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Nuclear isolation by a modified method of Hewish and Burgoyne: Implications for the study of nuclear enzymology
Life Sciences, Vol. 29, pp. 2709-2719 Printed in the U.S.A.
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NUCLEAR ISOLATION BY A MODIFIED METHOD OF HEWISH AND BURGOYNE: IMPLICATIONS FOR THE STUDY OF NUCLEAR ENZYMOLOGY W.W. WolI, J.J. Duffy, N.A. Giese, and T.J. Lindell Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 U.S .A. (Received in final form October 30, 1981)
Summary This study describes a rapid and convenient modification of the established procedure of Hewish and Burgoyne (1973) Biochem. Biophys. Res. Commun. 52, 475-481, for the preparation of r a t l i v e r nuclei. A procedure is also described for the i s o l a t i o n of nuclei from c e l l s in culture using modified buffers of Hewish and Burgoyne. Substitution of the polyamines spermine and spermidine for d i v a l e n t metal cations in the i s o l a t i o n solutions along with u t i l i z a t i o n of the chelators EDTA and EGTA distinguishes t h e i r technique from other well known methods. The modification described for r a t l i v e r nuclei u t i l i z e s a single ultracent r i f u g a t i o n step for separating the nuclei from the homogenate by sedimentation through a dense sucrose s o l u t i o n . The new method produces a high y i e l d of p u r i f i e d nuclei which are characterized to have highly polymerized DNA on a l k a l i n e sucrose gradients. However, when metal ion is added back to these nuclei for in v i t r o t r a n s c r i p t i o n , there is a time dependent reduction in DNA size as measured on a l k a l i n e sucrose gradients which correlates With the loss in l i n e a r i t y of UMP incorporation into RNA. I t is suggested that the modified nuclear i s o l a t i o n procedure described produces more i n t a c t nuclei which w i l l form the basis for further i n v e s t i g a t i o n of the nucleus as an isolated eukaryotic organelle and allow characterization of endogenous enzymology in s i t u . Over the years, numerous methods have been developed for the i s o l a t i o n of nuclei from c e l l s and tissues ( f o r reviews, see I - 3 ) . Many of these methods are used for the preparation of subnuclear components and for the i s o l a t i o n of nuclear enzymes. There are d i s t i n c t advantages and disadvantages of many of these methods. A method which appeared to produce nuclei with i n t a c t DNA, which would be suitable for the characterization of nuclear enzymology, is the method of Hewish and Burgoyne (4). This method substituted the polyamines spermine and spermidine for d i v a l e n t cations present in most other procedures. Divalent cations (Ca+2 and Mg+2) are required for an endogenous endonuclease which catalyzes the cleavage of nuclear DNA (5-7). The use of spermine and spermidine obviates t h i s problem since the endonuclease cannot function when d i v a l e n t cations are absent and polyamines do not substitute for these metal ions. In addition to the use of polyamines, these workers also used 0024-3205/81/262709-ii$02.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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EDTA and EGTA to sequester any endogenous divalent cations within the tissue homogenate. The procedure of Hewish and Burgoyne (4) results in rat l i v e r nuclei of high quality in good yield but i t requires a lengthy amount of time, primarily because more than one centrigugation step is involved. Since we had been using the method of Blobel and Potter (8) to isolate rat l i v e r nuclei, we thought i t would be possible to combine the best features of that procedure, a single centrifugation step through dense sucrose, with the buffers recommended by Hewish and Burgoyne (4). The resulting procedure described in this paper is rapid and produces a consistently high nuclear yield when compared with the original procedure, and contain highly polymerized DNA, METHODS Animals Male and female rats (Sprague-Dawley) weighing between 150 g and 250 g, about lO weeks old, were obtained from a breeding colony maintained by the Division of Animal Resources at the Arizona Health Sciences Center. Buffers and Solutions Solutions for homogenization and dilution prior to centrifugation contain l mM PMSF (9) and 15 mM B-mercaptoethanol. Both of these were added fresh prior to use.
Modified Homogenization Solution= 0.25 M sucrose (SchwartzMann enzyme grade), 2 mM Na~EDTA, 0.5 mM NapEGTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine (Sigma), 0.5 mM sper6idine (Sigma), and 15 mM i ris-HCl pH 7.4. This solution is similar to that o r i g i n a l l y described by Hewish and Burgoyne (4), except that sucrose concentration is reduced from 0.34 to 0.25M and the EDTA and EGTA concentrations are twice as high as in the original method, ~.l M Sucrose Dilution and Underlay Solution: This solution is identical to the "second underlay solution" of Hewish and Burgoyne (4). I t contains 2.1M sucrose, O.l mM Na2EDTA, O.l mM Na2EGTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine, and 15 mM Tris-HCl, pH 7.4. Isolation of Nuclei Rat l i v e r nuclei were isolated by the methods of Chauveau et al. (lO), Blobel and Potter (8) as modified by Lindell (9), and Hewish and Burgoyne (4). Rat l i v e r nuclei were also prepared by a modification of the method of Hewish and Burgoyne (4) as described below. Rats were sacrificed by spinal dislocation and decapitation. The livers were quickly removed and immediately placed in an ice-water slurry to cool them as rapidly as possible. Individual livers were then blotted dry and weighed. All subsequent procedures are performed at 0-40 •
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In the modified procedure, the ice-cold livers were minced with scissors and one mL of the modified homogenization solution per gram l i v e r wet weight was added to the finely chopped l i v e r . The mixture was poured into a chilled Potter-Elvehjem glass (Thomas, size C) homogenizing vessel and homogenized six strokes with a tear-drop shaped Teflon Dounce pestle by hand. The homogenate was then poured through two layers of cheesecloth into another Potter-Elvehjem vessel of the same size and homogenized ten strokes with a motor-driven standard Teflon pestle. The homogenate was then mixed with two volumes of 2.1M sucrose dilution solution, mixed well by s t i r r i n g , poured into nitrocellulose ultracentrifuge tubes (SW-27, 35 mL, Beckman), and underlayed with lO mL of the same 2.1M sucrose solution. This was accomplished with a 50-mL syringe f i t t e d with a blunt-ended number lO needle (Popper and Sons). The tubes were then centrifuged in a Beckman ultracentrifuge at 25,000 rpm (llO,O00 x g) for 40 min at 40 . The supernatant was discarded and wall of the tube carefully cleaned with a Kimwipe. The resulting nuclear pellet was then resuspended in 0.34 M sucrose, O.l mM Na~ EDTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine,=and 15 mM Tris-HCl, pH 7.4 as described by Hewish and Burgoyne (4). Nuclei were suspended in one-half volume per gram original wet weight tissue (~ l mg / mL) and b r i e f l y homogenized in a g~ass-Teflon homogenizer by hand. (ll),
DNA, protein and RNA were determined by the methods of Burton Lowry et al (12), and Fleck and Munro (13), respectively.
