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Chapter 19 Isolation and Characterization of Nonhistone Phosphoproteins
Chapter 19 Isolation and Characterir(uation of Nonhistone P hosphoproteins TUNG YUE WANG
AND NINA
C. KOSTRABA
Division of Cell and Molecular Biology. State University of New York at Buffalo. Buffalo, New York
I. Introduction The nonhistone chromosomal proteins are believed to play a regulatory role in the control of gene expression. One evidence supporting this regulatory role for the nonhistone proteins is their ability to alter transcription in vitro. In this chapter, we limit ourselves to the preparative procedures and characteristic descriptions of only those nonhistone phosphoproteins that directly stimulate or inhibit transcription in vitro from DNA or chromatin. At present, three nonhistone phosphoprotein fractions have been shown to exert such direct effects on transcription. For convenience, these three nonhistone protein fractions are referred to here as NHP-I, NHP-11, and NHP-111. One fraction (NHP-I) enhances the transcription from chromatin, as shown in liver, spleen, and kidney of rat ( 2 - 9 , in Walker 256 carcinosarcoma (2,3), in frog liver (6), in calf endometrium (7), and in cultured carrot cells (8). The second fraction (NHP-11) binds selectively to, and stimulates transcription from, homologous DNA (9-12). This nonhistone protein fraction has been demonstrated in rat liver ( I I , 2 3 ) ,in KB cells (24), and in Ehrlich ascites tumor cells (22). The third fraction (NHP-111), isolated from Ehrlich ascites tumor (15) and calf thymus inhibits DNA-directed RNA synthesis in vitro. NHP-I, the nonhistone protein fraction that stimulates transcription from chromatin in vitro, is a highly heterogeneous group of proteins containing RNA. It stimulates transcription from chromatin isolated from the same 317
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tissue of origin as the nonhistone protein fraction as well as that from other tissues. However, NHP-I stimulates the chromatin template of homologous tissue to a greater extent than heterologous chromatin (2,3,6). Like NHP-I, NHP-I1 is also heterogeneous but, as prepared, contains no nucleic acid. NHP-I1 selectively binds to, and enhances transcription of, homologous DNA (9-13). The specificity of NHP-I1 is such that it interacts with and activates transcription of only unique sequences in DNA (16).The mechanism of activation by NHP-11 is stimulation of the initiation of RNA synthesis (16,17). NHP-I11 has been isolated as a near-homogeneous nonhistone protein. This nonhistone protein binds to, and inhibits transcription of, reiterated sequences in DNA, but not unique DNA sequences, nor chromatin (17). It inhibits the transcription of repetitive DNA sequences by acting at the initiation step of RNA synthesis (15,17). In brief, two of the nonhistone protein fractions (I and 11) can be shown to stimulate transcription in vifro, one (NHP-I) by acting on chromatin and the other (NHP-11), on DNA. The third fraction (NHP-111) inhibits transcription from DNA. The varied but specific effects of these nonhistone proteins indicate the involvement of nonhistone chromosomal proteins in both positive and negative controls in the differential regulation of gene expression.
11. NHP-I. The Nonhistone Proteins That Stimulate Transcription of Chromatin A.
Preparative Procedure
Bufers: Buffer A. 0.02 M Tris-HC1 (pH 7.6)-0.15 M NaCl. Buffer B. 0.02 M Tris-HC1 (pH 7.6). Buffer C. 0.02 M Tris-HC1 (pH 8.0)-2.0 M NaCl. Buffer D. 0.02 M Tris-HC1 (pH 7.2)-0.4 M NaCl. Buffer E. 0.015 M Tris-HC1 (pH 8.0)-1 mM EDTA-1 mM p-mercaptoethanol. Buffer F. 0.015 M Tris-HC1 (pH 8.0)-0.05 MNaCl-1 mMEDTA-1 mM p-mercaptoethanol. Buffer G. 0.015 M Tris-HC1 (pH 8.0)-0.15 M NaC1-1 mM EDTA-1 mM p-mercaptoethanol. Buffer H. 0.015 M Tris-HCl (pH 8.0)-0.30 MNaCl-1 mMEDTA-1 mM p-mercaptoethanol. Buffer I. 0.02 M Tris-HCl (pH 8.0).
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Step 1. NHP-I is prepared from crude chromatin (saline-extracted nuclei). The method of Chauveau et al. (18) is applicable to the isolation of nuclei from rat and most other solid tissues, but other methods may also be used. For the isolation of nuclei, the tissue, trimmed and cleansed of fat and connective tissue, is forced through a Harvard press into a pulp. The macerated tissue is weighed and then homogenized with a Dounce homogenizer in 9 volumes of 2.4 M sucrose-10-4 M CaCl, to a 10% (w/w) homogenate. The suspension is centrifuged at 75,000 g for 1 hour. At the end of the centrifugation, the pellets are collected, resuspended in 10 volumes of 0.33 M sucrose, and centrifuged at 1000 g for 5 minutes. The isolated nuclei are recovered in the pellets. Step 2. The isolated nuclei are extracted with 100 volumes of Buffer A by first homogenizing them in a loose-fitting Dounce homogenizer and then stirring for 1 hour. The nuclear suspension is centrifuged at 25,000 g for 15 minutes, discarding the supernatant. This step of extraction with Buffer A is repeated twice. Step 3. The saline-extracted nuclei are suspended in 20 volumes of Buffer B and stirred. During the stirring, an equal volume of Buffer C is added slowly into it, and the stirring is continued for 4-6 hours. The mixture is then centrifuged at 75,000 g for 90 minutes. The supernatant is collected and its volume measured. To this supernatant solution 1 .5 volumes of Buffer B are added drop by drop with stirring to a final concentration of 0.4 M with respect to NaCl. The resulting mixture with its formed precipitate, which consists mostly of DNA and histones, is centrifuged at 75,000 g for 90 minutes. The supernatant solution from this centrifugation is recovered and dialyzed against Buffer D with three changes of the buffer. Step 4. Bio-Rex 70 (Na + form, minus 400 mesh) is suspended in Buffer D, and the pH of this mixture is adjusted to 7.2 with HC1. The mixture is centrifuged at 15,000 g for 5 minutes, and the sedimented resin is equilibrated with Buffer D for 30 minutes and again centrifuged to remove the buffer. The resin is then mixed with the dialyzed solution obtained from Step 3 in a protein-to-resin ratio of approximately 1/ 100(w/w). This mixture is stirred for 10 minutes and is allowed to settle for another 30 minutes. The unadsorbed acidic proteins are collected by centrifugation. The resin is washed with Buffer D, centrifuged, and the wash is combined with the first supernatant solution. The combined solution is dialyzed against 3 volumes of Buffer B for 2 hours and lyophilized. The lyophilized powder is taken up in a small volume of distilled water and dialyzed against several changes of Buffer E. Step 5. A 0-(diethylaminoethyl) cellulose (DEAE-cellulose) column is preequilibrated with Buffer E onto which the nonhistone protein sample obtained in Step 4 is charged. The column with its adsorbed proteins is then washed with two bed volumes of Buffer F and eluted with Buffer G until the
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absorbance at 280 nm of the eluate is less than 0.05. Both the wash and the eluate are discarded. The column is finally eluted with Buffer H. The eluate, representing NHP-I, is collected and the NaCl removed by dialysis against Buffer I. The NHP-I is then concentrated by negative pressure or by Sephadex G-200 and finally clarified at 20,000 g before use.
