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Biochemistry 1983,22, 13-21
A Rapid Method for Analysis of Ligand Binding to Deoxyribonucleic Acid and Soluble Nucleoproteins Using Streptomycin: Application to Steroid Receptor Ligands? Thomas C. Spelsberg
ABSTRACT:
A method is described which allows the rapid analysis of the binding of practically any molecules to DNA or to protein-DNA complexes (termed nucleoacidic protein or NAP). The antibiotic streptomycin sulfate, a soluble aminoglycoside, is used to precipitate the DNA after the ligand binding. Comparison of different sources and commercial batches of the antibiotic is described. Optimal conditions for precipitating DNA or NAP and the application of this method to the binding of the chick oviduct progesterone receptor to soluble NAP are described. The streptomycin method can be
used with DNA molecules whose size ranges from 750 base pairs to greater than 50000 base pairs. The method works with a DNA or NAP from a variety of sources, including synthetic homo- or heteropolymers. The precipitation of DNA or N A P by streptomycin occurs rapidly and has minimal effects on the steroid receptor complex or binding of the steroid receptor to DNA or NAP. The requirements and limitations of the method as well as the optimal conditions for binding of the progesterone receptor to DNA or NAP are described.
%e analysis of binding of soluble steroid receptors to soluble DNA or to specific protein-DNA complexes (termed nucleoacidic protein or NAP)’ has been plagued by difficulties in separating the DNA-bound steroid receptor complexes from the unbound steroid receptor (Thrall et al., 1978). A novel method has been developed whereby the soluble DNA or NAP bound with the chick oviduct progesterone receptor (PR) is precipitated with the antibiotic streptomycin sulfate, a soluble aminoglycoside. Streptomycin sulfate, on the other hand, displayed minimal damage to the PR and precipitated less than 1 .O% of the unbound PR while efficiently precipitating the DNA or N A P together with bound PR. Streptomycin is a water-soluble aminoglycoside derived from Streptomyces griseus. It was isolated as a bacteriostatin to Gram-negative bacteria (Waksman, 1953; Brock, 1966). Streptomycin binds to the cell membranes of bacteria and alters their membrane potential, causing a potassium efflux and later a magnesium efflux. These effects are now known to be minor and transitory to the bacteriostatin activity (Brock, 1966). The primary action of streptomycin is the inhibition of protein synthesis via binding to one or several proteins of the “30S”ribosomal subunit to terminate protein synthesis at some early stage after initiation (Brock, 1966;Balis, 1968; Hash, 1972). One of the first properties assigned to streptomycin was its precipitation of nucleic acids but not proteins or lipids (Cohen, 1947;Donovick et al., 1948;Moskowitz, 1963;Rybak & Gros, 1948;Euler & Heller, 1948;Waksman, 1949). It has been speculated that streptomycin acts as a polyvalent base to bind to polynucleotides (DNA or RNA) to form an intermolecular latticelike network (a cross-linking of distinct strands) sufficient to cause their precipitation (Cohen, 1947). The cationic diguanido group in the streptidine portion of streptomycin and the cationic iminium salt in the streptobiosamine portion of streptomycin are the suspected groups involved in the cross-linking and precipitation of nucleic acids. However, details of the optimal conditions for precip-
itation of nucleic acids, e.g., ionic strength, the DNA and streptomycin concentrations, DNA size, etc., have not been reported. This paper describes the requirements and limitations in the application of streptomycin to the analysis of the binding of PR to soluble DNA or NAP. The method has previously only been outlined briefly (Webster et al., 1976;Spelsberg et al., 1977). Studies were performed first to assess the efficiencies of different commercial preparations of streptomycin to precipitate avian DNA, second to determine the conditions needed to precipitate the DNA, e.g., size and species of DNA, ionic conditions, time, etc., third to determine the effects of the antibiotic on the integrity of the chick oviduct progesterone receptor (PR), and fourth to establish conditions whereby the streptomycin can be used to efficiently monitor PR binding to NAP. Lastly, a comparison is made between this streptomycin method and another method for PR binding to NAP.
