF binding activity was then eluted from the DNA affinity beads with 0.4 m NaCl in TEG buffer. Unless otherwise stated, all purification procedures were performed at 4 °C. Protein concentrations were measured by the Bradford assay (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar), and SEF activities were determined by electrophoretic mobility shift assays (EMSA). A 32P-labeled C element DNA containing a SEF-binding site (4 × 104 cpm) was incubated with protein samples from different stages of purification to assess SEF activity (20Huang J.H. Liao W.S.-L. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). Approximately 1–2 μg of protein was incubated with the radioactively labeled probe in TEG buffer containing 100 mm NaCl for 30 min at 4 °C. In assays with affinity-purified SEF samples, 5 μg/ml acetylated bovine serum albumin was included in the reaction buffer to minimize nonspecific loss of SEF protein. After incubation, the reaction mixtures were loaded onto a 5% polyacrylamide gel (19:1 cross-linking ratio) in glycine buffer and subjected to electrophoresis at 200 V for 90 min at 4 °C. The gel was dried before autoradiography. The SEF activity was quantified with a PhosphorImager (Molecular Dynamics). One unit of SEF binding activity was defined as the amount of protein required to retard 10% of the labeled DNA under our standard assay conditions. Rabbit polyclonal antibody (anti-LCL) raised against an N-terminal peptide, LPLADEVIESGLVQD, corresponding to amino acid residues 7 to 21 (30Yoon J.B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (99) Google Scholar) was used in antibody supershift experiments. DNA-affinity purified SEF was incubated with 32P-labeled C element in the presence of rabbit anti-LCL antiserum (1:1200 dilution) or preimmune serum for 30 min at 4 °C. The reaction mixtures were then subjected to electrophoresis as above. SDS-PAGE was performed as described by Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar), and protein sizes were determined by comparison with prestained molecular weight markers (Bio-Rad). Electrophoresis was performed at 165 V for 4.5 h. Silver staining was performed according to the instructions in the silver staining kit (Sigma). The gels for protein profiles were fixed in 30% ethanol and 10% glacial acetic acid. After exposure to silver nitrate, each gel was treated with developer to control the level of staining. When the desired staining intensity was reached, the gel was fixed and photographed. Affinity-purified SEF was incubated with 25 ng of poly(dI-dC) and 5 × 104 cpm of a 5-bromodeoxyuridine-subsitituted, uniformly labeled SEF-binding site in a 50-μl reaction mixture (25Wu C. Wilson S. Walker B. Dawid I. Paisley T. Zimarino V. Ueda H. Science. 1987; 238: 1247-1253Crossref PubMed Scopus (215) Google Scholar). The mixture was incubated at 4 °C for 30 min with or without a 100-fold molar excess of wild-type or mutant SEF-binding oligonucleotides. The incubation mixture was then either first separated on a 5% nondenaturing polyacrylamide gel and then exposed to UV radiation or directly exposed in solution to UV radiation for 7 min from a UV transilluminator (254 nm, 7000 milliwatts/cm2) at a distance of 4 cm. After separation on a 7.5% SDS-polyacrylamide gel, the proteins directly involved in binding to the DNA were identified by autoradiography. The southwestern assay was performed by the method of Philippe (26Philippe J. Methods Mol. Biol. 1994; 31: 349-361PubMed Google Scholar). Briefly, the DNA affinity-purified proteins were separated on a 7.5% SDS-PAGE, transferred to a nitrocellulose membrane, denatured, and then renatured in sequential dilutions of guanidine-HCl (3, 1.5, 0.75, 0.38, and 0.175m) and in binding buffer (25 mm HEPES, pH 7.9, 3 mm MgCl2, 50 mm KCl, and 0.1 mm dithiothreitol). Multimerized wild-type probe (containing 8 copies of SEF-binding sites) or mutant probe (containing 10 copies of mutated SEF-binding sites) (1 × 106 cpm) was added to the probe solution (0.25% nonfat milk and 250 ng of poly(dI-dC)/ml in binding buffer) in a heat-sealed bag after the membrane had been incubated with 40 ml of blocking buffer (5% nonfat milk in binding buffer) for 1 h to block the nonspecific sites. After gentle mixing for 2 h at 4 °C, the membranes were washed in binding buffer, and the protein that bound the probe was visualized by autoradiography. Protein