B. Assay The activity of NHP-I is measured by its stimulation of template activity of chromatin in an RNA polymerase reaction. The source of chromatin can be either the same tissue from which NHP-I is prepared (homologous) or a different tissue (heterologous). The homologous chromatin is preferred because it gives a greater activation than heterologous chromatin (2,3,6). The stimulatory activity of NHP-I varies among different preparations as well as different tissues. The activation is dose-related, increasing with the amount of NHP-I added to the chromatin-templated RNA polymerase system. Both bacterial RNA polymerase and eukaryotic RNA polymerase I1 (a-amanitin sensitive) can be used to provide the RNA synthesizing system in vitro. Two assay methods, one employing Micrococcus luteus RNA polymerase (19) and the other using rat liver nucleoplasmic RNA polymerase (20), are given here as examples.
1. ASSAYIN M. luteus RNA POLYMERASE REACTION The purification of RNA polymerase from M. luteus follows the procedure of Nakamoto et al. (19), and the assay is essentially that of the same authors. The reaction mixture, in a final volume of 0.50 ml, contains: Tris-HC1 (pH 8.0),50 pmol; KCl, 30 pmol; MgCl,, 2.5 pmol; MnCl,, 1.25pmol; ATP, CTP, GTP, and UTP, one of which is radioactively labeled, 400 nmol each; chromatin, 20 pg equivalent of DNA; RNA polymerase, 1 unit; NHP-I, 20-100 pg. The chromatin template is mixed with NHP-I, and the other components, except MnC1, and the enzyme, are then added. The reaction is initiated by addition of the enzyme and MnCl,, and the reaction mixture is incubated for 10 minutes at 30 "C. At the end of incubation, the reaction is stopped by chilling on ice and addition of 0.10 ml of cold 50% trichloroacetic acid. Processing of acid-insoluble radioactive precipitate for counting is carried out as routinely performed in laboratories. 2. ASSAYIN RAT LIVERRNA POLYMERASE I1 REACTION Form I1 RNA polymerase from rat liver is partially purified by theprocedure of Chesterton and Butterworth (20). The assay is conducted at 37 "C for 30 minutes in the following reaction mixture in a final volume of 0.25 ml: Tris-HC1 (pH 8.0),14 pmol; KCl, 2 pmol; MnCl,, 0.4 pmol; NaF,
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1.5 pmol; P-mercaptoethanol, 0.4 pmol; phosphoenol pyruvate, 1.O pmol; pyruvic kinase, 5 pg; ATP, CTP, and GTP, 150 nmol each; [ 'HJUTP, 15 nmol; chromatin, 20 pg equivalent of DNA; RNA polymerase, 0.5 units; NHP-I, 20-100 pg.
C. Properties The heterogeneity of NHP-I, some of the characteristics of the RNA product synthesized from chromatin activated by NHP-I, and the ability of NHP-I to activate transcription from homologous as well as heterologous chromatins all have been described previously. Regarding the latter, it also has been reported ( 8 )that nonhistone proteins from carrot cells of induced embryonic stages have a greater stimulatory activity than those from noninduced cells in promoting the transcription of chromatin. Nonhistone protein fractions also have been shown to restore histoneinhibited transcription from DNA (8,2I-23). In the assay procedure for NHP-I, a prior interaction between the nonhistone proteins and DNA template before the addition of histone results in a more effective counteraction toward histone inhibition (8,22).If NHP-I is added after DNA and histone have already been interacted, the restoration of histone-inhibited template activity of DNA for RNA synthesis is still effective but requires higher concentrations of the nonhistone proteins than those required if the nonhistone proteins are added after histone and DNA have reacted first ( 4 , 8 ) .This suggests that NHP-I stimulates transcription from chromatin by acting on chromatin proteins, perhaps by dissociating or displacing histones. The stimulatory action of NHP-I in promoting transcription from chromatin is tissue-specific. When chromatin-templated RNA synthesis is activated by NHP-I prepared from a different tissue, the synthesized product contains RNA speciesresemblingthe RNA transcribed from chromatin ofthe heterologous tissue, asdetermined by DNA-RNA hybridization (2,3,6).The RNA product transcribed from the activated chromatin is capable of stimulating amino acid incorporation into protein in a cell-free ribosomal system ( I ) .
111. NHP-11. The Nonhistone Proteins That Stimulate Transcription of DNA The activation of transcription from DNA by NHP-I1 was originally demonstrated by Teng et al. (13) and by Shea and Kleinsmith (ZZ) in rat liver nuclei. The former workers used phenol extraction and the latter used
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1.O M NaCl extraction, Bio-Rex 70 treatment, and calcium phosphate gel adsorption to isolate the active nonhistone proteins. In our laboratory, we have prepared NHP-I1 either by the phenolextraction method or by selective binding to homologous DNA (12). The starting material for the isolation of NHP-I1 is purified chromatin. There are a number of procedures for the isolation of chromatin. The advantages and disadvantages of several methods of preparation, in at least one aspect of the characteristics of chromatin (i.e., its template properties in RNA synthesis), have been discussed by DePomerai et al. (24). We have routinely used the procedure of Seligy and Myagi (25), which is modified after Marushige and Bonner (26),to prepare chromatin from isolated nuclei. The procedure uses minimal shearing and is detailed elsewhere (12).
