D I S T R I B U T I O N OF G L O B I N G E N E S IN CHICKEN R E T I C U L O C Y T E C H R O M A T I N F R A C T I O N A T E D ON U R O G R A F I N G R A D I E N T S
B. C. WILLETTSand J. R. E. WELLS Dept. of Biochemistry, University of Adelaide, Adelaide, South Australia, 5001
(Received 22 March, 1976) Abstract. Buoyant density centrifugation in Urografin solutions resolved French Pressure Cellsheared, and micrococcal nuclease-digested avian reticulocyte chromatin into a broad profile of two peaks. Hybridization experiments using a globin cDNA probe suggested minimal fractionation of transcriptionally active and inactive components with chromatin sheared at 6000 psi, while no evidence was obtained for any fractionation with chromatin sheared at lower or higher pressures, or with chromatin digested to various extents with micrococcal nuclease, despite a considerable spread of chromatin material across gradients. Abbreviations. cDNA, complementary DNA; psi, pounds per square inch; SDS, sodium dodecylsulphate; EDTA, ethylene diamine tetracetate; r.i., refractive index. I. INTRODUCTION Evidence for physical and chemical differences between transcriptionally active and inactive chromatin has stimulated the development of different procedures for attempting chromatin fractionation [1,2, for review see Ref. 3]. Several studies indicate that euchromatin is protein-rich compared with heterochromatin [4, 5, 6], and this difference suggests the possibility of fractionating chromatin by buoyant density centrifugation in a suitable medium. The density gradient media tested in this respect so far have been found to be unsuitable [7, 8]. With Metrizamide (a non-ionic compound which gives dense solutions of relatively low viscosity), gradients in which highly sheared chromatin shows a bimodal distribution have been described, but there was no evidence for fractionation ofheterochromatin and euchromatin [9]. Urografin (a mixed salt of 3,5-diacetamido-2,4,6-triiodobenzoic acid) is closely related to Metrizamide in chemical structure and has been found to have a higher resolving power than Metrizamide [ I 1]. Banding of Drosophila chromatin in Actinomycin D/Urografin gradients has resolved chromatin into fractions containing defined DNA satellites [ 11 ]. Here we report attempts to fractionate chicken reticulocyte chromatin in Urografin gradients. Chromatin was either sheared or partially digested with micrococcal nuclease prior to centrifugation and chicken globin cDNA was used to assay for 'active' genes in gradient fractions. 479 Molecular Biology Reports 2 (1976) 479-486. All Rights Reserved. Copyright 9 1976 by D. R eidel Publishing Company, Dordrecht-Holland.
II. MATERIALS AND METHODS
Source ofchromatin. Purified populations of chicken reticulocytes were obtained from highly anaemic hens [ 12]. Membrane-free nuclear bodies were prepared by the method of Harlow and Wells [13] and used as the source of chromatin. Preparation of chromatin for gradients. For sheared chromatin preparation, chromatin was solubilized by vigorous agitation in a solution of 38% Urografin (Schering Pty. Ltd., Germany), 5 mM Tris-HC1, pH 8. This solution was then sheared at the desired pressure in an Aminco French Pressure Cell. For nuclease-digested chromatin preparations, chromatin was suspended in Buffer A [ 14] to about 4 mg DNA m1-1, mixed with an equal volume of 2 mM CaCI2, 0.002%/3-mercaptoethanol, 0.2 mM EDTA in Buffer A on ice, then preincubated at 37~ for 5 min. The suspension was digested at 37 ~ with micrococcal nuclease (Staphylococcal aureus nuclease, 6000 units mgprotein: Worthington Bioehem. Corp.) at 100 units ml -~ and the reaction stopped with 0.25 vol of 100mM EDTA at 0~ Samples were mixed with Buffer A and added to an equal volume of 76% Urograf'm for centrifugation. Gradients and centrifugation. All Urografin/chromatin mixtures were adjusted to a refractive index of 1.3900 (20~ Polyallomer tubes containing 11 ml of liquid ( 2 - 5 mg of chromatinDNA per tube) were centrifuged in a Type 50 Ti rotor at 39000 rpm for 40 hr at 10~ Gradients were fractionated and the density of each fraction determined from