sequencing using mass spectrometry was carried out as described (27Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Briefly, DNA affinity-purified material accumulated from approximately 200 liters of HeLa cells was resolved by SDS-PAGE. The Coomassie Blue-stained 65-kDa protein band was in-gel-digested with trypsin, and the recovered peptides were analyzed using an electrospray ion trap mass spectrometer (LCQ, Finnigan MAT, San Jose, CA) coupled on-line with a capillary high performance liquid chromatograph (Magic 2002, Michrom BioResources, Auburn, CA). A 0.1 × 50-mm-MAGICMS C18 column (5-μm particle diameter, 200-Å pore size) with mobile phases of A (methanol:water:acetic acid, 5:94:1) and B (methanol:water:acetic acid, 85:14:1) was used with a gradient of 2–98% mobile phase B over 2.5 min followed by 98% B for 2 min at a flow rate of 50 μl/min. The flow was split with a Magic precolumn capillary splitter assembly (Michrom BioResources), and 1 μl/min was directed to the 100-μm column. The LC/MS was programmed to run in a data-dependent fashion. That is, the mass spectrometer was switched to the MS/MS mode to acquire collision-induced dissociation (CID) spectra once an ion signal was detected to exceed a preset value in the MS mode during the entire LC run. Data derived from the CID spectrum were used to search a compiled protein data base that was composed of the protein data base NR and a six-reading frame-translated Expressed Sequence Tag data base to identify the protein. A DNA fragment containing 306 bp of the 5′-flanking region and 45 bp of the untranslated exon 1 region of mouse SAA3 promoter was inserted into the Sma I site of the pGL3-Basic vector (Promega) to generate the pSAA3(−306)/Luc construct. The SEF cDNA was obtained by reverse transcription-polymerase chain reaction (Roche Molecular Biochemicals) and was inserted into the Xho I site of pCS2+MT vector (28Roth M.B. Zahler A.M. Stolk J.A. J. Cell Biol. 1991; 115: 587-596Crossref PubMed Scopus (267) Google Scholar), which contains six copies of the myc epitope fused in-frame at the N terminus of SEF. The integrity of this construct was confirmed by sequencing of the entire coding region. HepG2 cells were cultured in basal medium consisting of minimum essential medium and Waymouth MAB (3:1, v/v) plus 10% fetal calf serum (29Darlington G.J. Wilson D.R. Lachmann L.B. J. Cell Biol. 1986; 103: 787-793Crossref PubMed Scopus (292) Google Scholar) and were passaged at confluence by trypsinization once a week. pSAA3(−306)/Luc reporter was cotransfected with either SEF expression vector or empty vector into HepG2 cells using FuGENE method (Roche Molecular Biochemicals). Approximately 16 to 20 h after transfection, cells were stimulated with basal medium or 100 units of IL-1/ml. Cell extracts were assayed for protein content, and the luciferase activity was quantitated according to manufacturer's procedures. Originally identified in HepG2 and Hep3B cells, SEF activity was subsequently detected at high levels in several other cell types, including HeLa cells (20Huang J.H. Liao W.S.-L. Mol. Cell. Biol. 1994; 14: 4475-4484Crossref PubMed Scopus (53) Google Scholar). As HeLa cells can be easily cultured and grown as cell suspensions to a high cell density, we chose to use HeLa nuclear extracts as our starting material for the purification of SEF. Steps in the purification were carried out as described under “Experimental Procedures.” Protein eluates from each purification step were assayed for SEF binding activities using end-labeled C element containing the SEF-binding site as probe. As shown in Fig. 1, nuclear extracts and eluates from DEAE, heparin, and phenyl-Sepharose columns all showed strong SEF binding activities. Moreover, the binding activity is sequence-specific because the SEF-DNA complex could be completely inhibited by an excess of wild-type C element but not by the mutated C element. Although the DEAE-Sephacel and heparin steps only modestly increased the specific activity of SEF (TableI), they nevertheless efficiently concentrated the SEF activity and also eliminated some of the major contaminants in the crude nuclear extracts.Table IPurification of SEF from HeLa cell nuclear extractsPurification stepTotal proteinTotal activityYieldSpecific activityPurificationmg×103units1-aOne unit of SEF activity is defined as the amount of protein required to retard 10% of labeled DNA under standard assay conditions.