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1.
DNA-binding proteins in the cytoplasm of vaccinia virus-infected mouse L-cells. 总被引:4,自引:4,他引:0
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Mouse L cell fibroblasts were infected with vaccinia virus and labeled 2 to 3 h postinfection with [35S]methionine. Labeled proteins were fractionated on native and denatured DNA-cellulose columns and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four 90,000 to 12,500, were detected. VDP-12A (molecular weight, 29,750) had affinity for denatured but not native DNA, and its synthesis was dependent on viral DNA replication. VDP-20 (molecular weight, 41,000) bound very tightly to native and denatured DNA and was displaced only after boiling the protein-DNA-cellulose matrix in 1% sodium dodecyl sulfate. VDP-8,-11,-12,-13, -and-14 behaved electrophoretically like the polypeptide species previously shown to be present in DNA-protein complexes prepared from infected cells. The molecular weights of VDP-10 (50,000), VDP-11 (36,000), and VDP-8 (67,000) were similar to the polypeptide subunits of polyadenylate polymerase and phosphohydrolase I, enzymes purified from virions which have also been shown to have affinity for DNA. 相似文献
2.
DNA-binding proteins in human fibroblasts were examined by chromatography on DNA-cellulose columns. By successive chromatography on columns containing native, denatured, and UV-irradiated DNA-cellulose respectively the proteins binding to different types of DNA could be studied. Elution of the columns with sodium chloride followed by polyacrylamide gel electrophoresis allowed several DNA-binding proteins to be identified. All of the major DNA-binding proteins were present in strains of xeroderma pigmentosum cells respectively deficient in excision-repair and post-replication repair of ultraviolet-induced damage. 相似文献
3.
4.
PREVIOUS communications1,2 have described the synthesis in cultured cells of proteins with affinity for columns of denatured DNA-cellulose3. Polyacrylamide gel electrophoresis of the proteins eluted from such columns by 0.15 M NaCl has shown differences between the DNA-cellulose binding proteins synthesized by growing and resting fibroblasts1,2. One protein, P-6, later shown to have a molecular weight in SDS of 40,000, was synthesized only by growing cells, while P-1 and P-1′, much larger proteins, were very prominent only in cells resting due to serum limitation and contact inhibition. Investigation of the nature and role of P-6 and other proteins synthesized by growing cells is continuing; as for the DNA binding proteins synthesized by resting cells, we report here that P-1 and P-1′ do not seem to be related to growth control, as suggested by the earlier experiments, but are collagen type molecules, probably larger molecular weight protocollagen precursors of the α1 and α2-chains respectively. 相似文献
5.
Siu Sing Tsang Urs Kuhnlein 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,697(2):202-212
A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl2 and MnCl2 at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10?11 M. There are at least 1·105 DNA-binding protein molecules/HeLa cell. 相似文献
6.
Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus. 总被引:21,自引:0,他引:21
We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes. 相似文献
7.
DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells. 总被引:43,自引:32,他引:11
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Affinity chromatography on single-stranded and double-stranded DNA-cellulose indicates that 12 proteins previously identified from herpes simplex virus type 2-infected cells, ranging in molecular weight from 28 X 10(3) to 186 X 10(3), bind to DNA-cellulose. The DNA-binding proteins found in infected cells differed in relative binding strengths for denatured DNA-cellulose. The virus specificity of these DNA-binding proteins was further studied by comparison with DNA-binding proteins isolated from mock-infected cells, and by immunoprecipitation of infected-cell DNA-binding proteins with antisera specific for viral antigens. The promise this technique holds for the purification and study of polypeptides involved in virus DNA replication, recombination, or repair is discussed. 相似文献
8.
Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages 总被引:6,自引:5,他引:1
The isolation of deoxyribonucleic acid (DNA)-binding proteins from various stages of growth and sporulation of Bacillus subtilis is described. After adsorption and elution from phosphocellulose, the proteins were fractionated according to their ability to adsorb to denatured calf thymus DNA-cellulose or native B. subtilis DNA-cellulose. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purification was monitored by a nitrocellulose filter binding assay. Approximately 1% of the proteins in the crude extract adsorbed to denatured calf thymus DNA-cellulose and 0.1% adsorbed to native B. subtilis DNA-cellulose. Each class of proteins varied qualitatively and quantitatively as sporulation proceeded. Several proteins from the exponential phase of growth that bound to denatured DNA were lost by T(0), whereas at T(5) new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and T(0); however, the dominant species in these eluates were clearly different. 相似文献
9.
DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA. 相似文献
10.
