Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum.

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH₂[³²P]O₄ and [³H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.     

Binding of 5S estradiol receptor to poly-deoxynucleotides

Calf uterus cytosol was incubated with (³H)estradiol and fractionated on Sephadex G-200. Two (³H)estradiol-binding protein fractions were obtained with sedimentation coefficients of 5.1 S and 3.5 S, respectively. The 5.1 S fraction bound to poly dT, poly dA:dT and poly dG:dC to a higher extent than to calf thymus DNA. The 3.5 S fraction did not bind to DNA.     

Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.     

Chicken oviduct progesterone receptor: Location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes

We have used a DNA-cellulose competitive binding assay to measure the extent of displacement of the chicken oviduct progesterone-receptor complex from calf thymus DNA-cellulose by purified cloned fragments of genomic DNA. Several DNA fragments from hormonally responsive genes coding for egg-white proteins were found to be efficient competitors for either crude or partially purified receptor complexes when compared with calf thymus DNA. Data obtained with deletion mutations constructed in vitro allowed delineation of a specific region necessary for strong competition, located 250–300 bp upstream from the mRNA startsite of the oval-bumin gene. Sequence homologies with this 5′-upstream region were observed in other fragments of the ovalbumin, conalbumin, ovomucoid, X and Y genes, which were also efficient competitors. Based on a comparison of such sequences of homology, a consensus sequence that may constitute a region binding progesterone-receptor complex has been constructed: ATC_TT^CCATT_T^ATCTG_T^GTTGTA. The results suggest that specific double-stranded DNA sequences are recognized by. the oviduct progesterone-receptor complex in vitro, and are relevant to the question of whether specific DNA sequences are directly involved as genomic binding sites for steroid receptors.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[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.     

Extraction of DNA-cellulose-bound glucocorticoid-receptor complexes with sodium tungstate

Glucocorticoid-receptor complex from rat liver cytosol, activated by warming at 23°C or fractionation with (NH₄)₂SO₄, was adsorbed over DNA-cellulose. This DNA-cellulose-bound [³H]triamcinolone acetonide-receptor complex was extracted in a dose-dependent manner by incubation with different concentrations of sodium tungstate. A 50% recovery of receptor was achieved with 5 mM sodium tungstate. Almost the entire glucocorticoid-receptor complex bound to DNA-cellulose could be extracted with 20 mM sodium tungstate. The [³H]triamcinolone acetonide released from DNA-cellulose following tungstate and molybdate treatment was found to be associated with a macromolecule, as seen by analysis on a Sephadex G-75 column. The glucocorticoid-receptor complex extracted by both the compounds sedimented as a 4 S entity of 5–20% sucrose gradients under low- and high-salt conditions. Addition of tungstate or molybdate to the preparations containing activated receptor had no effect on the sedimentation rate of receptor. However, addition of tungstate to non-activated receptor preparation caused aggregates of larger size. The tungstate-extracted glucocorticoid-receptor complex failed to rebind to DNA-cellulose even after extensive dialysis, whereas receptor in molybdate-extract retained its DNA-cellulose binding capacity.     

Application of capillary electrophoresis for DNA-protein binding tests

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration of sodium chloride. Electropherograms of the crude protein extracts show two fractions of proteins specific for dsDNA (calf thymus) and three fractions specific for ssDNA (calf thymus). Four and five fractions of proteins specific for ssDNA (P. sativum) were identified in the material isolated from cytosolic and nuclear extracts, respectively. Both ds- and ssDNA (calf thymus) form complexes with ca. 4.0 % of the total amount of proteins, while ssDNA (P. sativum) binds to ca. 11.0 % of cytosolic and 5.0 % of nuclear proteins.     

Endogenous Thymic Factors Regulating Cell Proliferation and Analysis of Their Mechanism of Action

Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. an extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G₁ S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. > 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. the active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4–4) inhibits [³H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosol induces apparent selectivity of glucocorticoid receptor binding to nucleic acids of different secondary structure

Unpurified rat liver glucocorticoid-receptor complexes within cytosol show a distinct binding preference for double-stranded DNA over single-stranded DNA; the binding to Escherichia coli rRNA is negligible. Extensive purification of the receptor abolishes its ability to distinguish among DNAs of different secondary structure and the affinity of the purified receptor toward RNA is greatly enhanced, reaching 30–50% of that of DNA. The purification effect is reversible: after cytosol addition to purified receptor preparation the binding preference restores. NaCl does not mimic the effect of cytosol. The flow-through fraction of a phosphocellulose column retains the ability of crude cytosol to produce selective decrease in the receptor binding to single-stranded DNA. This effect may also be observed by using two types of DNA-cellulose bearing double-stranded or denatured DNA, pretreated with crude cytosol. Additionally, pretreatment of immobilized DNA with even low cytosol concentrations has been shown to markedly enhance receptor binding, although this enhancement was lacking specificity with respect to DNA secondary structure. The nature of cytosolic active principle and some possible regulatory implications are discussed.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Characterization of androgen receptors after photoaffinity labelling with [3H]methyltrienolone (R1881)

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in rat prostate, in a human transplantable prostatic adenocarcinoma (PC-82) and in calf uterus. Androgen receptors preparations were partially purified either via differential chromatography on 2',5'-ADP-Sepharose (rat prostate), via anion exchange fast protein liquid chromatography (rat prostate and PC-82) or via DNA-cellulose chromatography (calf uterus). Purification factors obtained with the three different methods were: 245, 75 and 40 respectively. Photolabelling of receptor preparations was performed via irradiation with a high pressure mercury lamp either before or after partial purification. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of approx 95 kD. The covalent attachment of [3H]R1881 to the 95 kD protein could be completely suppressed by a 200-fold molar excess of dihydrotestosterone. In rat prostate cytosol an androgen receptor with a molecular mass of approx 50 kD could be photoaffinity labelled with R1881. A similar size was found for the androgen receptor in the human prostatic adenocarcinoma. Our results show that photoaffinity labelling of androgen receptors with [3H]R1881 as ligand can be applied for characterization of partial purified androgen receptor preparations.     

Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages

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.     

Comparison of the crystallin mRNA populations from rat,calf and duck lens. Evidence for a longer αA2-mRNA and two distinct αB2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding α-, β-, γ- and δ-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two αA₂-mRNAs of approximately 1250 and 1350 nucleotides and the αA^Ins-mRNA with a size similar to that of the largest αA₂-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of αA₂-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with αA sequences. The duck lens αA₂-mRNA appears to be 400–450 bases longer than the rat and calf lens αA₂-mRNAs. Furthermore, in contrast to the single αB₂-mRNA in rat and calf lens, two αB₂-mRNAs have been identified in duck lens, one, the major species, similar in size to the αB₂-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the αA₂- and αB₂-mRNAs most likely reside in their 3′-untranslated sequences.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Characterization of multiple forms of the Ah receptor: recognition of a dioxin-responsive enhancer involves heteromer formation.

We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.