Isolation of Nuclei from Cells in Culture A procedure has also been developed for the isolation of nuclei from cells in culture using modified buffers of Hewish and Burgoyne (4). The procedure has been used to isolate nuclei from CHO, MCF-7, 3T3, Cloudman melanoma S-91, and 3T6 cells. The procedure described is for the isolation of nuclei from 3T6 cells. Cells were grown in Delbecco's MEM ~upplemented with I0% fetal calf serRm to a density of about 1.3 X lO D cells/cm L. Generally about l X lO° cells were used for nuclear isolations. The media is poured o f f and the cells are washed with ice cold Ca+z , Mg+z free phosphate buffered saline, and twice with modified homogenization solution (~3 mL/lO0 mm plate). The cells are then scraped from the plates with a rubber policeman and removed from the plate with about 3 mL of homogenization buffer. I f trypsin is employed, i t is important not to use PMSFuntil after trypsinization since PMSF inactivates trypsin. The cells are then centrifugedat 300-400 X g for 3 min in a benchtop centrifuge and the pellet resuspended in 5 mL of swelling buffer containing: 0.15 mM spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, and 15 mM Tris-HCl, pH 7.4 After standing on ice for I0 min, the cells are homogenized in a tight f i t t i n g glass Dounce (Kontes) homogenizer for 20 strokes (l stroke up and down), transferred to a plastic centrifuge tube, and nuclei are sedimented by centrigugation at 1300 X g for lO min. The nuclear pellet is then suspended in ~he resuspension buffer described for rat liver nuclei. From lOU cells, routinely, 600)g DNA is recovered. Alkaline Sucrose Gradients and DNA Assay Alkaline sucrose gradients were run in 15.6 mL of 5-20% sucrose (Mann) containing 0.4 M NaOH, O.l M NaEDTA, and 0.1% sarkosyl (Sigma) over a cushion of l mL 40% sucrose. Nuclei (150 ~L) were carefully pipetted onto the top of the gradient overlayed with 350 ~L of 2.5 mg/mL heparin and 2% sarkosyl as described by Walters and Hildebrand
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(14). A f t e r 15 min at room temperature, 450 ~L o f 0.8 M NaOH containing 0.2 M NaEDTA was added to lyse the n u c l e i . A f t e r an a d d i t i o n a l 45 min at room temperature, the gradients were centrifuged at 20o in a Beckman SW-27.1 r o t o r at I0,000 rpm (22,000 x g) f o r 24 hr. Gradients were c o l l e c t e d by pumping 60% sucrose from the bottom and c o l l e c t i n g 0.8 mL f r a c t i o n s from the top. Where i n d i c a t e d , 5-20% sucrose gradients were also run in volumes of 5 mL using the SW 50.1 r o t o r at I0,000 rpm (12,000 x g) f o r 24 hrs. The volumes given above were adjusted in proportion to the size o f the g r a d i e n t . DNA was determined by fluorimetry after reacting with 3,5-diaminobenzoic acid essentially by the method of Kissane and Robbins (15). The amount of DNA in each sample was determined on an Aminco-Bowman fluorimeter using 420 nm wavelength excitation and 520 nm wavelength emission. The amount of DNA corresponding to the percent fluorescence was determined from a standard curve using known amounts of calf thymus DNA (Sigma Type I) carried through the identical procedure. Standard curves routinely gave a value of 28% fluorescence per ~g DNA. Estimation of DNA Size The size of the DNA in the alkaline sucrose gr#dients was e s t i mated with Chinese hamster ovary (CHO) DNA as a mar'ker and using the relationship S = Bd/m2t where S is the sedimentation coefficient, d is the distance sedimented, ~ is the angular velocity and t is time (hrs) (16). The sedimentation coefficient of CHO DNA was 159S (14). The top of the gradient was estimated to begin 2/3rds into the f i r s t fraction to account for the volume of lysing layer employed. Electron Microscopy Nuclei (0.5 mL) suspended in 25% glycerol, l mM EDTA, and 0.05 M Tris-HCl, pH 7.9 were thawed after storage in l i q u i d nitrogen and centrifuged in a Brinkman microcentrifuge b r i e f l y . Nuclei were resuspended in 0.5 mL Hewish and Burgoyne final solution and recentrifuged as above. Nuclear pellets were fixed with Karnovsky's f i x a t i v e for 30 min at 40, gently rinsed three times in Hewish and Burgoyne final solution, and post-fixed for l hr in 2% OsO4 in the final buffer solution above at 40 . Pellets were rinsed lnlmL final buffer solution as previously mentioned and dehydration was accomplished through graded ethanol washes prior to propylene oxide. Pellets were f l a t embedded in Epon 812 (Ladd) and thin sections (approximately 80 ~m) were cut and stained with uranyl acetate prior to examination in a Philip's 300 transmission electron microscope. In Vitro Transcription In v i t r o transcription in isolated nuclei was performed based on the assay of Roeder and Rutter (17) as described by Lindell and Duffy (18). Results The u t i l i t y of a procedure for the isolation of nuclei depends on obtaining nuclei which are intact both morphologically and biochemically, thus reflecting in vivo conditions as closely as possible. In addition, the time of preparation of the nuclei should be s u f f i c i e n t l y rapid for convenience to allow the use of the isolated nuclei for assay while fresh. The nuclei isolation procedure described by Hewish and Burgoyne (4) p a r t i a l l y f u l f i l l s these c r i t e r i a . However, as described here, the time of preparation of nuclei by that method can be s i g n i f i c a n t l y reduced to
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permit rapid i s o l a t i o n (I hr for r a t l i v e r and the same for c e l l s in culture) allowing immediate use of the nuclei for studies of nuclear enzymology. For r a t l i v e r nuclei, we have used the one-step c e n t r i f u g a t i o n procedure and sucrose concentrations of Blobel and Potter (8), as modified by Lindell (9), with polyamines in place of d i v a l e n t metal ions in the solutions as described by Hewish and Burgoyne (4) to develop the nuclear i s o l a t i o n procedure described. The basic modifications from the method of Hewish and Burgoyne (4) are a reduction in the volume of the i n i t i a l homogenization solution (one mL homogenizing solution/gm weight of l i v e r ) and elimination of the low-speed c e n t r i f u g a t i o n step. Other a l t e r a t i o n s include a decrease in the sucrose concentration in the o r i g i n a l homogenization solution from 0.34 to 0.25 M and a doubling of the EDTA and EGTA concentrations. Although not complex, these changes allow for a very rapid means of nuclear i s o l a t i o n from r a t l i v e r without loss of the q u a l i t y obtainable with the Hewish and Burgoyne (4) method as seen in Table I . Nuclei prepared by the modified procedure are very s i m i l a r to those prepared by the method of Hewish and Burgoyne (4) in t h e i r content of macromolecules and y i e l d as measured by DNA, RNA and protein. TABLE I DNA, RNA and Protein Content and y i e l d of Rat Liver Nuclei Isolated by D i f f e r e n t Procedures
Type of Preparation
Experiment No.