refractive index readings [10]. DNA estimation. The fluorometric method described by Le Pecq [15] was used to measure the amount of DNA in the presence of Urograf'm. Preparation of chromatin fractions for hybridization. Aliquots of chromatin fractions were brought up to 150 #gm of DNA with calf thymus DNA, dialysed for 24 hr against 10 mM TrisHC1, pH 8.0 (2 changes), then Proteinase K buffer [16], in separate Collodion bags (Sartorius Membranf'dter : SM 13200). Samples were digested with Proteinase K (100/ag m1-1 added to each) for 2 hr at 37 ~ and after addition of NaOH to 0.3 N, the mixture was incubated for 1 hr at 37~ then neutralized to pH 7.0. DNA was recovered by ethanol precipitation and dried in vacuo. Hybridization and assay for globin genes. The DNA pellet was solubilized in 50 ~tl of hybridization buffer [ 17], and sonicated for 90 s with cooling ('Soniprobe', Dawe Instruments, setting 8, 3 4 mA). Each fraction was mixed with 3H-globin eDNA (1300 cpm, 5 • 107 dpm/ag-l: gift of Mr. R. C. Crawford) taken up into 100/al microcaps which were then sealed, heat denatured (100~ 5 min) and allowed to reassociate at 60~ to a Cot of 3000. The proportion of eDNA which had hybridized was determined by use of Sl-nuclease [ 17] as described by Kemp [ 18]. III. RESULTS AND DISCUSSION
Chromatin profiles in Urografin. Initially, samples of sheared reticulocyte chromatin were centri-
480
fuged in Metrizamide (Nyegaard, Oslo, Norway) and Urografin gradients for comparison. The potential for resolving sheared chromatin into 'active' and 'inactive' fractions appeared to be much greater in Urografin. All chromatin preparations used were well spread through Urografin gradients after centrifugation but formed a single sharp band in Metrizamide gradients. Although it is possible to obtain a spread in Metrizamide with highly sheared chromatin [9], our investigation suggested that similar profdes could be obtained in Urografin gradients for chromatin subjected to relatively gentle shearing conditions. We therefore used Urografin for further studies. On banding o f both sheared (2000, 6000, 12 000 and 18 000 psi) and nuclease-digested (30 s, 2, 5 and 30 min) reticulocyte chromatin in Urografin, it was found that in all cases very similar profiles were obtained with a general outline of two broad peaks spread across much of the gradient.
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Fig. 1. Banding of DNA, protein and chromatin in Urografin gradients. Chicken DNA (double-stranded, 400 ~g), [~4C]-histone (approx. 7000 cpm - prepared from [~4C]-lysinelabelled erythroblasts [191 ), and reticulocyte chromatin (2 mg chromatin-DNA) digested with micrococcal nuclease (100 units ml- ~) at 37 ~ for 2 min were each mixed with Urografin and centrifuged as described in Materials and Methods. O-----(3 density (g cm- 3) from refractive index (20~ A - - A chicken DNA, from fluorescence measurement; e - - o reticulocyte chromatin DNA, from fluorescence measurement; m - - u [~4C]-histone. The data in Figure 1 show the profdes for chromatin DNA, purified chicken DNA and chicken histone in fractions from Urografin gradients. The DNA preparation bands at a density of 1.17 g cm -3 and histone is found at the bottom of the gradient (density about 1.34 g cm-3). Most of the chromatin DNA bands between these positions, and the two broad peaks have median densities of 1.21 g cm -3 and 1.24 g cm -3. Unsheared and undigested chromatin had the same 481
general profile as sheared and nuclease-digested chromatin, but the spread across the gradient was reduced. In other experiments using cells pre-labelled with [aH]-leucine, we have examined the distribution of labelled proteins from chromatin preparations in Urografin gradients. Label is found in all fractions containing DNA, but about 25% of the label originally associated with the chromatin is found at the bottom of the gradient in which no DNA is detected. This clearly suggests displacement of protein from DNA in Urografin. This phenomenon may contribute to the relatively wide spread of chromatin DNA in Urograf'm gradients.
Position ofglobin genes in fract~'onated chromatin.