A. Preparative Procedure Buflers and solvents (all contain 1.O mM phenylmethylsulfonyl fluoride): Buffer A. 0.02 M Tris-HC1 (pH 7 3 4 . 3 5 M NaC1. Buffer B. 0.01 M Tris-HC1 (pH 7.0)4.4 M NaCI. Buffer C. 0.1 M Tris-HC1 (pH 8.4)4.01 M EDTA-0.14 M p-mercaptoethanol. Buffer D. 0.1 M acetic a c i d 4 1 4 M p-mercaptoethanol. Buffer E. 0.05 M acetic acid-9.0 M u r e a 4 1 4 M p-mercaptoethanol. Buffer F. 0.01 M Tris-HCl (pH 8.4)-8.6 M urea4.01 M ethylenediaminetetraacetic acid (EDTA)-0. 14 M p-mercaptoethanol. Buffer G. 0.01 M Tris-HC1 (pH 7.4)4.001 M EDTA. Buffer H. 0.01 M Tris-HC1 (PH 7.4)4.05 M NaC14.001 M EDTA. Buffer I. 0.01 M Tris-HC1 (PH 7.4)-0.6 M NaC14.001 M EDTA. Buffer J. 0.01 M Tris-HC1 (PH 7.4)4.05 M NaCl. Step 1. The isolated chromatin is suspended in 1000 volumes of Buffer A with the aid of a loose-fitting Dounce homogenizer, and the mixture is stirred for 20 minutes. The chromatin suspension is centrifuged at 105,000 g for 2 hours. The pellets obtained from this centrifugation are cut into small pieces with scissors and reextracted as in the first extraction. The two extracts are combined and dialyzed against Buffer B, with several changes of the buffer, and then centrifuged at 20,000 g for 15 minutes to remove a small amount of insoluble material. The supernatant solution is then mixed with Bio-Rex 70 (Na +)which has been previously equilibrated with Buffer B as described in Section II,A, Step 4. The Bio-Rex-treated nonhistone proteins are recovered by centrifugation at 12,000 g for 5 minutes and the supernatant solution is collected. Step 2(a) (13). The nonhistone proteins obtained from Step 1 areconcentrated by lyophilization, dissolved in a minimal volume of distilled water,
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and dialyzed against Buffer C. An equal volume of freshly redistilled phenol saturated with Buffer C is added to the protein solution. The mixture is kept at 2-4 oC. for 10-12 hours with occasional gentle swirling. This suspension is centrifuged at 12,000gfor 10 minutes to separate phenol from the aqueous layer. The phenol phase is saved, and the aqueous phase and interphase are collected and reextracted with phenol as above. The two phenol extracts are combined and dialyzed against 100 volumes of Buffer D until the phenol phase is reduced to one-fifth its original volume. The reduction of the phenol phase to no less than one-6fth the original volume is critical because the protein will irreversibly precipitate if the volume is further reduced. The resulting phenol phase is dialyzed for 24 hours against Buffer E, followed by dialysis against Buffer F for 4 hours. The urea is removed from the sample by exhaustive dialysis against 0.02 M Tris-HC1 (pH 8.0), and the sample is clarified at 20,000 g for 10 minutes prior to use. Step 2(b). An alternative procedure for preparing NHP-I1 is by selective binding to homologous DNA. DNA-cellulose is prepared essentiallyaccording to Alberts and Herrick (27); the method is as follows. The DNA, purified according to a modified procedure of Marmur (28) as described elsewhere (12), is dissolved in 24 ml of Buffer G to a concentration of 5 mg DNA/ml. Eight grams of Munktell410 cellulose, which have been washed three times with boiling ethanol and then washed successively with 0.1 NNaOH, 0.001 M EDTA, 0.01 N HC1, and thoroughly with distilled water, are mixed with the DNA solution. The resulting thick paste is spread on awatchglass, which is then covered with cheesecloth, and air-dried for 24 hours. The nearly dry mixture is ground to a fine powder and lyophilized to complete dryness. The powder is again mixed with 20 ml of DNA solution, and the above process is repeated. The dried DNA-cellulose powder is suspended in 200 volumes of r hours with occasional swirling.The Buffer G and left to stand at 2-4 ~ C f o24 buffer is then decanted and the DNA-cellulose is washed with Buffer H until no absorbance at 260 nm is detected in the wash. The amount of DNA bound to the cellulose is determined on an aliquot of the DNA-cellulose suspension in Buffer G. The suspension is heated in a boiling-water bath for 20 minutes, centrifuged, and the supernatant’s absorbance at 260 nm is read against the buffer. An absorbance at 260 nm of 32is taken as a DNA concentration of 1 mg/ml. To prepare the DNA-cellulose column, the DNA-cellulose suspension is poured into a short column 3 cm in diameter and packed to a height of 2-2.5 cm. Two such columns, each containing approximately 40 mg of DNA, are prepared: one contains E. coli DNA-cellulose, and the other, DNA-cellulose prepared with DNA purified from homologous tissue. Prior to use, the DNA-cellulose columns are once again washed with Buffer H to insure that no free DNA is present. Approximately 10-1 5 mg of the non-
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histone proteins, isolated as described in Step 1,in 10ml are dialyzed against Buffer H and are initially passed through a DNA-free cellulose column which has previously been equilibrated with Buffer H to remove nonspecific adsorbing material. The nonhistone proteins are then applied to the E. coli DNA-cellulose column. The column is washed with Buffer H until theA 280nm of the wash is less than 0.02. The nonadsorbed proteins in the wash are collected and applied to the homologous DNA-cellulose column. The column is washed with Buffer H. The flow-through and wash fractionsarediscarded. The column is then eluted with Buffer I at the rate of approximately 1 m1/3 minutes, Fractions of 1 ml of the eluate are collected until the A nm of the eluate is less than 0.02. The eluate fractions are pooled, dialyzed against Buffer J, and concentrated by Sephadex G-200. Prior to use, the sample is dialyzed against 0.01 M Tris-HC1 (pH 8.0) to remove the NaCl.
B. Assay The activity of NHP-I1 isolated from Ehrlich ascites tumor has been assayed with RNA polymerase I1 purified from homologous cells (29). Form I1 RNA polymerase from other eukaryotic sources presumably can be used with similar effectiveness, but this has not been tested. NHP-I1 shows no activity if assayed in M. luteus RNA polymerase reaction (22). Whether this applies to any otherprokaryotic RNApolymeraseis not known. The assay system of Ehrlich ascites tumor RNA polymerase I1 used in our laboratory is essentially that of Natori et al. (30) which varies somewhat from the rat liver enzyme assay. The reaction mixture, in a total volume of 0.25 ml, contains the following: Tris-HC1 (pH 8.0),10 pmol; (NH,),SO,, 12.5 pmol; MnCl,, 0.75 pmol; MgCl,, 1.15 pmol; p-mercaptoethanol, 1.0 pmol; ATP, CTP, and UTP, 62.5 nmol each; [3H]GTP, 6.25 nmol; DNA, 5.0 pg; RNA polymerase II,0.2-0.5 unit. The reaction is incubated at 37°C for 30 minutes or 1 hour.
C . Properties When analyzed by dodecylsulfate polyacrylamide gel electrophoresis, NHP-11, as prepared from Ehrlich ascites tumor chromatin either by phenol extraction or by specific DNA binding, is heterogeneous. Its molecular complexity, however, is relatively simplified as compared with the total nonhistone chromosomal proteins or the loosely bound nonhistone proteins obtained by extraction with 0.35 M NaCl. It consists of subunits mostly of molecular weights 36,000 and less and contains 0.90% (w/w) alkali-labile phosphorus (12). It has a higher acidic amino acids content than basic amino acid residues.
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Whether NHP-I1 prepared from the loosely bound nonhistone chromosomal proteins is identical to the activating fractions isolated from rat liver is unknown (9,10,11,13). However, the essential characteristics of these fractions prepared from various tissues and by different methods are similar. They all contain approximately 1% phosphorus and bind selectively to, and stimulate transcription of, only homologous DNA. As mentioned earlier, only unique homologous DNA sequences are involved in the interaction with NHP-I1 and subsequent stimulation of RNA synthesis. The mechanism of the activation of transcription is at the initiation step of RNA chain growth (16,31). The activation apparently requires an eukaryotic RNA polymerase (12)and depends on the phosphoprotein components (11). The gross amino acid compositions of the activating fractions isolated from rat liver, calf thymus, and Ehrlich ascites tumor cells are different. This is probably due to the heterogeneous nature of the fractions or to the different methods of preparation. If it is assumed that the activator fractions from different tissues are all derived from the loosely bound nonhistone chromosomal proteins, the chemical differences in various preparations may also suggest tissues variations for NHP-I1 (32).