Amounts (in mg) Recovered from I0 g Liver DNA RNA Protein
% Yield
Modified Procedure
1 2
13.2 10.6
1.62 2.87
74.9 83.5
65.4 60.6
HewishBurgoyne
1 2
13.8 13.6
1.49 2.32
80.1 82.0
69.7 68.3
DNA, RNA, and protein content of isolated nuclei were determined as described in Methods. The y i e l d of nuclei was calculated by comparing the DNA content of the homogenate to that of the isolated nuclei. Experiments 1 can be d i r e c t l y compared because the same tissue was used to prepare nuclei by both methods. Experiment 2 was done on separate tissue.
I t can be seen in the phase-contrast microqraph of (Fig. IA), that these nuclei q u a l i t a t i v e l y contain l i t t l e or no major cytoplasmic contamination. When compared with photographs of nuclei isolated by the Hewish and Burgoyne (4) procedure, no difference can be observed (data not shown). In a d d i t i o n , when these nuclei were examined by transmission electron microscopy, no cytoplasmic contaminants were observed (Fig. I B ) , although the outer nuclear envelope can be seen.
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The state of the DNA in nuclei isolated by various methods is an important consideration in the u t i l i t y of those nuclei for in v i t r o experiments or in the isolation of highly polymerized DNA. The size distribution of the DNA from four nuclear isolation procedures are compared in (Fig. 2, A-D) on alkaline sucrose dens}ty gradients. I t can be seen in (Fig. 2, A-B) that nuclei isolated by the method of Blobel and Potter (8) and Chauveau et a l . (lO) are not as highly polymerized as the DNA from nuclei isolated by the Hewish and Burgoyne (4) or the modified method (Fig. 2, C and D). The size distribution of these two l a t t e r profiles indicate that the DNA was not t o t a l l y denatured in these experiments, T h i s is not an unusual observation when alkaline sucrose gradients are run on the DNA from these nuclei and may be an index of the high degree of polymerization of the DNA from these nuclei.
FIG. l Phase Contrast and Transmission Electron Micrographs of Rat Liver Nuclei Isolated by the Modified Method. A. Nuclei photographed with a Zeiss phase contrast microscope. Magnification is 2200 fold. B. Electron micrograph of nuclei prepared and stained as described in Methods. Magnification is 6600-fold.
As a marker for estimating the sedimentation coefficient, Chinese hamster ovary (CHO) cells were lysed on top of the gradient and centrifuged through alkaline sucrose according to Walters and Hildebrand (14). The peak of CHO DNA sediments at 159S. Nuclei employed in (Fig. 2) (A-D) were stored frozen in l i q u i d N2 prior to performing alkaline sucrose gradients. There is no change in size when nuclei are used when fresh (Duffy, J J. unpublished).
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A major difference in the isolation of nuclei between the methods Hewish and Burgoyne (4) and the modified method described here with that of Blobel and Potter (8) is the use of the divalent metal ion Mg+z in the l a t t e r procedure. The state of the DNA during in vitro assays of the RNA polymerases is, therefore, of importance because transcription requires a divalent cation Although the modified procedure for nuclear +z isolation provides highly polymerized DNA, we were concerned about the Mn requirement in the RNA polymerase assay. Figure ~A shows that the nuclear DNA size is rapidly reduced under conditions employed for assaying RNA polymerases in nuclei. Within the f i r s t five minutes of incubation, the bulk of the nuclear DNA is reduced to a size (about 40 S) significantly smaller than that of the input DNA (llO S). Degradation continued beyond the f i f t h minute (see lO and 30 min), but the time period for major size reduction of the DNA occurs early in the incubation. Degradation of nuclear DNA was observed even when nuclei were stored on ice in the presence of the complete assay mixture for 30 min (data not shown).
I
I
I
I
15
A
B
C
D
10 5 < z
c}
15 10
5 top
10
15
20
5
Fraction Number
10
15
20 bottom
FIG. 2 Alkaline Sucrose Gradients of Rat Liver Nuclear DNA. Nuclei were prepared by the method described below and stored frozen in liquid nitrogen. Nuclei were thawed, and 150 ~L (60-80 ~g DNA) were used for alkaline sucrose gradients according to the procedure of Walters and Hildebrand as described in Methods The gradients were centrifuged in a Beckman SW27.1 rotor at lO,O00 rpm (22,000 x g) for 24 hours at 200 and fractions collected and assayed for DNA content as described in Methods. Nuclei were isolated by the methods of (A) Blobel and Potter (8), (B) Chauveau, et al. (lO), (C) Hewish and Burgoyne (4), and (D) the modified procedure described.
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4
I //"~.,,~ 10'
A
5
]
Top 5
5
10 15 Fraction Number
/
20 Bottom
'7 x
o
i
R.'