From the Section of Biochemistry, Department of Cell Biology, Mayo Clinic and Graduate School of Medicine, Rochester, Minnesota 55905. Received June 18, 1982. This work was supported in part by Grants HD 9140-B and HD 16705 from the National Institutes of Health and the Mayo Foundation.
0006-2960/83/0422-0013$01.50/0
Materials and Methods Preparation of 3H-LabeledProgesterone Receptor Complex. The detailed procedure for the isolation and labeling of the PR from estrogen-stimulated chick oviduct has been described previously (Webster et al., 1976). Briefly, immature chicks were treated with diethylstilbestrol for 4-5 weeks, and the developed oviducts were excised. These oviducts contain cytosolic receptor specific for progesterone with little endogenous progesterone present. The freshly excised oviducts were immediately homogenized in 3 volumes (v/w) of Tesh buffer (10 mM Tris-HC1, 1 mM EDTA, and 12 M thioglycerol, pH 7.4) at 4 “C, and a lOOOOOg supernatant or Kcytosol”was prepared.
’
Abbreviations: NAP, nucleoacidic protein (this term refers to the residual DNA-protein complex after extraction of chromatin with 4.0 M guanidine hydrochloride, pH 6.0); [3H]PR, ”-labeled progesterone receptor from chick oviduct; Gdn-HCI, guanidine hydrochloride; Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; Tesh buffer, 10 mM Tris-HC1, 1 mM EDTA, and 12 mM thioglycerol, pH 7.4;TKM buffer, 50 mM Tris-HCI, 25 mM KC1, and 2 mM MgC12, pH 7.5; chromatin 1 , 80 mM NaCl 20 mM EDTA, pH 6.3; chromatin 2, 300 M NaC1; chromatin 3, 2 mM Tris-HC1 + 0.1 mM EDTA, pH 7.5; 1 X SSC, 15 mM NaCl 1.5 mM trisodium citrate, pH 7.0; NaDodS04, sodium dodecyl sulfate; PPO, 2,s-diphenyloxazole;POPOP, 1,4-bis[2-(5-phenyloxazolyl)]benzene.
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0 1983 American Chemical Society
14
BIOCHEMISTRY
The resulting cytosol was incubated for 2 h at 4 OC with 2 X M [3H]progesterone at 1 pCi/mL. The [3H]PR was then precipitated with 35% (w/w) (NH4)*S04,and the pellets were stored at -80 OC until needed (Schrader & O'Malley, 1972). The specifically bound [3H]progesterone (to receptor) was quantitated by using the Dextran-coated charcoal assay as described previously (Korenman, 1968; Boyd & Spelsberg, 1979a). Preparation of Chromatin and Nucleoacidic Protein (NAP). Shortly after the adult hens were sacrificed, the oviducts were excised, cleaned, and frozen in dry ice. When needed, the tissue was homogenized while still partly frozen in a Waring Blendor in buffered sucrose solutions, and the nuclei and chromatin were purified as described previously (Spelsberg et al., 1972; Pikler et al., 1976). All steps were performed at 0-4 OC. Briefly, the nuclei were sediiented in a 0.5 M sucrose solution in TKM buffer (50 mM Tris-HC1, 25 mM KCl, and 2 mM MgC12, pH 7.5) at lOOOOg,, for 10 min. The pellets were resuspended in a 2.0 M sucrose solution in TKM buffer by using a Teflon pestle-glass homogenizer and were resedimented at 60000ga, for 1 h. The nuclei were resuspended in a solution with 0.5 M sucrose in TKM buffer with 0.2% (w/w) Triton X-100, passed through 100-mesh organza cloth, and resedimented at 15000gavfor 20 min. These nuclei were used to isolate chromatin via suspension in a series of solutions as follows: chromatin 1 solution (80 mM NaCl and 20 mM EDTA, pH 6.3),chromatin 2 solution (300 mM NaCl), and chromatin 3 solution (2 mM Tris-HC1 and 0.1 mM EDTA, pH 7.5) as described elsewhere (Spelsberg & Hnilica, 1971; Spelsberg et al., 1971). The chromatin was then used either to prepare NAP-cellulose (described below) or to prepare unbound NAP. For the latter, the chromatin was extracted with 4 M Gdn-HC1 solution in 50 mM phosphate buffer, pH 6.0. The solution was centrifuged for 24 h at 4 OC at 80000gav. The pellet was resuspended in chromatin 3 solution and dialyzed against the same solution for 4 h, and the insoluble protein was removed by centrifugation for 10 min at 5000ga,. The supernatant represents the nucleoacidic protein (NAP) which has been the subject of previous studies of PR binding (Webster et al., 1976; Spelsberg et al., 1976a-q 1977, 1979; Thrall et al., 1978; Boyd & Spelsberg, 1979a,b). Preparation of NAP-Cellulose. The procedure for preparation of NAP-cellulose starting