IV. NHP-111. The Nonhistone Protein That Inhibits Transcription of DNA A. Preparative Procedure Buffers (all contain 1.O mM phenylmethylsulfonyl fluoride): Buffer A. 0.05 M Tris-HC1 (pH 8.0). Buffer B. 0.05 M Tris-HC1 (pH 8.0)4.0 M NaCl. Buffer C. 0.01 M Tris-HC1 (pH 8.0). Buffer D. 0.1 M Tris-HC1 (pH 8.4). Buffer E. 0.02 M Tris-HC1 (pH 7.0) -0.4 M NaCI. NHP-I11 was isolated originally from the DNA-protein complex of Ehrlich ascites tumor chromatin (15) and, employing the same procedure, recently from calf thymus (17). The method for preparation seems to be applicable to other tissues. The starting material is chromatin. Step 1 . The isolated chromatin is suspended in 500 volumes of Buffer A with the aid of a Dounce homogenizer. To this suspension an equal volume of Buffer B is added slowly with stirring. The stirring is continued for 6-12 hours, and the mixture is centrifuged at 76,000gfor 90minutes. The viscous supernatant solution is collected and dialyzed against 13 volumes of Buffer C. During dialysis, the content of the dialysis tubing is occasionally
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mixed. Dialysis is carried out for 6-12 hours until heavy precipitation of the DNA-protein complex occurs. The DNA-protein precipitate is recovered after centrifugation of the dialyzed mixture at 76,000 g for 1 hour. Step 2. The DNA-protein pellets are cut into small pieces, suspended in 1000 volumes of 0.4 NH,SO,, blended at 4400 rpm in an Omni-mixer, and stirred in a beaker for 1 hour. The acid-soluble proteins, mostly histones, are removed by centrifugation at 20,000 g for 10 minutes. The pellet is reextracted with H,SO, twice in a similar manner, except that the blending is omitted. Step 3. The DNA-protein complex extracted 0.4 N H,SO, is washed twice with 10 volumes of Buffer D and extracted with phenol as described in Section 111,A, Step 2(a), except that the final removal ofureais accomplished by dialysis against Buffer E, with three changes of the buffer. The dialyzed sample is then treated with Bio-Rex 70, as described in Section II,A, Step4. The NHP-I11 thus obtained is dialyzed against Buffer C and concentrated by Sephadex (3-200. NHP-I11 may be further purified by DNA-cellulose chromatography according to Section III,A, Step 2@), but this is generally not necessary. In the case of Ehrlich ascites tumor cells, large amounts of starting ascites fluid from 1000 mice are required to give a yield of 50-100 pg of NHP-111. Yield from calf thymus is approximately 100 pg/lOO gm of tissue.
B. Assay NHP-I11 has thus far been assayed with only form I1 RNA polymerase purified from homologous tissues. RNA polymerase from other eukaryotic sources has not been investigated, but it is presumed to be equally operative. The assay system using Ehrlich ascites tumor RNA polymerase I1 is described in Section 111, B.
C. Properties The NHP-I11 as prepared above is nearly homogeneous when subjected to polyacrylamide gel electrophoresis. The NHP-I11 isolated from Ehrlich ascites tumor cells contains 2.7% alkali-labile phosphorus and is isoelectrically focused at pH 5.3, having an acidic-to-basic amino acid residues ratio of 1.42. It consists of one polypeptide of molecular weight 10,273,which is determined by dodecylsulfate polyacrylamide gel electrophoresis and calculated from its amino acid composition (IS). The NHP-111prepared from calf thymus has a sedimentation coefficient of s20,w = 3.0. Equilibrium ultra-
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centrifugation of the nonhistone protein yielded a molecular weight of 30,800 k 2,400. It consists of two subunits, with approximate molecular weights of 16,000 and 13,000 (17). The amino acid composition of the calf thymus complete protein is quite different from that of the Ehrlich ascites tumor cells, notably in the relative molar contents of proline, glycine, isoleucine, leucine, tyrosin, and phenylalanine. The NHP-I11 from both tissues lacks cystine and methionine which, if present, are only in trace amounts. As mentioned previously, NHP-I11 inhibits the in vitro transcription of DNA at the initiation step; only reiterated sequences in DNA interact with NHP-I11 and are inhibited for transcription (15,17). ACKNOWLEDGMENTS
This work was supported in part by research grants from the National Foundation-March of Dimes and the National Institute of Health (HD-09443).
REFERENCES 1. Kamiyama, M., and Wang, T. Y . ,Biochim. Biophys. Acta 228, 563 (1971). 2. Kostraba, N. C., and Wang, T. Y., Biochim. Biophys. Acta 262, 169 (1972). 3. Kostraba, N. C., and Wang, T. Y . , Cancer Res. 32, 2348 (1972). 4. Wang, T. Y., Exp. CeNRes. 61, 455 (1970). 5. Wang, T. Y . ,Exp. Cell Res. 69, 217 (1971). 6. Wang, T. Y.,and Kostraba, N. C., in “The Role of RNA in Reproduction and Development” (M. C. Niu and s. Segal, ed~.),p. 324. North-Holland Pub]., Amsterdam, 1973. 7. Teng, C. S., and Hamilton, T. H., Proc. Natl. Acad. Sci. U.S.A. 63,465 (1969). 8. Matsumoto, H., Gregor, D., and Reinert, H., Phytochemisrry 14.41 (1975). 9. Teng, C. T., Teng, C. S., and Allfrey, V. G., Biochem. Biophys. Res. Commun. 41,690 (1970). 10. Kleinsmith, L. J., Heidema, J., and Carroll, A., (London)266, 1025 (1970). 2 1 . Shea, M., and Kleinsmith, L. J., Biochem. Biophys. Res. Commun. 50,473 (1973). 12. Kostraba, N. C., Montagna, R. A., and Wang, T. Y . , J. Biol. Chem. 250 1548 (1975). 13. Teng, C. S . , Teng, C. T., and Allfrey, V. G., J. Biol. Chem. 246, 3597 (1971). 14. Conrad, D., and Wang, T. Y.,Unpublished results (1976). 15. Kostraba, N. C., and Wang, T. Y . ,J. Biol. Chem. 250, 8938 (1975). 16. Kostraba, N. C., Montagna, R. A., and Wang, T. Y . , Biochem. Biophys. Res. Commun., 72, 334 (1976). 17. Wang, T. Y . ,Kostraba, N. C., and Newman, R. S . , Progr. NucleicAcidRes. 19,447 (1977). 18. Chauveau, J . , MoulC, Y.,and Roueller, C., Exp. CellRes. 11, 317 (1956). 19. Nakamoto, T., Fox, F. C., and Weiss, S., J . Biol. Chem. 239, 169 (1964). 20. Chesterton, C. J., and Butterworth, P. H. W., FEBS Leu. 15, 181 (1971). 21. Langan, T., in “Regulation of Nucleic Acid and Protein Synthesis” (V. V. Koningsberger and L. Bosch, eds.), p. 233. Elsevier, Amsterdam, 1967. 22. Spelsberg, T. C., and Hnilica, L. S., Biochim. Biophys. Acta 195, 63 (1969). 23. Wang, T. Y . , Exp. CeNRes. 53, 288 (1968). 24. DePomerai, D. I., Chesterton, C. J., and Butterworth, P. H. W., Eur. J. Biochem. 46, 461 (1974). 25. Seligy, V., and Miyagi, M., Exp. Cell Res. 58, 27 (1%9). 26. Marushige, K., and Bonner, J., J. Mol. Biol. 15, 160 (1966).