Up to seven early poxvirus-specific proteins have been isolated from vaccinia-WR-infected and cowpox-virus-infected chick embryo fibroblasts by affinity chromatography on native DNA-cellulose columns. The proteins have been characterized by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by nonequilibrium pH-gradient electrophoresis. The molecular weights of the viral proteins were determined by comparison with proteins of known molecular weight and are comparable to several of the vaccinia-WR-specific DNA-binding proteins isolated previously from infected L-929 cells by Solosky J. M., Esteban M. and Holowczak J.A. [J. Virol. 25, 263-273 (1978)]. The viral proteins binding reversibly to native DNA have been classified as immediate early viral gene products. Synthesis of cowpox-virus-induced early DNA-binding proteins is inhibited in chick cells pretreated with homologous interferon at a concentration of 500--1000 units/ml. 相似文献
11.
Summary A salt-extraction procedure was used to isolate a nucleolar nonhistone protein fraction, containing [32P]phosphoserine, from the nucleoli of Novikoff hepatoma ascites cells. These proteins are similar in amino-acid composition to whole nuclear (chromosomal) nonhistone proteins. DNA-cellulose column chromatography showed that this fraction contains DNA-binding phosphoproteins, some of which will bind only to homologous (Novikoff) nucleolar or nuclear DNA. 相似文献
12.
A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity. 相似文献
13.
DNA-binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA-specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg(2+) at pH 7.4. Enzyme activity was completely inhibited by EDTA. 相似文献
14.
DNA of acidothermophilic archaebacterium Sulfolobus acidocaldarius has a base composition of about 40 mol% G + C content. A low intracellular salt concentration has been inferred for this organism. These features and the high optimal temperature of growth (75°C) would have a destabilising effect on the helical structure of the intracellular DNA. Hence, the nucleoid of this organism has been isolated in order to analyse its proteins composition and to identify any protein factors responsible for stabilisation of the organism's DNA at its growth temperature. The acid-soluble fraction of the nucleoid contains four low-molecular-weight basic proteins. The four proteins have been purified to homogeneity and antibodies to these proteins have been raised in rabbits. Immunodiffusion results suggest that the proteins are antigenically distinct. Three proteins (A, C and C′) stabilise different double-stranded DNA during thermal denaturation and increase Tm of DNA by about 25 C°. These proteins are referred to as helix-stabilising nucleoid proteins (HSNP). Protein B (referred to a DNA-binding nucleoid protein, DBNP-B) does not show helix-stabilising effect. None of the four proteins stabilises double-stranded RNA. The four proteins bind to native and denatured DNA to different extents as measured by DNA-cellulose chromatography and [3H]DNA binding by filtration. We suggest, based on the DNA binding, histone-like and helix-stabilising properties, that the intracellular function of these proteins is to prevent strand separation of DNA at the optimal temperature of growth (75°C). 相似文献
15.
Jan M.B. Carlstedt-Duke Ulla-Britt Harnemo Bertil Högberg Jan-Åke Gustafsson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,672(2):131-141
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to a specific, high-affinity, low-capacity protein in rat liver cytosol. The TCDD-receptor complex is a large molecule with a Stokes radius of 6.6 nm as determined by gel filtration on calibrated columns. The receptor complex sediments at 5.0 S on glycerol gradients. The calculated molecular weight from the physical parameters was 136 000 and the frictional ratio 1.79.The TCDD-receptor complex binds to DNA-cellulose without preceding heat activation or incubation at high ionic strength. The receptor must first bind TCDD before it can interact with DNA. The DNA-binding ability can be removed from the TCDD receptor by limited proteolysis with trypsin. This treatment does not affect the TCDD-binding site of the receptor. The proteolytic fragment of the TCDD-receptor complex containing the TCDD-binding site but not the ability to bind to DNA appears to be approximately the same size as the native receptor, as judged from chromatography of Sepharose CL-6B and glycerol gradient centrifugation. 相似文献
16.
M H Cake J A Goidl L G Parchman G Litwack 《Biochemical and biophysical research communications》1976,71(1):45-52
[3H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [3H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur. 相似文献
17.
