NONHISTONE
CHROMOSOMAL PROTEINS
Isolation, Purification, and Fractionation of Nonhistone Chromosomal Proteinst H. W. J. van den Brock,$ Larry D. NoodCn,§ J. Sanders Sevall,* and James Bonner
ABSTRACT: We describe a mild method for the separation and fractionation of nonhistone chromosomal proteins (NHC proteins) on a preparative scale. Rat liver chromatin is dissociated in 3 M NaCI-10 mM Tris-HCI (pH 8), and is resolved into DNA and protein by gel filtration on Bio-Gel A-50m. The resulting mixture of chromosomal proteins is concentrated by ammonium sulfate precipitation and histones are then removed by cation exchange chromatography on Bio-Rex
T
he histone components of chromatin are now relatively well understood, separated, and even to a considerable degree sequenced (Elgin et a/., 1971 ; Wilhelm et a/., 1971). It remains of interest to characterize the nonhistone protein components of chromatin in a similar way. Many approaches to the study of this class of proteins have already been reported (Wang, 1966, 1967; Benjamin and Gellhorn, 1968; Marushige and Bonner, 1966; Marushige et a/., 1968; Shelton and Allfrey, 1970; Elgin and Bonner, 1970; Elgin et a/., 1971). In all of these cases the methods used have been denaturing ones, and in several cases also cross-contaminations with histones and/or DNA have complicated the findings. More recent studies conducted simultaneously with the present one have described milder isolation conditions in which no denaturants stronger than urea or guanidinium chloride were employed (Shaw and Huang, 1970; Graziano and Huang, 1971 ; MacGillivray er a/., 1971, 1972; Arnold and Young, 1972; Levy et a/., 1972; Richter and Sekeris, 1972; Hill et a/., 1972). The procedure for the preparation of the NHCl proteins which we describe below employs very mild conditions and is particularly suited to the large scale preparation of these materials. By its use it may prove possible to separate and characterize individual N H C proteins in the same way that individual histones have been separated and characterized in the past. Methods Preparation of Chromatin. Chromatin was isolated from frozen male rat livers from Pel-Freeze Biologicals, Rogers, Ark. (Bonner et a/., 1968). All steps throughout the fractionaf' F r o m the Division of Biology, California Institute of Technology, Pasadena, California 91109. Receiaed July 27, 1972. Report of work supported in part by U. S. Public Health Service G r a n t No. GM-13762, in p a r t by a National Institutes of Health special research fellowship t o L. D. N., a Damon Runyon Memorial F u n d Research Fellowship to J. S . S . , and a research fellowship from the Netherlands Organization to H . W. J. v. d. B. for the Advancement of Pure Research (2.W. 0.) $ Present address' Agricultural University, Department of Genetics, Wageningen, T h e Netherlands. 5 Present address: Botany Department, University of Michigan, Ann Arbor, Mich. Abbreviation used is: N H C protcins, nonhistone chromosomal proteins.
70 in the presence of 0.4 M NaCI. The N H C proteins which are free of detectable amounts of histones include those proteins which are detected by sodium dodecyl sulfate or acidic urea polyacrylamide gel electrophoresis of the proteins of the intact chromatin. We show that the N H C proteins include species which do not bind to DNA at low ionic strength, as well as species which have an affinity for heterologous DNA and a minor component which binds to homologous DNA.
tion of the nonhistone proteins on DNA-cellulose were carried out at 0-4". The livers were homogenized in a Waring Blendor in 0.075 M NaC1-0.025 M EDTA (pH €9,and filtered through cheesecloth and llliracloth. The crude nuclear pellet obtained by low speed centrifugation was washed with saline-EDTA once, and 10 mM Tris-HCI buffer, pH 8, six times. Each time the pellet was resuspended with a Potter-Elvehjem homogenizer. In the later stages special care was taken to remove the opaque subpellet that formed below the gel. The chromatin was further purified by centrifugation through 1.7 M sucrose10 mM Tris-HCI (pH 8) followed by two washes with 10 mM Tris-HC1 (pH 8) solution to remove the sucrose. This pellet was then suspended in 3 M NaCl (final concentration)-10 m M Tris-HCI (pH 8) to yield an absorbance at 260 nm of 18-20, and then sheared in a Virtis 45 homogenizer at 60 V for 60 sec with about 0.5 2-octanol (to prevent foaming). The preparation was then stirred for 45 min, followed by a centrifugation at 30,OOOg for 10 min to remove the