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27. Alberts, B. M., and Hemck, G., in “Methods in Enzymology,” Vol. 21: Nucleic Acids, Part D (L. Grossman and K. Moldave, eds.) p. 198. Academic Press, New York, 1971. 28. Marmur, J., J. Mol. Biol. 3, 208 (1961). 29. Chan, J. Y. H., Loor, R. M., and Wang, T. Y., Arch. Biochem. Biophys. 173, 564(1976). 30. Natori, S., Takeuchi, K., and Mizuno, D., J. Biochem. (Tokyo) 73,345 (1973). 31. Kostraba, N. C., and Wang, T. Y., unpublished results. 32. Burckard, J., Mazen, A., and Champagne, M., Biachim. Biaphys. Acta 405, 434 (1975).
AND NINA
C. KOSTRABA
Division of Cell and Molecular Biology. State University of New York at Buffalo. Buffalo, New York
I. Introduction The nonhistone chromosomal proteins are believed to play a regulatory role in the control of gene expression. One evidence supporting this regulatory role for the nonhistone proteins is their ability to alter transcription in vitro. In this chapter, we limit ourselves to the preparative procedures and characteristic descriptions of only those nonhistone phosphoproteins that directly stimulate or inhibit transcription in vitro from DNA or chromatin. At present, three nonhistone phosphoprotein fractions have been shown to exert such direct effects on transcription. For convenience, these three nonhistone protein fractions are referred to here as NHP-I, NHP-11, and NHP-111. One fraction (NHP-I) enhances the transcription from chromatin, as shown in liver, spleen, and kidney of rat ( 2 - 9 , in Walker 256 carcinosarcoma (2,3), in frog liver (6), in calf endometrium (7), and in cultured carrot cells (8). The second fraction (NHP-11) binds selectively to, and stimulates transcription from, homologous DNA (9-12). This nonhistone protein fraction has been demonstrated in rat liver ( I I , 2 3 ) ,in KB cells (24), and in Ehrlich ascites tumor cells (22). The third fraction (NHP-111), isolated from Ehrlich ascites tumor (15) and calf thymus inhibits DNA-directed RNA synthesis in vitro. NHP-I, the nonhistone protein fraction that stimulates transcription from chromatin in vitro, is a highly heterogeneous group of proteins containing RNA. It stimulates transcription from chromatin isolated from the same 317
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tissue of origin as the nonhistone protein fraction as well as that from other tissues. However, NHP-I stimulates the chromatin template of homologous tissue to a greater extent than heterologous chromatin (2,3,6). Like NHP-I, NHP-I1 is also heterogeneous but, as prepared, contains no nucleic acid. NHP-I1 selectively binds to, and enhances transcription of, homologous DNA (9-13). The specificity of NHP-I1 is such that it interacts with and activates transcription of only unique sequences in DNA (16).The mechanism of activation by NHP-11 is stimulation of the initiation of RNA synthesis (16,17). NHP-I11 has been isolated as a near-homogeneous nonhistone protein. This nonhistone protein binds to, and inhibits transcription of, reiterated sequences in DNA, but not unique DNA sequences, nor chromatin (17). It inhibits the transcription of repetitive DNA sequences by acting at the initiation step of RNA synthesis (15,17). In brief, two of the nonhistone protein fractions (I and 11) can be shown to stimulate transcription in vifro, one (NHP-I) by acting on chromatin and the other (NHP-11), on DNA. The third fraction (NHP-111) inhibits transcription from DNA. The varied but specific effects of these nonhistone proteins indicate the involvement of nonhistone chromosomal proteins in both positive and negative controls in the differential regulation of gene expression.
11. NHP-I. The Nonhistone Proteins That Stimulate Transcription of Chromatin A.
Preparative Procedure
Bufers: Buffer A. 0.02 M Tris-HC1 (pH 7.6)-0.15 M NaCl. Buffer B. 0.02 M Tris-HC1 (pH 7.6). Buffer C. 0.02 M Tris-HC1 (pH 8.0)-2.0 M NaCl. Buffer D. 0.02 M Tris-HC1 (pH 7.2)-0.4 M NaCl. Buffer E. 0.015 M Tris-HC1 (pH 8.0)-1 mM EDTA-1 mM p-mercaptoethanol. Buffer F. 0.015 M Tris-HC1 (pH 8.0)-0.05 MNaCl-1 mMEDTA-1 mM p-mercaptoethanol. Buffer G. 0.015 M Tris-HC1 (pH 8.0)-0.15 M NaC1-1 mM EDTA-1 mM p-mercaptoethanol. Buffer H. 0.015 M Tris-HCl (pH 8.0)-0.30 MNaCl-1 mMEDTA-1 mM p-mercaptoethanol. Buffer I. 0.02 M Tris-HCl (pH 8.0).