with chromatin, covalently attached to cellulose by ultraviolet light, has been described in detail elsewhere (Webster et al., 1976; Spelsberg et al., 1978). Briefly, a high-intensity UV light treatment in absolute alcohol was used to covalently couple chromatin to cellulose (Litman, 1968). This yielded a resin bound with a high amount of DNA which was stable to treatments of high concentrations of salt, urea, or Gdn.HC1 (Spelsberg et al., 1976b, 1978). DNA bound to resins which were prepared without UV light treatment resulted in the dissociation of the DNA from the resins in these reagents. The chromatincellulose was resuspended in 4 M Gdn-HC1 in 50 mM phosphate buffer, pH 6.0, at 1 mL/mg of cellulose for 2 h at 4 OC. This resin was collected by filtration and then washed again with the same Gdn-HC1solution. Lastly, the resin was washed extensively with chromatin 3 solution. This resin now consists of cellulose covalently linked to the nucleoacidic protein, which contains the nuclear binding sites (acceptor sites) for PR (Webster et al., 1976; Spelsberg et al., 1976a, 1977, 1979; Thrall et al., 1978; Boyd & Spelsberg, 1979a,b). Isolation of DNA. DNA was isolated from hen spleen nuclei as described previously (Spelsberg et al., 1972). All procedures were performed at room temperature unless oth-
SPELSBERG
erwise specified. Briefly, the nuclei were suspended in a solution containing 150 mM NaCl and 100 mM EDTA, brought to 1 M in NaClO,, and deproteinized repeatedly with chloroform-isoamyl alcohol. The DNA was precipitated with 2 volumes of cold ethanol, spooled on a glass rod, dried, and resuspended in a 1 X SSC solution (containing 15 mM NaCl and 1.5 mM trisodium citrate, pH 7.0) at 4 "C for 12 h. The solution was then made 0.33 M in sodium acetate and 0.1 mM in EDTA and the DNA precipitated with 0.6 volume of isopropyl alcohol. The precipitate was resuspended in 1 X SSC and was treated with RNase and Pronase. After enzyme treatment, the solution was made 1 M in NaC104 and 1% (w/w) in sodium dodecyl sulfate and incubated overnight at 22 "C. The solution was then extracted repeatedly with chloroform-isoamyl alcohol. The DNA solution was made 70% (w/w) in ethanol at -20 OC, and the DNA was spooled and resuspended in 0.1 X SSC overnight. This solution was adjusted to 0.33 M sodium acetate and precipitated again with isopropyl alcohol. This pellet of DNA was resuspended in 1 X SSC buffer and represented the purified "native" DNA used in these studies. Care was taken not to homogenize or stir the DNA solution too vigorously to prevent damage to the DNA or NAP which affects PR binding (Thrall & Spelsberg, 1980). The final product was analyzed for purity by measuring DNA with the diphenylamine reaction (Burton, 1956), RNA by the orcinol reaction (Cerriotti, 1955), and protein by the Lowry method (Lowry et al., 1951). An acceptable DNA preparation contained less than 0.5% (w/w) protein or RNA with respect to DNA. If > 1% protein was found, further deproteinization steps were carried out by repeating treatments of Pronase, chloroform-isoamyl alcohol, and NaDodS04. Fragmentation of DNA. Native DNA at 25 pg/mL in dilute buffer (Le., 1 X SSC or chromatin 3 solution) was physically sheared in a Virtis 60 homogenizer at 40 000 rpm at 4 OC with five blades attached to the rotating shaft. The time of shear varied to obtain different sizes of DNA. The extent of shearing was monitored by acid precipitation and by gel electrophoresis in 2.0% acrylamide and 0.5% agarose (Peacock & Dingman, 1968). More than 90% of the DNA was rendered acid soluble under the more extensive shearing. DNA fragments with.a mean size range of 750-12000 base pairs could be generated, depending on the degree of shear. The size was determined by polyacrylamide gel electrophoresis with Hind11 restriction fragments of phage X for reference (Landy et al., 1974). Binding of [ 3H]PR Complex to DNA or to Nucleoacidic Protein Using the Streptomycin Assay. Two methods were used in this paper for measuring the interaction of PR with DNA or with NAP (Webster et al., 1976;Thrall & Spelsberg, 1980; Spelsberg et al., 1979; Thrall et al., 1978). Both binding techniques allow rapid processing of multiple samples. The methods render the DNA