%units/mgfoldNuclear extracts140561004.0 × 1021DEAE-Sephacel5052951.0 × 1032.6Heparin-agarose3751911.4 × 1033.5Phenyl-Sepharose225451.3 × 10432DNA affinity6 × 10−411201.8 × 1064500SEF activity was measured by EMSA.1-a One unit of SEF activity is defined as the amount of protein required to retard 10% of labeled DNA under standard assay conditions. Open table in a new tab SEF activity was measured by EMSA. The steps that achieved the most significant purification were the phenyl-Sepharose and DNA affinity chromatography steps. More than 90% of the protein from the heparin-agarose column either did not bind to the phenyl-Sepharose column or was eluted in the 30% ethylene glycol, 0.25 mNaCl wash (Fig. 2 A). Only about 6% of the protein loaded remained on the column and was eluted with 65% ethylene glycol. Some of the C element binding activity that apparently migrated at the same position as SEF was found in the flow-through fraction. There are two possible explanations for this observation, which are that the column capacity was insufficient for the amount of protein loaded, or the C element binding activity in the flow-through fraction may be not SEF but some interfering protein or proteins. To test the first possibility, we collected the flow-through and reloaded it onto a freshly prepared phenyl-Sepharose column. The binding activity was again recovered in the flow-through fraction; no binding activity was detected in the 30 and 65% ethylene glycol eluates (data not shown). The binding activity in the flow-through fraction was therefore not due to overloading of the column but rather may be due to another binding protein or proteins with properties different from those of SEF. To determine the sequence specificities of this binding activity, competition analysis was performed with32P end-labeled C element as probe and wild-type and mutant C elements as competitor DNAs. As shown in Fig. 2 B, both wild-type and mutant C elements competed for this C element binding activity, indicating that this activity was due to nonspecific DNA binding. In contrast to the flow-through fractions, fractions from the 65% ethylene glycol eluate contained specific SEF binding activity as they were specifically competed by the wild-type but not by the mutated C element oligonucleotides (Fig. 2 B). A second nonspecific binding activity (Fig. 2 B, lower band) that could be competed by both wild-type and mutated C elements was efficiently removed by the 30% ethylene glycol wash. Therefore, the phenyl-Sepharose column step not only resulted in a nearly 10-fold purification of SEF but, more importantly, efficiently removed two major nonspecific DNA-binding proteins that could have severely interfered with subsequent DNA affinity chromatography. To facilitate DNA affinity purification, we sought to define some parameters that would minimize protein degradation, preserve the integrity of the DNA affinity beads, and at the same time maintain maximum SEF binding. We examined the effects of various concentrations of EDTA, NaCl, and poly(dI-dC) on the ability of SEF to bind DNA. Our results showed that SEF binding activities were at or near optimal levels under a wide range of concentrations (2 to 18 mm EDTA, 50 to 110 mmNaCl, and 50 to 100 μg of poly(dI-dC)) (data not shown). Therefore, buffers used in DNA affinity chromatography included 10 mmEDTA, 100 mm NaCl, and 50 μg o" @default.
• W1998354003 created "2016-06-24" @default.
• W1998354003 creator A5000640302 @default.
• W1998354003 creator A5022614068 @default.
• W1998354003 creator A5028206626 @default.
• W1998354003 creator A5071531520 @default.
• W1998354003 creator A5077474550 @default.
• W1998354003 date "1999-08-01" @default.
• W1998354003 modified "2023-10-18" @default.
• W1998354003 title "Purification and Characterization of the Serum Amyloid A3 Enhancer Factor" @default.
• W1998354003 cites W1500915940 @default.
• W1998354003 cites W1520052348 @default.
• W1998354003 cites W1549520365 @default.
• W1998354003 cites W1597030361 @default.
• W1998354003 cites W1598873313 @default.
• W1998354003 cites W1861730213 @default.
• W1998354003 cites W1968424865 @default.
• W1998354003 cites W1968451804 @default.
• W1998354003 cites W1969677431 @default.
• W1998354003 cites W1978604713 @default.
• W1998354003 cites W1980014349 @default.
• W1998354003 cites W1982668809 @default.