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Step 1. NHP-I is prepared from crude chromatin (saline-extracted nuclei). The method of Chauveau et al. (18) is applicable to the isolation of nuclei from rat and most other solid tissues, but other methods may also be used. For the isolation of nuclei, the tissue, trimmed and cleansed of fat and connective tissue, is forced through a Harvard press into a pulp. The macerated tissue is weighed and then homogenized with a Dounce homogenizer in 9 volumes of 2.4 M sucrose-10-4 M CaCl, to a 10% (w/w) homogenate. The suspension is centrifuged at 75,000 g for 1 hour. At the end of the centrifugation, the pellets are collected, resuspended in 10 volumes of 0.33 M sucrose, and centrifuged at 1000 g for 5 minutes. The isolated nuclei are recovered in the pellets. Step 2. The isolated nuclei are extracted with 100 volumes of Buffer A by first homogenizing them in a loose-fitting Dounce homogenizer and then stirring for 1 hour. The nuclear suspension is centrifuged at 25,000 g for 15 minutes, discarding the supernatant. This step of extraction with Buffer A is repeated twice. Step 3. The saline-extracted nuclei are suspended in 20 volumes of Buffer B and stirred. During the stirring, an equal volume of Buffer C is added slowly into it, and the stirring is continued for 4-6 hours. The mixture is then centrifuged at 75,000 g for 90 minutes. The supernatant is collected and its volume measured. To this supernatant solution 1 .5 volumes of Buffer B are added drop by drop with stirring to a final concentration of 0.4 M with respect to NaCl. The resulting mixture with its formed precipitate, which consists mostly of DNA and histones, is centrifuged at 75,000 g for 90 minutes. The supernatant solution from this centrifugation is recovered and dialyzed against Buffer D with three changes of the buffer. Step 4. Bio-Rex 70 (Na + form, minus 400 mesh) is suspended in Buffer D, and the pH of this mixture is adjusted to 7.2 with HC1. The mixture is centrifuged at 15,000 g for 5 minutes, and the sedimented resin is equilibrated with Buffer D for 30 minutes and again centrifuged to remove the buffer. The resin is then mixed with the dialyzed solution obtained from Step 3 in a protein-to-resin ratio of approximately 1/ 100(w/w). This mixture is stirred for 10 minutes and is allowed to settle for another 30 minutes. The unadsorbed acidic proteins are collected by centrifugation. The resin is washed with Buffer D, centrifuged, and the wash is combined with the first supernatant solution. The combined solution is dialyzed against 3 volumes of Buffer B for 2 hours and lyophilized. The lyophilized powder is taken up in a small volume of distilled water and dialyzed against several changes of Buffer E. Step 5. A 0-(diethylaminoethyl) cellulose (DEAE-cellulose) column is preequilibrated with Buffer E onto which the nonhistone protein sample obtained in Step 4 is charged. The column with its adsorbed proteins is then washed with two bed volumes of Buffer F and eluted with Buffer G until the
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absorbance at 280 nm of the eluate is less than 0.05. Both the wash and the eluate are discarded. The column is finally eluted with Buffer H. The eluate, representing NHP-I, is collected and the NaCl removed by dialysis against Buffer I. The NHP-I is then concentrated by negative pressure or by Sephadex G-200 and finally clarified at 20,000 g before use.
B. Assay The activity of NHP-I is measured by its stimulation of template activity of chromatin in an RNA polymerase reaction. The source of chromatin can be either the same tissue from which NHP-I is prepared (homologous) or a different tissue (heterologous). The homologous chromatin is preferred because it gives a greater activation than heterologous chromatin (2,3,6). The stimulatory activity of NHP-I varies among different preparations as well as different tissues. The activation is dose-related, increasing with the amount of NHP-I added to the chromatin-templated RNA polymerase system. Both bacterial RNA polymerase and eukaryotic RNA polymerase I1 (a-amanitin sensitive) can be used to provide the RNA synthesizing system in vitro. Two assay methods, one employing Micrococcus luteus RNA polymerase (19) and the other using rat liver nucleoplasmic RNA polymerase (20), are given here as examples.
1. ASSAYIN M. luteus RNA POLYMERASE REACTION The purification of RNA polymerase from M. luteus follows the procedure of Nakamoto et al. (19), and the assay is essentially that of the same authors. The reaction mixture, in a final volume of 0.50 ml, contains: Tris-HC1 (pH 8.0),50 pmol; KCl, 30 pmol; MgCl,, 2.5 pmol; MnCl,, 1.25pmol; ATP, CTP, GTP, and UTP, one of which is radioactively labeled, 400 nmol each; chromatin, 20 pg equivalent of DNA; RNA polymerase, 1 unit; NHP-I, 20-100 pg. The chromatin template is mixed with NHP-I, and the other components, except MnC1, and the enzyme, are then added. The reaction is initiated by addition of the enzyme and MnCl,, and the reaction mixture is incubated for 10 minutes at 30 "C. At the end of incubation, the reaction is stopped by chilling on ice and addition of 0.10 ml of cold 50% trichloroacetic acid. Processing of acid-insoluble radioactive precipitate for counting is carried out as routinely performed in laboratories. 2. ASSAYIN RAT LIVERRNA POLYMERASE I1 REACTION Form I1 RNA polymerase from rat liver is partially purified by theprocedure of Chesterton and Butterworth (20). The assay is conducted at 37 "C for 30 minutes in the following reaction mixture in a final volume of 0.25 ml: Tris-HC1 (pH 8.0),14 pmol; KCl, 2 pmol; MnCl,, 0.4 pmol; NaF,
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1.5 pmol; P-mercaptoethanol, 0.4 pmol; phosphoenol pyruvate, 1.O pmol; pyruvic kinase, 5 pg; ATP, CTP, and GTP, 150 nmol each; [ 'HJUTP, 15 nmol; chromatin, 20 pg equivalent of DNA; RNA polymerase, 0.5 units; NHP-I, 20-100 pg.
C. Properties The heterogeneity of NHP-I, some of the characteristics of the RNA product synthesized from chromatin activated by NHP-I, and the ability of NHP-I to activate transcription from homologous as well as heterologous chromatins all have been described previously. Regarding the latter, it also has been reported ( 8 )that nonhistone proteins from carrot cells of induced embryonic stages have a greater stimulatory activity than those from noninduced cells in promoting the transcription of chromatin. Nonhistone protein fractions also have been shown to restore histoneinhibited transcription from DNA (8,2I-23). In the assay procedure for NHP-I, a prior interaction between the nonhistone proteins and DNA template before the addition of histone results in a more effective counteraction toward histone inhibition (8,22).If NHP-I is added after DNA and histone have already been interacted, the restoration of histone-inhibited template activity of DNA for RNA synthesis is still effective but requires higher concentrations of the nonhistone proteins than those required if the nonhistone proteins are added after histone and DNA have reacted first ( 4 , 8 ) .This suggests that NHP-I stimulates transcription from chromatin by acting on chromatin proteins, perhaps by dissociating or displacing histones. The stimulatory action of NHP-I in promoting transcription from chromatin is tissue-specific. When chromatin-templated RNA synthesis is activated by NHP-I prepared from a different tissue, the synthesized product contains RNA speciesresemblingthe RNA transcribed from chromatin ofthe heterologous tissue, asdetermined by DNA-RNA hybridization (2,3,6).The RNA product transcribed from the activated chromatin is capable of stimulating amino acid incorporation into protein in a cell-free ribosomal system ( I ) .
111. NHP-11. The Nonhistone Proteins That Stimulate Transcription of DNA The activation of transcription from DNA by NHP-I1 was originally demonstrated by Teng et al. (13) and by Shea and Kleinsmith (ZZ) in rat liver nuclei. The former workers used phenol extraction and the latter used
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1.O M NaCl extraction, Bio-Rex 70 treatment, and calcium phosphate gel adsorption to isolate the active nonhistone proteins. In our laboratory, we have prepared NHP-I1 either by the phenolextraction method or by selective binding to homologous DNA (12). The starting material for the isolation of NHP-I1 is purified chromatin. There are a number of procedures for the isolation of chromatin. The advantages and disadvantages of several methods of preparation, in at least one aspect of the characteristics of chromatin (i.e., its template properties in RNA synthesis), have been discussed by DePomerai et al. (24). We have routinely used the procedure of Seligy and Myagi (25), which is modified after Marushige and Bonner (26),to prepare chromatin from isolated nuclei. The procedure uses minimal shearing and is detailed elsewhere (12).