As the extent of shearing may be an important factor in separation of heterochromatin and euchromatin, chromatin sheared over a wide range of pressures (unsheared, 2000, 6000, 12 000 and 18 000 psi) was tested for fractionation after centrifugation in Urografin. The effectiveness of fractionation was assayed with globin cDNA probe. Since globin genes are actively transcribed in the chicken reticulocyte [ 13], the globin genes should be present in 'active' chromatin. The results of hybridization experiments are presented in Figure 2. The hybridization profile obtained for 6000 psi sheared chromatin (Figure 2C), shows slight enrichment of the globin genes under the more dense (presumably protein-rich) chromatin peak where euchromatin theoretically should band. Unsheared chromatin
B -80
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-60 10C
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-40
B. C. WILLETTSand J. R. E. WELLS Dept. of Biochemistry, University of Adelaide, Adelaide, South Australia, 5001
(Received 22 March, 1976) Abstract. Buoyant density centrifugation in Urografin solutions resolved French Pressure Cellsheared, and micrococcal nuclease-digested avian reticulocyte chromatin into a broad profile of two peaks. Hybridization experiments using a globin cDNA probe suggested minimal fractionation of transcriptionally active and inactive components with chromatin sheared at 6000 psi, while no evidence was obtained for any fractionation with chromatin sheared at lower or higher pressures, or with chromatin digested to various extents with micrococcal nuclease, despite a considerable spread of chromatin material across gradients. Abbreviations. cDNA, complementary DNA; psi, pounds per square inch; SDS, sodium dodecylsulphate; EDTA, ethylene diamine tetracetate; r.i., refractive index. I. INTRODUCTION Evidence for physical and chemical differences between transcriptionally active and inactive chromatin has stimulated the development of different procedures for attempting chromatin fractionation [1,2, for review see Ref. 3]. Several studies indicate that euchromatin is protein-rich compared with heterochromatin [4, 5, 6], and this difference suggests the possibility of fractionating chromatin by buoyant density centrifugation in a suitable medium. The density gradient media tested in this respect so far have been found to be unsuitable [7, 8]. With Metrizamide (a non-ionic compound which gives dense solutions of relatively low viscosity), gradients in which highly sheared chromatin shows a bimodal distribution have been described, but there was no evidence for fractionation ofheterochromatin and euchromatin [9]. Urografin (a mixed salt of 3,5-diacetamido-2,4,6-triiodobenzoic acid) is closely related to Metrizamide in chemical structure and has been found to have a higher resolving power than Metrizamide [ I 1]. Banding of Drosophila chromatin in Actinomycin D/Urografin gradients has resolved chromatin into fractions containing defined DNA satellites [ 11 ]. Here we report attempts to fractionate chicken reticulocyte chromatin in Urografin gradients. Chromatin was either sheared or partially digested with micrococcal nuclease prior to centrifugation and chicken globin cDNA was used to assay for 'active' genes in gradient fractions. 479 Molecular Biology Reports 2 (1976) 479-486. All Rights Reserved. Copyright 9 1976 by D. R eidel Publishing Company, Dordrecht-Holland.
II. MATERIALS AND METHODS
Source ofchromatin. Purified populations of chicken reticulocytes were obtained from highly anaemic hens [ 12]. Membrane-free nuclear bodies were prepared by the method of Harlow and Wells [13] and used as the source of chromatin. Preparation of chromatin for gradients. For sheared chromatin preparation, chromatin was solubilized by vigorous agitation in a solution of 38% Urografin (Schering Pty. Ltd., Germany), 5 mM Tris-HC1, pH 8. This solution was then sheared at the desired pressure in an Aminco French Pressure Cell. For nuclease-digested chromatin preparations, chromatin was suspended in Buffer A [ 14] to about 4 mg DNA m1-1, mixed with an equal volume of 2 mM CaCI2, 0.002%/3-mercaptoethanol, 0.2 mM EDTA in Buffer A on ice, then preincubated at 37~ for 5 min. The suspension was digested at 37 ~ with micrococcal nuclease (Staphylococcal aureus nuclease, 6000 units mgprotein: Worthington Bioehem. Corp.) at 100 units ml -~ and the reaction stopped with 0.25 vol of 100mM EDTA at 0~ Samples were mixed with Buffer A and added to an equal volume of 76% Urograf'm for centrifugation. Gradients and centrifugation. All Urografin/chromatin mixtures were adjusted to a refractive index of 1.3900 (20~ Polyallomer tubes containing 11 ml of liquid ( 2 - 5 mg of chromatinDNA per tube) were centrifuged in a Type 50 Ti rotor at 39000 rpm for 40 hr at 10~ Gradients were fractionated and the density of each fraction determined from refractive index readings [10]. DNA estimation. The fluorometric method described by Le Pecq [15] was used to measure the amount of DNA in the presence of Urograf'm. Preparation of chromatin fractions for hybridization. Aliquots of chromatin fractions were brought up to 150 #gm of DNA with calf thymus DNA, dialysed for 24 hr against 10 mM TrisHC1, pH 8.0 (2 changes), then Proteinase K buffer [16], in separate Collodion bags (Sartorius Membranf'dter : SM 13200). Samples were digested with Proteinase K (100/ag m1-1 added to each) for 2 hr at 37 ~ and after addition of NaOH to 0.3 N, the mixture was incubated for 1 hr at 37~ then neutralized to pH 7.0. DNA was recovered by ethanol precipitation and dried in vacuo. Hybridization and assay for globin genes. The DNA pellet was solubilized in 50 ~tl of hybridization buffer [ 17], and sonicated for 90 s with cooling ('Soniprobe', Dawe Instruments, setting 8, 3 4 mA). Each fraction was mixed with 3H-globin eDNA (1300 cpm, 5 • 107 dpm/ag-l: gift of Mr. R. C. Crawford) taken up into 100/al microcaps which were then sealed, heat denatured (100~ 5 min) and allowed to reassociate at 60~ to a Cot of 3000. The proportion of eDNA which had hybridized was determined by use of Sl-nuclease [ 17] as described by Kemp [ 18]. III. RESULTS AND DISCUSSION
Chromatin profiles in Urografin. Initially, samples of sheared reticulocyte chromatin were centri-
480
fuged in Metrizamide (Nyegaard, Oslo, Norway) and Urografin gradients for comparison. The potential for resolving sheared chromatin into 'active' and 'inactive' fractions appeared to be much greater in Urografin. All chromatin preparations used were well spread through Urografin gradients after centrifugation but formed a single sharp band in Metrizamide gradients. Although it is possible to obtain a spread in Metrizamide with highly sheared chromatin [9], our investigation suggested that similar profdes could be obtained in Urografin gradients for chromatin subjected to relatively gentle shearing conditions. We therefore used Urografin for further studies. On banding o f both sheared (2000, 6000, 12 000 and 18 000 psi) and nuclease-digested (30 s, 2, 5 and 30 min) reticulocyte chromatin in Urografin, it was found that in all cases very similar profiles were obtained with a general outline of two broad peaks spread across much of the gradient.
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Number
Fig. 1. Banding of DNA, protein and chromatin in Urografin gradients. Chicken DNA (double-stranded, 400 ~g), [~4C]-histone (approx. 7000 cpm - prepared from [~4C]-lysinelabelled erythroblasts [191 ), and reticulocyte chromatin (2 mg chromatin-DNA) digested with micrococcal nuclease (100 units ml- ~) at 37 ~ for 2 min were each mixed with Urografin and centrifuged as described in Materials and Methods. O-----(3 density (g cm- 3) from refractive index (20~ A - - A chicken DNA, from fluorescence measurement; e - - o reticulocyte chromatin DNA, from fluorescence measurement; m - - u [~4C]-histone. The data in Figure 1 show the profdes for chromatin DNA, purified chicken DNA and chicken histone in fractions from Urografin gradients. The DNA preparation bands at a density of 1.17 g cm -3 and histone is found at the bottom of the gradient (density about 1.34 g cm-3). Most of the chromatin DNA bands between these positions, and the two broad peaks have median densities of 1.21 g cm -3 and 1.24 g cm -3. Unsheared and undigested chromatin had the same 481
general profile as sheared and nuclease-digested chromatin, but the spread across the gradient was reduced. In other experiments using cells pre-labelled with [aH]-leucine, we have examined the distribution of labelled proteins from chromatin preparations in Urografin gradients. Label is found in all fractions containing DNA, but about 25% of the label originally associated with the chromatin is found at the bottom of the gradient in which no DNA is detected. This clearly suggests displacement of protein from DNA in Urografin. This phenomenon may contribute to the relatively wide spread of chromatin DNA in Urograf'm gradients.
Position ofglobin genes in fract~'onated chromatin.
As the extent of shearing may be an important factor in separation of heterochromatin and euchromatin, chromatin sheared over a wide range of pressures (unsheared, 2000, 6000, 12 000 and 18 000 psi) was tested for fractionation after centrifugation in Urografin. The effectiveness of fractionation was assayed with globin cDNA probe. Since globin genes are actively transcribed in the chicken reticulocyte [ 13], the globin genes should be present in 'active' chromatin. The results of hybridization experiments are presented in Figure 2. The hybridization profile obtained for 6000 psi sheared chromatin (Figure 2C), shows slight enrichment of the globin genes under the more dense (presumably protein-rich) chromatin peak where euchromatin theoretically should band. Unsheared chromatin
B -80
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-60 10C
5O 40 3O 20 10
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