13. £3 I
5
I
10 Time (min)
I
I
15
20
FIG. 3 Comparison of The Rate of Degradation of Nuclear DNA and the Rate of Total RNA Synthesis Upon Incubation In V i t r o . Frozen nuclei prepared by the modified method of Hewish and Burgoyne (4) were thawed, diluted 2.4 fold into the RNA polymerase assay mixture. (Final concentration: 0.05 M Tris HCI, pH 7.9,6 mM NaF, 0.6 mM each Rf GTP, CTP, ATP, O.l mM UTP, and 1.6 mM M~CI2) and incubated at 30V. At the times indicated, aliquots were removed and treated one of two ways: (A) 25 ~L aliquots were transferred to the top of alkaline sucrose gradients, lysed, sedimented at lO,O00 rpm (max. 12,000 x g) for 24 hrs and the DNA d i s t r i b u t i o n assayed as described in Methods except that a SW 50.I rotor was used. Incubation times were 5 min (0), lO min (A) and 30 min (A). The size of the input DNA (Q) was determined from the same nuclei diluted 2.4 fold with water and stored 30 min on ice before lysis and c e n t r i fugation. Input DNA was 22 ~g and recovery ranged from lO pg to 16 pg. (B) 36 ~L aliquots of the incubation mixture were spotted on DE81 f i l t e r s and the product RNA quantitated as described by Lindell and Duffy. (18). Each sample contained 34 ~9 DNA. The points shown are the mean value of t r i p l i c a t e determinatiQns.
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When the time course of UMP incorporation into RNA is measured in nuclei isolated by the modified method, it can be seen (Fig. 3B) that there is a rapid loss of linearity which appears to correlate with the reduction in DNA size as observed with time in (Fig. 3A). The experiment depicted in (Fig. 3A, and 3B) employed nuclei which had been frozen in liquid N2. Nuclei which have been frozen are not as good for in vitro transcription as those which are assayed while nuclei have linear incorporation of UMP into fresh. Freshlymated RNA for 5 to 1.0min. Therefore, when nuclei isolated by this procedure (or any procedure) are used for in vitro transcription, it is important to determine the time-course of mP= RNA.
Discussion We have described a modification of the nuclear isolation procedure of Hewish and Burgoyne (4) for rat liver nuclei which is rapid and allows a good yield of nuclei containing highly polymerized DNA. The brevity and convenience of this modified procedure makes it more attractive than the original method. This is especially convenient when nuclei are isolated from individual animals for the assay of nuclear RNA polymerases within the same day of preparation. This procedure was developed for rat liver nuclei but we have also used similar buffers to isolate nuclei from cells in culture. Nuclei isolated by this method are free of attached cytoplasm as seen by phase microscopy, and yield of DNA is good. However, without a sucrose cushion and additional centrifugation, considerable cell debris is present. This procedure can also be employed to isolate nuclei from tissues other than liver. If this is done, the density of nuclei from other tissues is often lower than those of rat liver nuclei. For example, we have previously observed that good yields of nuclei can be obtained from chick intestine by employing a 1.6 M sucrose underlay in the centrifugation step (19). By lowering the molarity of the sucrose underlay, higher nuclear yields can be obtained although often times cytoplasmic contamination is greater. When optimizing nuclear yield in a new tissue, we often vary the molarity of the underlay in separate tubes of the same homogenate during ultracentrifugation step. The yield and purity of nuclei are then assessed by phase microscopy and the most acceptable combination of these parameters adopted for standard use. Despite the usefulness of the procedure developed, it is still apparent that the endogenous endonuclease is still a problem when divalent metal ion(s) are added for in vitro transcription. For rat liver nuclei, the loss in linearity ??UMPncorporation correlates with the rapid reduction in DNA size (Figs. 3A, 38). This short time for linearity of -in vitro transcription has also been observed in nuclei isolated from cells in culture (data not shown).
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I t has been reported that poly (ADP-ribose) attachment to the Ca+2 -Mg+2 dependent endonuclease may i n h i b i t this enzyme (20, 21). We have, however, found that incubation of these nuclei with NAD+ under conditions where poly (ADP-ribose) is formed does not prevent the breakdown of DNA in the presence of divalent cations (Duffy J . J . , and L i n d e l l , T . J . , unpublished). One major problem in many nuclear isolation procedures is the well documented loss of nuclear enzymes including RNA polymerases (9,22-27). Since most nuclear isolation procedures u t i l i z e divalent cations, which results in the activation of the endogenous endonuclease, i t is possible that the loss of nuclear enzymes is due to this endonuclease action. We have also observed that nuclei isolated by the procedure described have less "free" RNA polymerase (28) than nuclei isolated by other traditional procedures. Two additional observations appear to r e l a t e to the amount of endogenous endonuclease action in nuclei. The f i r s t is that nuclei isolated by the procedure described are not good for the i s o l a t i o n of nucleoli by sonication. Second, we have observed that i t is d i f f i c u l t to s o l u b i l i z e nuclear protein from them by sonication in the presence of (NHa)pSOa(17). We suspect that these nuclei could be b r i e f l y treated 6i~h ~ divalent cation, l i k e Mgt~prior to the preparation of nucleoli or s o l u b i l i z a t i o n of nuclear protein. Treatment of isolated nuclei from c e l l s in culture with DNase has previously been used as a method to i s o l a t e nucleoli (29). In addition, Beebee (30) has described a similar procedure for the preparation of nuclear RNA polymerases~ We are, t h e r e f o r e , l e f t with a highly desirable, rapid nuclear i s o l a t i o n procedure which yields i n t a c t DNA. These nuclei are p o t e n t i a l l y suitable for the study of this isolated organelle. However, p r i o r to characterization of nuclear enzymology in s i t u , i t w i l l be important to find a way to i n h i b i t this endogenous endonuclease. We are, however, impressed enough with the nuclear i s o l a t i o n system developed to begin the search for the putative modulator or i n h i b i t o r of the endogenous endonuclease which w i l l allow us the opportunity to study t r a n s c r i p t i o n for extended periods of time without the degradation of endogenous template.
Acknowledgements We thank Dr. Eugene Gerner and Mr. David Holms for the Chinese hamster ovary c e l l s and Dr. C. Ward Kischer for the electron microscopy. We acknowledge the expert technical assistance of Mr. Russell Beaver, Mr. Bernard Byrnes, Ms. Lois O'Brien and Ms. Susan Bovair. Supported by GM-22897, AG-01289, and CA-27502.