A. Preparative Procedure Buflers and solvents (all contain 1.O mM phenylmethylsulfonyl fluoride): Buffer A. 0.02 M Tris-HC1 (pH 7 3 4 . 3 5 M NaC1. Buffer B. 0.01 M Tris-HC1 (pH 7.0)4.4 M NaCI. Buffer C. 0.1 M Tris-HC1 (pH 8.4)4.01 M EDTA-0.14 M p-mercaptoethanol. Buffer D. 0.1 M acetic a c i d 4 1 4 M p-mercaptoethanol. Buffer E. 0.05 M acetic acid-9.0 M u r e a 4 1 4 M p-mercaptoethanol. Buffer F. 0.01 M Tris-HCl (pH 8.4)-8.6 M urea4.01 M ethylenediaminetetraacetic acid (EDTA)-0. 14 M p-mercaptoethanol. Buffer G. 0.01 M Tris-HC1 (pH 7.4)4.001 M EDTA. Buffer H. 0.01 M Tris-HC1 (PH 7.4)4.05 M NaC14.001 M EDTA. Buffer I. 0.01 M Tris-HC1 (PH 7.4)-0.6 M NaC14.001 M EDTA. Buffer J. 0.01 M Tris-HC1 (PH 7.4)4.05 M NaCl. Step 1. The isolated chromatin is suspended in 1000 volumes of Buffer A with the aid of a loose-fitting Dounce homogenizer, and the mixture is stirred for 20 minutes. The chromatin suspension is centrifuged at 105,000 g for 2 hours. The pellets obtained from this centrifugation are cut into small pieces with scissors and reextracted as in the first extraction. The two extracts are combined and dialyzed against Buffer B, with several changes of the buffer, and then centrifuged at 20,000 g for 15 minutes to remove a small amount of insoluble material. The supernatant solution is then mixed with Bio-Rex 70 (Na +)which has been previously equilibrated with Buffer B as described in Section II,A, Step 4. The Bio-Rex-treated nonhistone proteins are recovered by centrifugation at 12,000 g for 5 minutes and the supernatant solution is collected. Step 2(a) (13). The nonhistone proteins obtained from Step 1 areconcentrated by lyophilization, dissolved in a minimal volume of distilled water,
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and dialyzed against Buffer C. An equal volume of freshly redistilled phenol saturated with Buffer C is added to the protein solution. The mixture is kept at 2-4 oC. for 10-12 hours with occasional gentle swirling. This suspension is centrifuged at 12,000gfor 10 minutes to separate phenol from the aqueous layer. The phenol phase is saved, and the aqueous phase and interphase are collected and reextracted with phenol as above. The two phenol extracts are combined and dialyzed against 100 volumes of Buffer D until the phenol phase is reduced to one-fifth its original volume. The reduction of the phenol phase to no less than one-6fth the original volume is critical because the protein will irreversibly precipitate if the volume is further reduced. The resulting phenol phase is dialyzed for 24 hours against Buffer E, followed by dialysis against Buffer F for 4 hours. The urea is removed from the sample by exhaustive dialysis against 0.02 M Tris-HC1 (pH 8.0), and the sample is clarified at 20,000 g for 10 minutes prior to use. Step 2(b). An alternative procedure for preparing NHP-I1 is by selective binding to homologous DNA. DNA-cellulose is prepared essentiallyaccording to Alberts and Herrick (27); the method is as follows. The DNA, purified according to a modified procedure of Marmur (28) as described elsewhere (12), is dissolved in 24 ml of Buffer G to a concentration of 5 mg DNA/ml. Eight grams of Munktell410 cellulose, which have been washed three times with boiling ethanol and then washed successively with 0.1 NNaOH, 0.001 M EDTA, 0.01 N HC1, and thoroughly with distilled water, are mixed with the DNA solution. The resulting thick paste is spread on awatchglass, which is then covered with cheesecloth, and air-dried for 24 hours. The nearly dry mixture is ground to a fine powder and lyophilized to complete dryness. The powder is again mixed with 20 ml of DNA solution, and the above process is repeated. The dried DNA-cellulose powder is suspended in 200 volumes of r hours with occasional swirling.The Buffer G and left to stand at 2-4 ~ C f o24 buffer is then decanted and the DNA-cellulose is washed with Buffer H until no absorbance at 260 nm is detected in the wash. The amount of DNA bound to the cellulose is determined on an aliquot of the DNA-cellulose suspension in Buffer G. The suspension is heated in a boiling-water bath for 20 minutes, centrifuged, and the supernatant’s absorbance at 260 nm is read against the buffer. An absorbance at 260 nm of 32is taken as a DNA concentration of 1 mg/ml. To prepare the DNA-cellulose column, the DNA-cellulose suspension is poured into a short column 3 cm in diameter and packed to a height of 2-2.5 cm. Two such columns, each containing approximately 40 mg of DNA, are prepared: one contains E. coli DNA-cellulose, and the other, DNA-cellulose prepared with DNA purified from homologous tissue. Prior to use, the DNA-cellulose columns are once again washed with Buffer H to insure that no free DNA is present. Approximately 10-1 5 mg of the non-
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histone proteins, isolated as described in Step 1,in 10ml are dialyzed against Buffer H and are initially passed through a DNA-free cellulose column which has previously been equilibrated with Buffer H to remove nonspecific adsorbing material. The nonhistone proteins are then applied to the E. coli DNA-cellulose column. The column is washed with Buffer H until theA 280nm of the wash is less than 0.02. The nonadsorbed proteins in the wash are collected and applied to the homologous DNA-cellulose column. The column is washed with Buffer H. The flow-through and wash fractionsarediscarded. The column is then eluted with Buffer I at the rate of approximately 1 m1/3 minutes, Fractions of 1 ml of the eluate are collected until the A nm of the eluate is less than 0.02. The eluate fractions are pooled, dialyzed against Buffer J, and concentrated by Sephadex G-200. Prior to use, the sample is dialyzed against 0.01 M Tris-HC1 (pH 8.0) to remove the NaCl.
B. Assay The activity of NHP-I1 isolated from Ehrlich ascites tumor has been assayed with RNA polymerase I1 purified from homologous cells (29). Form I1 RNA polymerase from other eukaryotic sources presumably can be used with similar effectiveness, but this has not been tested. NHP-I1 shows no activity if assayed in M. luteus RNA polymerase reaction (22). Whether this applies to any otherprokaryotic RNApolymeraseis not known. The assay system of Ehrlich ascites tumor RNA polymerase I1 used in our laboratory is essentially that of Natori et al. (30) which varies somewhat from the rat liver enzyme assay. The reaction mixture, in a total volume of 0.25 ml, contains the following: Tris-HC1 (pH 8.0),10 pmol; (NH,),SO,, 12.5 pmol; MnCl,, 0.75 pmol; MgCl,, 1.15 pmol; p-mercaptoethanol, 1.0 pmol; ATP, CTP, and UTP, 62.5 nmol each; [3H]GTP, 6.25 nmol; DNA, 5.0 pg; RNA polymerase II,0.2-0.5 unit. The reaction is incubated at 37°C for 30 minutes or 1 hour.