insoluble to facilitate its separation from soluble unbound [3H]PR. As described above, the PR was partially purified by ammonium sulfate precipitation (Webster et al., 1976; Schrader & O'Malley, 1972; Boyd & Spelsberg, 1979a,b) since it was found that excessive protein in the binding assays can affect the P R binding to DNA or NAP. One method involves DNA or NAP coupled to cellulose (prepared from chromatin-cellulose as described above) in the binding assays which renders the DNA insoluble. This method is described only briefly in the next section since it has been described in detail (Webster et al., 1976; Boyd & Spelsberg, 1979a,b; Spelsberg et al., 1976a-c, 1977; Thrall et al., 1978). The second method (the streptomycin method) utilizes the antibiotic streptomycin sulfate at the end of incubation to
RAPID METHOD FOR LIGAND BINDING TO D N A
precipitate the DNA or NAP together with any bound PR. The procedure has only briefly been outlined previously (Thrall et al., 1978; Boyd & Spelsberg, 1979a,b). The optimal conditions, which are the subject of this paper, for this method are described as follows. A minimum of 50 pg of DNA (as pure DNA or as NAP) per mL of assay was incubated in a buffered 0.1 M KCl solution with a given amount of receptor for 90 min at 4 OC. Only partially purified steroid receptor complexes were used since excess soluble protein interfered with the streptomycin-induced precipitation of DNA (probably by binding the streptomycin). In contrast, when DNA itself was bound by chromosomal proteins, the precipitation by streptomycin was enhanced. After incubation with [3H]PR, streptomycin was then added to a final concentration of 1.0 mg/mL or 0.1% (w/w) in the assay, which rendered the DNA or NAP insoluble within a few minutes. As will be shown in this paper, this level of antibiotic had minimal effects on the integrity of the progesterone receptor while precipitating maximal amounts of the DNA or NAP. However, some differences in the effect on PR were found between different commercial sources of the compound. The source of the antibiotic or the size of the DNA (from 750 to 50000 base pairs) had little effect on the efficiency of DNA precipitation. After the addition of 0.1% streptomycin, the solutions were incubated for 15 min and were centrifuged at 5000g for 10 min at 4 OC. The pellets of the streptomycin-DNA or -NAP complexes were then washed twice (by resuspending) in a wash solution containing 0.02% (w/w) streptomycin in chromatin 3 solution. Each wash was followed by centrifugation of the streptomycin-NAP complex at 5000gav. Lower centrifugations resulted in lower recoveries of DNA if sizes of less than 5000 base pairs were used. The pellets of N A P were resuspended in the same wash solution and were collected on membrane filters of mixed ester cellulose (type HA, 24 mm, 0.45-pm pore size; Millipore Corp., Bedford, MA). The filters were then dried and were counted for radioactivity in a standard toluene-PPO-POPOP scintillation cocktail. After the filters were counted, they were removed and dried, and the DNA was quantitated by using the diphenylamine reaction of Burton (1956) as described elsewhere (Webster et al., 1976; Thrall & Spelsberg, 1980; Spelsberg et al., 1971; Boyd & Spelsberg, 1979a,b). The blanks represented either assays containing no NAP or complete assays with free [3H]progesterone. When fragmented DNA was used, modification of the streptomycin method was necessary to improve the efficiency of the DNA recovery. In this instance, a 50% increase in DNA and streptomycin concentrations per milliliter of assay was employed, and the centrifugation was performed at 5000g for 10 min to enhance the recovery of the DNA precipitation. Binding of [3H]PRto NAP Using the Cellulose Method. Since this method has been described in detail previously (Webster et al., 1976; Spelsberg et al., 1978), it will only be briefly outlined here. In the cellulose method, a 25 pg/mL sample of the cellulose-bound NAP or DNA was incubated in Tesh buffer containing 0.15 M KCl at 4 "C. The NAP or DNA was sedimented by centrifugation at lOOOg for 10 min, and the pellets were washed twice by resuspensions in chromatin 3 solution. Pure cellulose was added in the same amount to the blank assays as was added to the experimental assays. After the incubation, the blank assay tubes were washed in a manner similar to that of the experimental assay tubes with the last wash transferred to the filters. The radioactivity per blank filter was subtracted from the respective experimental filter values. The residual values of bound radioactivity were