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D. B. ROODYN, in Subcellular Components (G.D. Birne and S.M. Fox, Eds.), pp. 15-42, Plenum Press, New York (1969). E.A. SMUCKLER, M.KOPLITZ, and D.E. SMUCKLER, in Subnuclear Components: P_reparation and Fractionation (G.D. Birne, ed.) pp. 1-57 Butterworths, London (1976). H. BUSCH, and Y. DASKAL, in Methods in Cell Biology XVI (G. Stein and J. Stein, eds.) pp. 1-43, Academic Press, New York (1978). D.R. HEWISH, and L.A. BURGOYNE,Biochem. Biophys. Res. Commun. 52, 475-481 (1973). L.A. BURGOYNE,M.A. WAQAR, and M.R. ATKINSON, Biochem. Biophys. Res. Commun. 39, 254-259, (1970). L.A. BURGOYNE,M.A. WAQAR, and M.R. ATKINSON, Biochem. Biophys. Res. Commun. 39, 918-922 (1970). R. ISHIDA, H. AKIYOSHI, and T. TAKANASHI, Biochem. Biophys. Res. Commun. 56, 704-710 (1974). G. BLOBEL, and V.R. POTTER, Science 154, 1662-1665 (1966). T.J. LINDELL, Arch. Biochem. Biophys. 171, 268-275 (1975). J. CHAUVEAU, Y. MOULE, and C.H. ROUILLER, Expl. Cell Res. l l , 317-321 (I 956). K. BURTON, Biochem. J. 62, 315-323 (1956). O.H. LOWRY, N.J. ROSEBROUGH,A.L. FARR, and R.J..RANDALL, J. Biol. Chem. 193, 265-275 (1951). A. FLECK--~-H.N. MUNRO, Biochem. Biophys. Acta 55, 571-583 (1962). R.A. WALTERS, and C.E. HILDEBRAND, Biochem. Biophys. Acta 407, 120-124 (1975). J.M. KISSANE, and E. ROBINS, J. Biol. Chem. 233, 184-188 (1968). R. PAVLIS, R.W. YATSCOFF, R. SRIDHAR, and I.G. WALKER, Chem. Biol. Interactions 23, 31-44 (1978). R.G. ROEDER,and W.J. RUTTER, Nature 224, 231-237 (1969). T.J..LINDELL, and J.J. DUFFY, J. Biol. Chem. 254, 1454-1456 (1979). J.E. ZERWEKH, T.j. LINDELL, and M.R. HAUSSLER, J. Biol. Chem. 251,2388-2394 (1976). M. YAMADA, M. NAGAU, M. MIWA, and T. SUGIMURA, Biochem. Biophys. Res. Commun. 56, I093-I099 (1974). K. YOSHIHARA, Y. TAUGAWAand S.S. KOIDE, Biochem. Biophys. Res. Commun. 59, 658-665 (1974). S. LIAO,~. SAGHER, and S.M. FRAN, Nature 220, 1336-1337 (1968). S.T. JACOB, E.M. SAJDEL, and H.N. MUNRO, Biochem. Biophys. Res. Commun. 32, 831-838 (1968). C.j. CHESTERTON,and P.H.W. BUTTERWORTH, FEBS Lett. 12, 301-308 (1971). J.P. MONJARDINO, and L.J. CRAWFORD,Cold Spring Harbor Symp. Quant. Biol. 35, 659-662 (1970). B. SUGDEN, and J. SAMBROOK, Cold Spring HarbOr Symp. Quant. Biol. 35, 663-669 (1970). W. KELLER, and R. GOOR, Cold Spring Harbor Symp. Quant. Biol. 35, 671-680(1970). F.-~L. YU, Nature (London) 251, 344-346 (1974). S. PENMAN, J. Mol. Biol. ]]_7, I17-130 (1966). T.J.C. BEEBEE, Biochem. J. 183, 43-54 (1979).
Pergamon Pre
NUCLEAR ISOLATION BY A MODIFIED METHOD OF HEWISH AND BURGOYNE: IMPLICATIONS FOR THE STUDY OF NUCLEAR ENZYMOLOGY W.W. WolI, J.J. Duffy, N.A. Giese, and T.J. Lindell Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 U.S .A. (Received in final form October 30, 1981)
Summary This study describes a rapid and convenient modification of the established procedure of Hewish and Burgoyne (1973) Biochem. Biophys. Res. Commun. 52, 475-481, for the preparation of r a t l i v e r nuclei. A procedure is also described for the i s o l a t i o n of nuclei from c e l l s in culture using modified buffers of Hewish and Burgoyne. Substitution of the polyamines spermine and spermidine for d i v a l e n t metal cations in the i s o l a t i o n solutions along with u t i l i z a t i o n of the chelators EDTA and EGTA distinguishes t h e i r technique from other well known methods. The modification described for r a t l i v e r nuclei u t i l i z e s a single ultracent r i f u g a t i o n step for separating the nuclei from the homogenate by sedimentation through a dense sucrose s o l u t i o n . The new method produces a high y i e l d of p u r i f i e d nuclei which are characterized to have highly polymerized DNA on a l k a l i n e sucrose gradients. However, when metal ion is added back to these nuclei for in v i t r o t r a n s c r i p t i o n , there is a time dependent reduction in DNA size as measured on a l k a l i n e sucrose gradients which correlates With the loss in l i n e a r i t y of UMP incorporation into RNA. I t is suggested that the modified nuclear i s o l a t i o n procedure described produces more i n t a c t nuclei which w i l l form the basis for further i n v e s t i g a t i o n of the nucleus as an isolated eukaryotic organelle and allow characterization of endogenous enzymology in s i t u . Over the years, numerous methods have been developed for the i s o l a t i o n of nuclei from c e l l s and tissues ( f o r reviews, see I - 3 ) . Many of these methods are used for the preparation of subnuclear components and for the i s o l a t i o n of nuclear enzymes. There are d i s t i n c t advantages and disadvantages of many of these methods. A method which appeared to produce nuclei with i n t a c t DNA, which would be suitable for the characterization of nuclear enzymology, is the method of Hewish and Burgoyne (4). This method substituted the polyamines spermine and spermidine for d i v a l e n t cations present in most other procedures. Divalent cations (Ca+2 and Mg+2) are required for an endogenous endonuclease which catalyzes the cleavage of nuclear DNA (5-7). The use of spermine and spermidine obviates t h i s problem since the endonuclease cannot function when d i v a l e n t cations are absent and polyamines do not substitute for these metal ions. In addition to the use of polyamines, these workers also used 0024-3205/81/262709-ii$02.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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EDTA and EGTA to sequester any endogenous divalent cations within the tissue homogenate. The procedure of Hewish and Burgoyne (4) results in rat l i v e r nuclei of high quality in good yield but i t requires a lengthy amount of time, primarily because more than one centrigugation step is involved. Since we had been using the method of Blobel and Potter (8) to isolate rat l i v e r nuclei, we thought i t would be possible to combine the best features of that procedure, a single centrifugation step through dense sucrose, with the buffers recommended by Hewish and Burgoyne (4). The resulting procedure described in this paper is rapid and produces a consistently high nuclear yield when compared with the original procedure, and contain highly polymerized DNA, METHODS Animals Male and female rats (Sprague-Dawley) weighing between 150 g and 250 g, about lO weeks old, were obtained from a breeding colony maintained by the Division of Animal Resources at the Arizona Health Sciences Center. Buffers and Solutions Solutions for homogenization and dilution prior to centrifugation contain l mM PMSF (9) and 15 mM B-mercaptoethanol. Both of these were added fresh prior to use.