DNA-binding activity of retinoblastoma protein is intrinsic to its carboxyl-terminal region 总被引:4,自引:0,他引:4
N P Wang P L Chen S Huang L A Donoso W H Lee E Y Lee 《Cell growth & differentiation》1990,1(5):233-239
The retinoblastoma (RB) gene encodes a nuclear phosphoprotein with a molecular weight of 110,000 (pp110RB) associated with DNA-binding activity. This sequence-nonspecific DNA binding activity was further studied by Southwestern and DNA-cellulose chromatography using purified fusion proteins expressed in Escherichia coli. Three fusion proteins, containing amino acids 612-775, 776-928, and 612-928 of pp110RB, bound to DNA; the binding affinity of the latter was approximately 20-fold higher than those of either smaller region. Other regions of pp110RB had no detectable binding activity, indicating that the carboxyl-terminal region of the RB protein is the major domain responsible for interacting with DNA. Since several potential phosphorylation sites reside within this region, isoforms of RB protein from cellular lysates with various degrees of phosphorylation were compared with respect to their DNA-binding affinity. The hyperphosphorylated form was eluted from DNA-cellulose columns at 0.1-0.3 M NaCl, whereas the hypophosphorylated form appeared in the eluates only at salt concentrations of 0.4-0.7 M, implying that phosphorylation of RB protein may affect its DNA-binding activity. That pp110RB can bind DNA intrinsically, and that this activity can be modulated by phosphorylation, is consistent with the proposed regulatory role of the RB protein in cell growth and differentiation. 相似文献
18.
DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125–0.15M potassium phosphate. PC fractions containing electrophoretically pure 34KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3–5 mM Mg2+ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate. 相似文献
19.
Changes in synthesis of DNA-binding proteins during the onset of transformation in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.
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Synthesis of cytoplasmic DNA-binding proteins was investigated after a shift from the nonpermissive to the permissive temperature in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus [ts339(RSV)]. Cells were labeled for several generations in [3H]leucine and were pulse-labeled with [35S]methionine for 1 h at the nonpermissive temperature (39 degrees C) and at the permissive temperature (33 degrees C, 5 h after shift from 39 degrees C). Proteins binding to sequential columns of double-stranded and single-stranded DNA-cellulose were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the 35S/3H ratios were obtained for each column fraction and for individual polypeptides. The protein fractions binding to single-stranded, but not double-stranded, DNA and eluting at high salt concentrations (greater than 0.60 M NaCl) showed elevated 35S/3H ratios. This indicated increased synthesis of these proteins within 5 h after the onset of transformation. The majority of the polypeptides in these fractions showed increased synthesis as a consequence of transformation. One prominent polypeptide among them constituted 0.1% of the cytosol protein and had a molecular weight of 93,000. We conclude that the synthesis of proteins binding tightly to single-stranded DNA is increased early after the onset of transformation. 相似文献
20.
Katsumi Yoshida Paul J. Davis Marion Schoenl 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(2):332-345
Dog kidney cytosol contains a high molecular weight (50 000–70 000) and a low molecular weight (approx. 6000) thyronine-binding protein. Low molecular weight cytosol thyronine-binding protein has not been previously recognized in cytoplasm. Binding of thyroxine (tetraiodothyronine, T4) by the low molecular weight protein has a half-time of association of more than 24 h and accounts for 32% of bound cytoplasmic tetraiodothyronine after 48 h of incubation. Binding of labeled tetraiodothyronine and triiodothyronine by this moiety is non-dissociable in the presence of 1 · 10?5 M unlabeled tetra- or triiodothyronine. The low molecular weight protein exists in a dispersed and apparently aggregated form; the latter elutes in the void volume on Sephadex G-100 and its generation is minimized by 2 mM Ca2+. This binding protein elutes in a fraction which has a high A260nm : A280nm ratio, is pentose enriched (orcinol method) and which, because of these characteristics and low susceptibility to digestion by nuclease, is postulated to be a ribosylated cytoplasmic protein or polypeptide.Binding of tetra- and triiodothyronine by the high molecular weight protein has a half-time of association of 2 h and is saturable. Displacement of labeled triiodothyronine from this cytosol thyronine-binding protein is more readily effected with excess unlabeled tetra- than with triiodothyronine, indicating the absence of a triiodothyronine-specific cytosol thyronine-binding protein site. 3,3′,5′-Triiodothyronine (reverse triiodothyronine) is bound with low avidity. Uptake of high molecular weight protein by isolated kidney cell nuclei cannot be demonstrated.Binding of tetraiodothyronine by cytosol proteins is independent of pH in the pH range 6.8–8.9, but binding of triiodothyronine is minimized at pH 7.4 and enhanced at alkaline pH to the point of equivalency of tetra- and triiodothyronine binding at pH 8.9.At concentrations of tetraiodothyronine calculated to exist intracellularly, essentially all soluble fraction tetraiodothyronine is bound to cytosol thyronine-binding protein, restricting access of this iodothyronine to binding sites in nucleus and mitochondria. Cytosol removes labeled tetra- and triiodothyronine previously reacted in vitro with isolated cell nuclei; such removal is a linear function of cytosol protein concentration and is blocked by saturation of cytosol thyronine-binding protein with unlabeled iodothyronines. Only the high molecular weight protein accounts for unbinding by cytosol of nuclear hormone. 相似文献