C . Properties When analyzed by dodecylsulfate polyacrylamide gel electrophoresis, NHP-11, as prepared from Ehrlich ascites tumor chromatin either by phenol extraction or by specific DNA binding, is heterogeneous. Its molecular complexity, however, is relatively simplified as compared with the total nonhistone chromosomal proteins or the loosely bound nonhistone proteins obtained by extraction with 0.35 M NaCl. It consists of subunits mostly of molecular weights 36,000 and less and contains 0.90% (w/w) alkali-labile phosphorus (12). It has a higher acidic amino acids content than basic amino acid residues.
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Whether NHP-I1 prepared from the loosely bound nonhistone chromosomal proteins is identical to the activating fractions isolated from rat liver is unknown (9,10,11,13). However, the essential characteristics of these fractions prepared from various tissues and by different methods are similar. They all contain approximately 1% phosphorus and bind selectively to, and stimulate transcription of, only homologous DNA. As mentioned earlier, only unique homologous DNA sequences are involved in the interaction with NHP-I1 and subsequent stimulation of RNA synthesis. The mechanism of the activation of transcription is at the initiation step of RNA chain growth (16,31). The activation apparently requires an eukaryotic RNA polymerase (12)and depends on the phosphoprotein components (11). The gross amino acid compositions of the activating fractions isolated from rat liver, calf thymus, and Ehrlich ascites tumor cells are different. This is probably due to the heterogeneous nature of the fractions or to the different methods of preparation. If it is assumed that the activator fractions from different tissues are all derived from the loosely bound nonhistone chromosomal proteins, the chemical differences in various preparations may also suggest tissues variations for NHP-I1 (32).
IV. NHP-111. The Nonhistone Protein That Inhibits Transcription of DNA A. Preparative Procedure Buffers (all contain 1.O mM phenylmethylsulfonyl fluoride): Buffer A. 0.05 M Tris-HC1 (pH 8.0). Buffer B. 0.05 M Tris-HC1 (pH 8.0)4.0 M NaCl. Buffer C. 0.01 M Tris-HC1 (pH 8.0). Buffer D. 0.1 M Tris-HC1 (pH 8.4). Buffer E. 0.02 M Tris-HC1 (pH 7.0) -0.4 M NaCI. NHP-I11 was isolated originally from the DNA-protein complex of Ehrlich ascites tumor chromatin (15) and, employing the same procedure, recently from calf thymus (17). The method for preparation seems to be applicable to other tissues. The starting material is chromatin. Step 1 . The isolated chromatin is suspended in 500 volumes of Buffer A with the aid of a Dounce homogenizer. To this suspension an equal volume of Buffer B is added slowly with stirring. The stirring is continued for 6-12 hours, and the mixture is centrifuged at 76,000gfor 90minutes. The viscous supernatant solution is collected and dialyzed against 13 volumes of Buffer C. During dialysis, the content of the dialysis tubing is occasionally
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mixed. Dialysis is carried out for 6-12 hours until heavy precipitation of the DNA-protein complex occurs. The DNA-protein precipitate is recovered after centrifugation of the dialyzed mixture at 76,000 g for 1 hour. Step 2. The DNA-protein pellets are cut into small pieces, suspended in 1000 volumes of 0.4 NH,SO,, blended at 4400 rpm in an Omni-mixer, and stirred in a beaker for 1 hour. The acid-soluble proteins, mostly histones, are removed by centrifugation at 20,000 g for 10 minutes. The pellet is reextracted with H,SO, twice in a similar manner, except that the blending is omitted. Step 3. The DNA-protein complex extracted 0.4 N H,SO, is washed twice with 10 volumes of Buffer D and extracted with phenol as described in Section 111,A, Step 2(a), except that the final removal ofureais accomplished by dialysis against Buffer E, with three changes of the buffer. The dialyzed sample is then treated with Bio-Rex 70, as described in Section II,A, Step4. The NHP-I11 thus obtained is dialyzed against Buffer C and concentrated by Sephadex (3-200. NHP-I11 may be further purified by DNA-cellulose chromatography according to Section III,A, Step 2@), but this is generally not necessary. In the case of Ehrlich ascites tumor cells, large amounts of starting ascites fluid from 1000 mice are required to give a yield of 50-100 pg of NHP-111. Yield from calf thymus is approximately 100 pg/lOO gm of tissue.
B. Assay NHP-I11 has thus far been assayed with only form I1 RNA polymerase purified from homologous tissues. RNA polymerase from other eukaryotic sources has not been investigated, but it is presumed to be equally operative. The assay system using Ehrlich ascites tumor RNA polymerase I1 is described in Section 111, B.
C. Properties The NHP-I11 as prepared above is nearly homogeneous when subjected to polyacrylamide gel electrophoresis. The NHP-I11 isolated from Ehrlich ascites tumor cells contains 2.7% alkali-labile phosphorus and is isoelectrically focused at pH 5.3, having an acidic-to-basic amino acid residues ratio of 1.42. It consists of one polypeptide of molecular weight 10,273,which is determined by dodecylsulfate polyacrylamide gel electrophoresis and calculated from its amino acid composition (IS). The NHP-111prepared from calf thymus has a sedimentation coefficient of s20,w = 3.0. Equilibrium ultra-
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centrifugation of the nonhistone protein yielded a molecular weight of 30,800 k 2,400. It consists of two subunits, with approximate molecular weights of 16,000 and 13,000 (17). The amino acid composition of the calf thymus complete protein is quite different from that of the Ehrlich ascites tumor cells, notably in the relative molar contents of proline, glycine, isoleucine, leucine, tyrosin, and phenylalanine. The NHP-I11 from both tissues lacks cystine and methionine which, if present, are only in trace amounts. As mentioned previously, NHP-I11 inhibits the in vitro transcription of DNA at the initiation step; only reiterated sequences in DNA interact with NHP-I11 and are inhibited for transcription (15,17). ACKNOWLEDGMENTS
This work was supported in part by research grants from the National Foundation-March of Dimes and the National Institute of Health (HD-09443).
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27. Alberts, B. M., and Hemck, G., in “Methods in Enzymology,” Vol. 21: Nucleic Acids, Part D (L. Grossman and K. Moldave, eds.) p. 198. Academic Press, New York, 1971. 28. Marmur, J., J. Mol. Biol. 3, 208 (1961). 29. Chan, J. Y. H., Loor, R. M., and Wang, T. Y., Arch. Biochem. Biophys. 173, 564(1976). 30. Natori, S., Takeuchi, K., and Mizuno, D., J. Biochem. (Tokyo) 73,345 (1973). 31. Kostraba, N. C., and Wang, T. Y., unpublished results. 32. Burckard, J., Mazen, A., and Champagne, M., Biachim. Biaphys. Acta 405, 434 (1975).