Modified Homogenization Solution= 0.25 M sucrose (SchwartzMann enzyme grade), 2 mM Na~EDTA, 0.5 mM NapEGTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine (Sigma), 0.5 mM sper6idine (Sigma), and 15 mM i ris-HCl pH 7.4. This solution is similar to that o r i g i n a l l y described by Hewish and Burgoyne (4), except that sucrose concentration is reduced from 0.34 to 0.25M and the EDTA and EGTA concentrations are twice as high as in the original method, ~.l M Sucrose Dilution and Underlay Solution: This solution is identical to the "second underlay solution" of Hewish and Burgoyne (4). I t contains 2.1M sucrose, O.l mM Na2EDTA, O.l mM Na2EGTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine, and 15 mM Tris-HCl, pH 7.4. Isolation of Nuclei Rat l i v e r nuclei were isolated by the methods of Chauveau et al. (lO), Blobel and Potter (8) as modified by Lindell (9), and Hewish and Burgoyne (4). Rat l i v e r nuclei were also prepared by a modification of the method of Hewish and Burgoyne (4) as described below. Rats were sacrificed by spinal dislocation and decapitation. The livers were quickly removed and immediately placed in an ice-water slurry to cool them as rapidly as possible. Individual livers were then blotted dry and weighed. All subsequent procedures are performed at 0-40 •
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Modified Nuclear Isolation Procedure
In the modified procedure, the ice-cold livers were minced with scissors and one mL of the modified homogenization solution per gram l i v e r wet weight was added to the finely chopped l i v e r . The mixture was poured into a chilled Potter-Elvehjem glass (Thomas, size C) homogenizing vessel and homogenized six strokes with a tear-drop shaped Teflon Dounce pestle by hand. The homogenate was then poured through two layers of cheesecloth into another Potter-Elvehjem vessel of the same size and homogenized ten strokes with a motor-driven standard Teflon pestle. The homogenate was then mixed with two volumes of 2.1M sucrose dilution solution, mixed well by s t i r r i n g , poured into nitrocellulose ultracentrifuge tubes (SW-27, 35 mL, Beckman), and underlayed with lO mL of the same 2.1M sucrose solution. This was accomplished with a 50-mL syringe f i t t e d with a blunt-ended number lO needle (Popper and Sons). The tubes were then centrifuged in a Beckman ultracentrifuge at 25,000 rpm (llO,O00 x g) for 40 min at 40 . The supernatant was discarded and wall of the tube carefully cleaned with a Kimwipe. The resulting nuclear pellet was then resuspended in 0.34 M sucrose, O.l mM Na~ EDTA, 60 mM KCI, 15 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine,=and 15 mM Tris-HCl, pH 7.4 as described by Hewish and Burgoyne (4). Nuclei were suspended in one-half volume per gram original wet weight tissue (~ l mg / mL) and b r i e f l y homogenized in a g~ass-Teflon homogenizer by hand. (ll),
DNA, protein and RNA were determined by the methods of Burton Lowry et al (12), and Fleck and Munro (13), respectively.
Isolation of Nuclei from Cells in Culture A procedure has also been developed for the isolation of nuclei from cells in culture using modified buffers of Hewish and Burgoyne (4). The procedure has been used to isolate nuclei from CHO, MCF-7, 3T3, Cloudman melanoma S-91, and 3T6 cells. The procedure described is for the isolation of nuclei from 3T6 cells. Cells were grown in Delbecco's MEM ~upplemented with I0% fetal calf serRm to a density of about 1.3 X lO D cells/cm L. Generally about l X lO° cells were used for nuclear isolations. The media is poured o f f and the cells are washed with ice cold Ca+z , Mg+z free phosphate buffered saline, and twice with modified homogenization solution (~3 mL/lO0 mm plate). The cells are then scraped from the plates with a rubber policeman and removed from the plate with about 3 mL of homogenization buffer. I f trypsin is employed, i t is important not to use PMSFuntil after trypsinization since PMSF inactivates trypsin. The cells are then centrifugedat 300-400 X g for 3 min in a benchtop centrifuge and the pellet resuspended in 5 mL of swelling buffer containing: 0.15 mM spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, and 15 mM Tris-HCl, pH 7.4 After standing on ice for I0 min, the cells are homogenized in a tight f i t t i n g glass Dounce (Kontes) homogenizer for 20 strokes (l stroke up and down), transferred to a plastic centrifuge tube, and nuclei are sedimented by centrigugation at 1300 X g for lO min. The nuclear pellet is then suspended in ~he resuspension buffer described for rat liver nuclei. From lOU cells, routinely, 600)g DNA is recovered. Alkaline Sucrose Gradients and DNA Assay Alkaline sucrose gradients were run in 15.6 mL of 5-20% sucrose (Mann) containing 0.4 M NaOH, O.l M NaEDTA, and 0.1% sarkosyl (Sigma) over a cushion of l mL 40% sucrose. Nuclei (150 ~L) were carefully pipetted onto the top of the gradient overlayed with 350 ~L of 2.5 mg/mL heparin and 2% sarkosyl as described by Walters and Hildebrand