octanol pellicle from the solution. The resulting opalescent supernatant contains the dissociated DNA and chromosomal proteins. In general 1 g of liver yielded 18-20 AZG0units of dissociated chromatin. Separation of DNA and Chromosomal Proteins. The sheared, dissociated chromatin was resolved into its DNA and protein components by exclusion gel chromatography (Bio-Gel A50m, 50-100 mesh) preequilibrated with 3 M NaCI-IO mM Tris-HCI (pH 8). In general about 1000 AZ60units in 70 ml of solution was loaded onto each column (bed diameter 3.3 cm, length 97 cm). These coluinns were run overnight at a flow rate of about 50 ml/hr; both upward and downward flowing columns were used with similar results. Concentration of the Chromosonzal Proteins with Ammonium Sulfate and Remocal of Histones with Bio-Rex 70. The protein pool obtained from the Bio-Gel column was dialyzed for 3 hr against 20 vol of 10 mM Tris-HC1 (pH 7) to lower the NaCl concentration. This solution was then dialyzed overnight against ammonium sulfate containing 30 mM Tris-HCI (pH 7) to a final concentration of ammonium sulfate of ca. 9 5 z saturation. The precipitated protein was collected by centrifugation, redissolved in 0.4 M NaC1-10 mM Tris-HCI (pH 7) at a concentration of about 2-3 mg of protein/ml, and dialyzed against 20 vol of 0.4 M NaCI-10 mM Tris-HC1 (pH 7) for 6 hr. After centrifugation this solution was applied to a Bio-Rex 70 column (200-400 mesh), precycled with 3 M NaCI-IO mM BIOCHEMISTRY,
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Tris-HCI (pH 7) and equilibrated with 0.4 M NaCI-10 mM Tris-HCI (pH 7). The column contained at least 1 g of BioRex 70 resin for each 10 mg of protein. The run-off protein. that eluted with 0.4 M NaCI, contains the N H C protein components. The histones which are retained by the Bio-Rex under these conditions were eluted with 1 \i NaCl-10 m v Tris-HCI (pH 7) containing 1 mbi NaHSOiI.The run-off from the Bio-Rex column was dialyzed against 20 vol of 0.05 M NaCI--lO mM Tris-HCI (pH 7). In general, no precipitate was formed at this stage, while scattering data (&o) indicate the absence of any considerable amount of insoluble material. Preparution of' DNA-Cellulose ColLimns. (1) PRTP.!RAION OF T H E DNA. DNA was prepared from frozen mid- or latelog phase E. coli cells, strain B, from General Biochemicals and from frozen male rat livers. The bacterial cells were lysed with lysozyme and sodium dodecyl sulfate (Marmur. 1961). The DNA was purified by sequential treatments with phenol. chloroform, amylase, RNase, and Pronase (WLIr t (11.. 1972). Rat liver DNA was extracted in the same way from crude rat liver chromatin. (2) PREPARATION OF DNA-CELLULOSI:. The DNA-cellulose was prepared by the method previously described (Litman, 1968). Four milliliters of a 1L3 mg,"l of DNA solution was mixed with 1 g of acid-washed cellulose (Whatman F-ll), spread into a thin layer, and allowed.to dry by evaporation at room temperature. To ensure dryness the DNA-celltilose was cooled to -20", frozen, and lyophilized overnight. The cellulose cake was suspended in 10 vol of absolute ethanol and stirred slowly under a L I V (ultraviolet) lamp (G15T8 Sylvania) for 30 min at a distance of 5 cm. After removal of the ethanol by filtration, the DNA-cellulose was washed with 3 M NaCI-10 mi%Tris-HCI (pH 7) twice, and 0.05 kt NaCl-10 msi Tris-HCI (pH 7) three times. The washed DNAcellulose was then air-dried or lyophilized and stored at 4". Its DNA content was determined to be 8-10 mg of DNA per g of cellulose. This was done by hydrolyzing a known weight of cellulose with 5 % perchloric acid for 10 min at 100" and determination of A?wagainst an appropriate blank. Fraciionution o / NHC Proteins on DNA-Cdlirlose. The dry DNA-cellulose containing an amount of DNA equal to twice the amount of protein to be applied was suspended in 3 M NaCI-IO msi Tris-HCI (pH 7 ) about 24 hr before use. A column I .5 cm in diameter was then packed with the material and washed for 2--3 hr with the same but'fer, at which time a change to 0.05 h% NaCI-10 mM Tris-HCI (pH 7) was made. The wash was continued overnight. Before the column was ~ised,the absorbance at 260 nm and the conductivity of the eluent from it were recorded using the wash bufer as a refcrence. The N H C proteins, usually 0.2-0.3 mg/ml, were then applied to ii tandem series of E . coli DNA-cell~ilo~e and rat liver DNA-cellulose columns. The absorbance of the eluent at 230, 260, and 280 nm was recorded. After sample application the tandem series was washed with 0.05 M NaCI-10 nni Tris-HC1 (pH 7 ) until absorbance at 230 nm was below 0.2. The columns were then separated, washed with 2 void volumes of 0.05 $1 NaCI-I0 mM Tris-HCI (pH 7 ) , and eluted with 0.6 M NaCI-10 mM Tris-HCI (pH 7). Controls were run as previously described (Alberts and Herrick. 