• W1998354003 cites W1989497089 @default.
• W1998354003 cites W1993243592 @default.
• W1998354003 cites W2005397075 @default.
• W1998354003 cites W2013002081 @default.
• W1998354003 cites W2013890168 @default.
• W1998354003 cites W2024018102 @default.
• W1998354003 cites W2027569621 @default.
• W1998354003 cites W2037540469 @default.
• W1998354003 cites W2037872996 @default.
• W1998354003 cites W2040412368 @default.
• W1998354003 cites W2052151378 @default.
• W1998354003 cites W2075840772 @default.
• W1998354003 cites W2080928368 @default.
• W1998354003 cites W2100837269 @default.
• W1998354003 cites W2126726439 @default.
• W1998354003 cites W2139996002 @default.
• W1998354003 cites W2162545255 @default.
• W1998354003 cites W2340683598 @default.
• W1998354003 cites W2409785618 @default.
• W1998354003 cites W2417995055 @default.
• W1998354003 cites W314190729 @default.
• W1998354003 cites W4293247451 @default.
• W1998354003 cites W87340914 @default.
• W1998354003 doi "https://doi.org/10.1074/jbc.274.35.24649" @default.
• W1998354003 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10455131" @default.
• W1998354003 hasPublicationYear "1999" @default.
• W1998354003 type Work @default.
• W1998354003 sameAs 1998354003 @default.
• W1998354003 citedByCount "40" @default.
• W1998354003 countsByYear W19983540032012 @default.
• W1998354003 countsByYear W19983540032013 @default.
• W1998354003 countsByYear W19983540032014 @default.
• W1998354003 countsByYear W19983540032016 @default.
• W1998354003 crossrefType "journal-article" @default.
• W1998354003 hasAuthorship W1998354003A5000640302 @default.
• W1998354003 hasAuthorship W1998354003A5022614068 @default.
• W1998354003 hasAuthorship W1998354003A5028206626 @default.
• W1998354003 hasAuthorship W1998354003A5071531520 @default.
• W1998354003 hasAuthorship W1998354003A5077474550 @default.
• W1998354003 hasBestOaLocation W19983540031 @default.
• W1998354003 hasConcept C104317684 @default.
• W1998354003 hasConcept C111936080 @default.
• W1998354003 hasConcept C179104552 @default.
• W1998354003 hasConcept C185592680 @default.
• W1998354003 hasConcept C2777633098 @default.
• W1998354003 hasConcept C43617362 @default.
• W1998354003 hasConcept C55493867 @default.
• W1998354003 hasConcept C86339819 @default.
• W1998354003 hasConceptScore W1998354003C104317684 @default.
• W1998354003 hasConceptScore W1998354003C111936080 @default.
• W1998354003 hasConceptScore W1998354003C179104552 @default.
• W1998354003 hasConceptScore W1998354003C185592680 @default.
• W1998354003 hasConceptScore W1998354003C2777633098 @default.
• W1998354003 hasConceptScore W1998354003C43617362 @default.
• W1998354003 hasConceptScore W1998354003C55493867 @default.
• W1998354003 hasConceptScore W1998354003C86339819 @default.
• W1998354003 hasIssue "35" @default.
• W1998354003 hasLocation W19983540031 @default.
• W1998354003 hasOpenAccess W1998354003 @default.
• W1998354003 hasPrimaryLocation W19983540031 @default.
• W1998354003 hasRelatedWork W1531601525 @default.
• W1998354003 hasRelatedWork W2319480705 @default.
• W1998354003 hasRelatedWork W2384464875 @default.
• W1998354003 hasRelatedWork W2606230654 @default.
• W1998354003 hasRelatedWork W2607424097 @default.
• W1998354003 hasRelatedWork W2748952813 @default.
• W1998354003 hasRelatedWork W2899084033 @default.
• W1998354003 hasRelatedWork W2948807893 @default.
• W1998354003 hasRelatedWork W4387497383 @default.
• W1998354003 hasRelatedWork W2778153218 @default.
• W1998354003 hasVolume "274" @default.
• W1998354003 isParatext "false" @default.
• W1998354003 isRetracted "false" @default.
• W1998354003 magId "1998354003" @default.
• W1998354003 workType "article" @default.