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(14). A f t e r 15 min at room temperature, 450 ~L o f 0.8 M NaOH containing 0.2 M NaEDTA was added to lyse the n u c l e i . A f t e r an a d d i t i o n a l 45 min at room temperature, the gradients were centrifuged at 20o in a Beckman SW-27.1 r o t o r at I0,000 rpm (22,000 x g) f o r 24 hr. Gradients were c o l l e c t e d by pumping 60% sucrose from the bottom and c o l l e c t i n g 0.8 mL f r a c t i o n s from the top. Where i n d i c a t e d , 5-20% sucrose gradients were also run in volumes of 5 mL using the SW 50.1 r o t o r at I0,000 rpm (12,000 x g) f o r 24 hrs. The volumes given above were adjusted in proportion to the size o f the g r a d i e n t . DNA was determined by fluorimetry after reacting with 3,5-diaminobenzoic acid essentially by the method of Kissane and Robbins (15). The amount of DNA in each sample was determined on an Aminco-Bowman fluorimeter using 420 nm wavelength excitation and 520 nm wavelength emission. The amount of DNA corresponding to the percent fluorescence was determined from a standard curve using known amounts of calf thymus DNA (Sigma Type I) carried through the identical procedure. Standard curves routinely gave a value of 28% fluorescence per ~g DNA. Estimation of DNA Size The size of the DNA in the alkaline sucrose gr#dients was e s t i mated with Chinese hamster ovary (CHO) DNA as a mar'ker and using the relationship S = Bd/m2t where S is the sedimentation coefficient, d is the distance sedimented, ~ is the angular velocity and t is time (hrs) (16). The sedimentation coefficient of CHO DNA was 159S (14). The top of the gradient was estimated to begin 2/3rds into the f i r s t fraction to account for the volume of lysing layer employed. Electron Microscopy Nuclei (0.5 mL) suspended in 25% glycerol, l mM EDTA, and 0.05 M Tris-HCl, pH 7.9 were thawed after storage in l i q u i d nitrogen and centrifuged in a Brinkman microcentrifuge b r i e f l y . Nuclei were resuspended in 0.5 mL Hewish and Burgoyne final solution and recentrifuged as above. Nuclear pellets were fixed with Karnovsky's f i x a t i v e for 30 min at 40, gently rinsed three times in Hewish and Burgoyne final solution, and post-fixed for l hr in 2% OsO4 in the final buffer solution above at 40 . Pellets were rinsed lnlmL final buffer solution as previously mentioned and dehydration was accomplished through graded ethanol washes prior to propylene oxide. Pellets were f l a t embedded in Epon 812 (Ladd) and thin sections (approximately 80 ~m) were cut and stained with uranyl acetate prior to examination in a Philip's 300 transmission electron microscope. In Vitro Transcription In v i t r o transcription in isolated nuclei was performed based on the assay of Roeder and Rutter (17) as described by Lindell and Duffy (18). Results The u t i l i t y of a procedure for the isolation of nuclei depends on obtaining nuclei which are intact both morphologically and biochemically, thus reflecting in vivo conditions as closely as possible. In addition, the time of preparation of the nuclei should be s u f f i c i e n t l y rapid for convenience to allow the use of the isolated nuclei for assay while fresh. The nuclei isolation procedure described by Hewish and Burgoyne (4) p a r t i a l l y f u l f i l l s these c r i t e r i a . However, as described here, the time of preparation of nuclei by that method can be s i g n i f i c a n t l y reduced to
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permit rapid i s o l a t i o n (I hr for r a t l i v e r and the same for c e l l s in culture) allowing immediate use of the nuclei for studies of nuclear enzymology. For r a t l i v e r nuclei, we have used the one-step c e n t r i f u g a t i o n procedure and sucrose concentrations of Blobel and Potter (8), as modified by Lindell (9), with polyamines in place of d i v a l e n t metal ions in the solutions as described by Hewish and Burgoyne (4) to develop the nuclear i s o l a t i o n procedure described. The basic modifications from the method of Hewish and Burgoyne (4) are a reduction in the volume of the i n i t i a l homogenization solution (one mL homogenizing solution/gm weight of l i v e r ) and elimination of the low-speed c e n t r i f u g a t i o n step. Other a l t e r a t i o n s include a decrease in the sucrose concentration in the o r i g i n a l homogenization solution from 0.34 to 0.25 M and a doubling of the EDTA and EGTA concentrations. Although not complex, these changes allow for a very rapid means of nuclear i s o l a t i o n from r a t l i v e r without loss of the q u a l i t y obtainable with the Hewish and Burgoyne (4) method as seen in Table I . Nuclei prepared by the modified procedure are very s i m i l a r to those prepared by the method of Hewish and Burgoyne (4) in t h e i r content of macromolecules and y i e l d as measured by DNA, RNA and protein. TABLE I DNA, RNA and Protein Content and y i e l d of Rat Liver Nuclei Isolated by D i f f e r e n t Procedures
Type of Preparation
Experiment No.
Amounts (in mg) Recovered from I0 g Liver DNA RNA Protein
% Yield
Modified Procedure
1 2
13.2 10.6
1.62 2.87
74.9 83.5
65.4 60.6
HewishBurgoyne
1 2
13.8 13.6
1.49 2.32
80.1 82.0
69.7 68.3
DNA, RNA, and protein content of isolated nuclei were determined as described in Methods. The y i e l d of nuclei was calculated by comparing the DNA content of the homogenate to that of the isolated nuclei. Experiments 1 can be d i r e c t l y compared because the same tissue was used to prepare nuclei by both methods. Experiment 2 was done on separate tissue.
I t can be seen in the phase-contrast microqraph of (Fig. IA), that these nuclei q u a l i t a t i v e l y contain l i t t l e or no major cytoplasmic contamination. When compared with photographs of nuclei isolated by the Hewish and Burgoyne (4) procedure, no difference can be observed (data not shown). In a d d i t i o n , when these nuclei were examined by transmission electron microscopy, no cytoplasmic contaminants were observed (Fig. I B ) , although the outer nuclear envelope can be seen.