VOL. 22, NO. 1, 1983
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then adjusted to a per milligram of DNA basis by using the quantity of DNA per filter estimated as described in the previous section. Results Initial studies to identify compounds which would precipitate the soluble DNA or N A P along with the bound steroid receptor without precipitating the unbound steroid receptor involved a variety of compounds including quaternary ammonium salts, cationic polypeptides, and antibiotics. Of these, only streptomycin sulfate effectively performed the above. The quaternary ammonium salt cetyltrimethylammonium bromide, while efficiently precipitating DNA or NAP, readily destroyed the chick oviduct PR. The basic proteins not only precipitated the DNA or the NAP but also precipitated much of the PR whether bound or unbound to DNA. Further, the conditions for effective analysis of steroid receptor binding to NAP were found to be critical. Preliminary studies were performed to establish optimal conditions only for streptomycin precipitation of NAP. These studies are described in this paper for the potential application of this technique to other ligand binding studies. Conditions which may be optimal for binding of steroid receptors to DNA or NAP may not be optimal for the analysis of other ligand interactions. Early characterization of different preparations of streptomycin from two sources, Calbiochem-Behring Corp. (La Jolla, CA) and Eli Lilly (Indianapolis, IN), showed no differences in their ability to precipitate DNA or NAP or in their elution patterns from reverse-phase chromatography using high-pressure liquid chromatography (data not shown). Only slight differences in their absorption spectra (data not shown) and marked differences in their effects on the progesterone receptor (described below) were observed. The recoveries of DNA or NAP by streptomycin precipitation were found to be improved by using a higher centrifugal force (5000ga,) compared to a lower centrifugal force (2000g,,). The particular buffer one uses in the precipitation assay was found not to be critical as long as the ionic strength was controlled (discussed below). The pH of the buffers between pH 6 and 8 showed little effect on the precipitation of DNA (data not shown). Above pH 8.0, the efficiency decreased rapidly. Figure 1A shows the effect of increasing streptomycin concentration on the recovery of pure DNA or NAP by using approximately 50 pg of DNA (or NAP) per mL of Tesh buffer and with centrifugation at 2000gav. At this DNA concentration, 0.1% (w/w) streptomycin [giving a streptomycin/ DNA (w/w) ratio of around 201 resulted in maximal (80-95%) precipitation of hen oviduct NAP or hen spleen DNA. As shown in Figure lB, the concentrations of DNA and NAP in the assay solutions were also critical for recovery of DNA. A minimal concentration of 50 pg of DNA (as NAP) was found to be required for optimal precipitation. The recovery of DNA was the same whether the streptomycin concentration in the assay was kept constant or varied with the DNA concentration to maintain a constant ratio (see Figure 1B). Above 50 pg of DNA/mL, there was little improvement in recovery. The ionic strength was also found to be important. Figure 2A shows that the precipitation of the DNA as well as integrity of the streptomycin-DNA complex decreased in a linear pattern from 0.001 M KCl and was abolished by 0.2 M KC1. This effect of ionic strength was independent of the salt used [Le., monovalent, divalent, NaCI, KCl, (NH4)2S04,etc.]. The effect of the ionic strength was moderately dependent on the concentration of streptomycin in the assay. Figure 2B shows that at higher concentrations of streptomycin and in the presence of the progesterone re-
16 B I O C H E M I S T R Y
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pg streptornycin/pg DNA 44.47 9.5 I 48.9
O0
28.4
.