1971). A DNA-free cellulose column was inserted as the first column in tandem with the DNA-cellulose column. When it was eluted with 2 hi NaCI, no spurious binding of NHC proteins could bc detected. To ensure that the DNAcellulose columns were not being saturated, N H C proteins were passed over two rat liver DNA-celltilose columns in tandem both containing a 2:1 ratio of DNA- protein. Al-
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though protein was bound to the initial DNA-cellulose column in abundant amounts. only a slight amount, less than 1 % of the total. was found to bind to the second column. Pol,~~ncr,~~lumide Gel Elecrroplioresis. (1) SODIUMD O D ~ C Y L SULF4Tt-TRIS GEL LLECTROPHORESIS. Samples O f the proteins taken at various stages of purification were analyzed elcctrophoretically in gels containing sodium dodecyl sulfate (Laemmli, 1970). The running gels (0.5 X 5-6 cm) were made LIP with Tris-HCI (pH 8.8) plus 10% acrylamide, while the stacking gels (0.5 X 0.5 cm) contained Tris-HCI (pH 6.8) plus 3% acrylamide. The samples were lyophilized (if the salt concentration was very high, they were first dialyzed against a 10-20-fold volume of 10 msr Tris-HCI ( p H 7) for 2-3 hr) and dissolved in 0.1 sodium dodecyl stilfrite-65 mhi TrisHCI (pH 6.8)- 0.1 2-mercaptoethanol. After dialyzing against this medium for 24 hr at room temperature, the saniples were further dialyzed against the same medium containing 10% glycerol at 37' for another 12 hr. The samples were run at 1.5 mA/gel for about 5 hr, then fixed with 1 5 % trichloroacetic acid for at least 40 min at room temperature, stained with 0.25 % Coomassie Brilliant BILK in 7 acetic acid . 5 0 7; methanol for 3 - - 5 hr. and destained by diffusion in 10% acetic acid-5 % methanol. (7) , 4 C ' I ~ l l ( . URtA GEL ELEC TROPHORCSIS. l h e presence Of histones at iarious stages in the purification was monitored by electrophoresis in pH 3.2 and 4.3 tma gels. The former gels (0.6 x 7 cm) were prepared and run according to the method of Panyim and Chalkley (1969) using 2.5 h i urea a n d 15 % acrylamide. The samples were prepared by lyophilization. dissolution in 7 hi tireti, and dialysis against 7 st urea. Mercaptoethanol (to give about 5 75 by volume) was added before use. After 2-3 hr of preelectrophoresis at 2 mA;'gel. the samples were loaded and rtin for about 3 hr at the same amperage. The gels were stained with 1 Amido-Schwarr. and destained electrophoretically at low amperage in 40% ethanoI-7Z acetic acid. The pH 4.3 gels (0.6 X 5 cm) were made by the method of Reisfeld (Bonner v r d..1968) with 10% acrylamide and rtin for about 2 hr at 4 niA.!gel. The saniples were prepared and the gels stained a s above. An7ino Acid ,4nci/)~sis.Samples containing 100-200 pg of protein were dialyzed against 0.01 mbi NH,OH for 2 days, lyophilized, and then hydrolyzed in 6 ;\j HCI at 108" for 20 hr. The resulting amino acids were analyzed in a Beckman-Spinco 120R amino acid analyzer converted to a one-column system with a Bio-Rad Aminex A6 resin. Genc.rtrl Mc.rhod.s. Protein concentrations were determined by the method of Lowry ~t r d . (1951) or by the filter method of Brainhall ~'i d.(1969) with bovine serum albumin (Sigma) as 21 standard. Analysis of chromatin samples was performed as previously described (Bonner o r d . , 1968). The total protein content was determined after mlubilization of the chromatin in I N NaOH. Separate analysis of histone and NHC proteins was done b y first extracting chromatin two times with 0.1 LI H,SOI at 0' followed by centrifugation at 20,000g. The concentration of histones in the combined extracts was determined by ultraviolet absorption using an extinction coellicicnt of ii solution containing 1 nig of histonelm1 of 4.35 at 230 nni ( R . H . Jensen. 1966, unpublished data quoted by Elgin and Bonner, 1970) a n d by the method of Loury o r r d . (1951). The pellet froin the acid extraction was homogenized in 1 .O ?I NaOH and its protein content determined by the Lowry proccd tire. R N A was scparatcd from DNA I>y the method of Schmidt a n d Tannhatrser a\ modified b y Ts'o a n d Sat0 (1959). RNA \ c a s determined hy the orcinol method of Dische and Schwarr