37.9
L,,a2500 base pairs. This marked selectivity was not observed with the transcribed X and Y genes nor with transcriptionally inert DNA sequences adjacent to the ovalbumin and X genes. Since the X and Y genes are transcribed at low rates as compared to ovalbumin, these results suggest that micrococcal nuclease can distinguish between genes that exhibit different transcriptional rates. So that a more complete description of the transcriptional domain of the ovalbumin chromatin could be provided, the endogenous nuclease sensitivity of the ovalbumin gene and regions im-
A
model system for the study of transcriptional regulation is the chicken oviduct which synthesizes ovalbumin and other egg white proteins in response to steroid hormones [for a review, see OMalley et al. (1979)l. The ovalbumin gene exists within a multigene family that is composed of three genes, X,
From the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 (J.N.A. and J.N.V.), and the Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 (G.M.L., M.-J.T.,and B.W.O.). Received June 14, 1982. This work was supported by Grant R01-CA25799-03 from the National Cancer Institute (J.N.A.),National Institutes of Health Grant HD-08188 (B.W.O.), and Grant HD-07495 (B.W.O.) from the Baylor Center for Reproductive Biology. J.N.V. is supported by a predoctoral NIH traineeship. *Present address: Clinic Chemistry Section, Mayo Clinic, Rochester, MN.
0006-2960/83/0422-0021$01 SO10
mediately adjacent to this gene was also examined by blot hybridization analysis. The entire coding region of the ovalbumin gene in hen and hormone-treated chick oviduct nuclei was preferentially attacked by this nuclease as compared to the inactive sequences flanking the ovalbumin transcriptional unit and the ovalbumin gene sequences in nuclei from hormone-withdrawn chicks. The endogenous nuclease also introduced a series of site-specific cleavages upstream from the ovalbumin gene in oviduct nuclei from the hen, hormonestimulated, and hormone-withdrawn chicks. These nuclease cutting sites were not detected in digested nuclei from erythrocytes or kidney. The selective nuclease cutting sites flanking the ovalbumin gene are present, therefore, in oviduct cells prior to the transcriptional activation of the gene whereas the coding region is rendered nuclease sensitive only after hormone treatment.
Y, and ovalbumin (Royal et al., 1979; Colbert et al., 1980). These genes, which exhibit limited sequence homology, are expressed in a tissue-specific manner by the oviduct and are coordinately induced by steroid hormones. The maximal rates of transcription of genes X and Y , however, are no more than about 3% and 9%, respectively, of the transcriptional rate of ovalbumin (Colbert et al., 1980; LeMeur et al., 1981). Recent studies using extensive DNase I digestion to examine the chromatin configuration of the ovalbumin gene family have revealed that the ovalbumin, X, and Y genes exist within a contiguous DNase I sensitive domain containing over 100 kilobase(s) (kb) of DNA in hen oviduct chromatin whereas the entire domain is resistant to digestion in spleen, liver, and erythrocyte nuclei (Lawson et al., 1980, 1982). The packaging of the ovalbumin and related X and Y genes in this common 0 1983 American Chemical Society