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The state of the DNA in nuclei isolated by various methods is an important consideration in the u t i l i t y of those nuclei for in v i t r o experiments or in the isolation of highly polymerized DNA. The size distribution of the DNA from four nuclear isolation procedures are compared in (Fig. 2, A-D) on alkaline sucrose dens}ty gradients. I t can be seen in (Fig. 2, A-B) that nuclei isolated by the method of Blobel and Potter (8) and Chauveau et a l . (lO) are not as highly polymerized as the DNA from nuclei isolated by the Hewish and Burgoyne (4) or the modified method (Fig. 2, C and D). The size distribution of these two l a t t e r profiles indicate that the DNA was not t o t a l l y denatured in these experiments, T h i s is not an unusual observation when alkaline sucrose gradients are run on the DNA from these nuclei and may be an index of the high degree of polymerization of the DNA from these nuclei.
FIG. l Phase Contrast and Transmission Electron Micrographs of Rat Liver Nuclei Isolated by the Modified Method. A. Nuclei photographed with a Zeiss phase contrast microscope. Magnification is 2200 fold. B. Electron micrograph of nuclei prepared and stained as described in Methods. Magnification is 6600-fold.
As a marker for estimating the sedimentation coefficient, Chinese hamster ovary (CHO) cells were lysed on top of the gradient and centrifuged through alkaline sucrose according to Walters and Hildebrand (14). The peak of CHO DNA sediments at 159S. Nuclei employed in (Fig. 2) (A-D) were stored frozen in l i q u i d N2 prior to performing alkaline sucrose gradients. There is no change in size when nuclei are used when fresh (Duffy, J J. unpublished).
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A major difference in the isolation of nuclei between the methods Hewish and Burgoyne (4) and the modified method described here with that of Blobel and Potter (8) is the use of the divalent metal ion Mg+z in the l a t t e r procedure. The state of the DNA during in vitro assays of the RNA polymerases is, therefore, of importance because transcription requires a divalent cation Although the modified procedure for nuclear +z isolation provides highly polymerized DNA, we were concerned about the Mn requirement in the RNA polymerase assay. Figure ~A shows that the nuclear DNA size is rapidly reduced under conditions employed for assaying RNA polymerases in nuclei. Within the f i r s t five minutes of incubation, the bulk of the nuclear DNA is reduced to a size (about 40 S) significantly smaller than that of the input DNA (llO S). Degradation continued beyond the f i f t h minute (see lO and 30 min), but the time period for major size reduction of the DNA occurs early in the incubation. Degradation of nuclear DNA was observed even when nuclei were stored on ice in the presence of the complete assay mixture for 30 min (data not shown).
I
I
I
I
15
A
B
C
D
10 5 < z
c}
15 10
5 top
10
15
20
5
Fraction Number
10
15
20 bottom
FIG. 2 Alkaline Sucrose Gradients of Rat Liver Nuclear DNA. Nuclei were prepared by the method described below and stored frozen in liquid nitrogen. Nuclei were thawed, and 150 ~L (60-80 ~g DNA) were used for alkaline sucrose gradients according to the procedure of Walters and Hildebrand as described in Methods The gradients were centrifuged in a Beckman SW27.1 rotor at lO,O00 rpm (22,000 x g) for 24 hours at 200 and fractions collected and assayed for DNA content as described in Methods. Nuclei were isolated by the methods of (A) Blobel and Potter (8), (B) Chauveau, et al. (lO), (C) Hewish and Burgoyne (4), and (D) the modified procedure described.
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4
I //"~.,,~ 10'
A
5
]
Top 5
5
10 15 Fraction Number
/
20 Bottom
'7 x
o
i
R.'
13. £3 I
5
I
10 Time (min)
I
I
15
20
FIG. 3 Comparison of The Rate of Degradation of Nuclear DNA and the Rate of Total RNA Synthesis Upon Incubation In V i t r o . Frozen nuclei prepared by the modified method of Hewish and Burgoyne (4) were thawed, diluted 2.4 fold into the RNA polymerase assay mixture. (Final concentration: 0.05 M Tris HCI, pH 7.9,6 mM NaF, 0.6 mM each Rf GTP, CTP, ATP, O.l mM UTP, and 1.6 mM M~CI2) and incubated at 30V. At the times indicated, aliquots were removed and treated one of two ways: (A) 25 ~L aliquots were transferred to the top of alkaline sucrose gradients, lysed, sedimented at lO,O00 rpm (max. 12,000 x g) for 24 hrs and the DNA d i s t r i b u t i o n assayed as described in Methods except that a SW 50.I rotor was used. Incubation times were 5 min (0), lO min (A) and 30 min (A). The size of the input DNA (Q) was determined from the same nuclei diluted 2.4 fold with water and stored 30 min on ice before lysis and c e n t r i fugation. Input DNA was 22 ~g and recovery ranged from lO pg to 16 pg. (B) 36 ~L aliquots of the incubation mixture were spotted on DE81 f i l t e r s and the product RNA quantitated as described by Lindell and Duffy. (18). Each sample contained 34 ~9 DNA. The points shown are the mean value of t r i p l i c a t e determinatiQns.
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Modified Nuclear Isolation Procedure
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When the time course of UMP incorporation into RNA is measured in nuclei isolated by the modified method, it can be seen (Fig. 3B) that there is a rapid loss of linearity which appears to correlate with the reduction in DNA size as observed with time in (Fig. 3A). The experiment depicted in (Fig. 3A, and 3B) employed nuclei which had been frozen in liquid N2. Nuclei which have been frozen are not as good for in vitro transcription as those which are assayed while nuclei have linear incorporation of UMP into fresh. Freshlymated RNA for 5 to 1.0min. Therefore, when nuclei isolated by this procedure (or any procedure) are used for in vitro transcription, it is important to determine the time-course of mP= RNA.
Discussion We have described a modification of the nuclear isolation procedure of Hewish and Burgoyne (4) for rat liver nuclei which is rapid and allows a good yield of nuclei containing highly polymerized DNA. The brevity and convenience of this modified procedure makes it more attractive than the original method. This is especially convenient when nuclei are isolated from individual animals for the assay of nuclear RNA polymerases within the same day of preparation. This procedure was developed for rat liver nuclei but we have also used similar buffers to isolate nuclei from cells in culture. Nuclei isolated by this method are free of attached cytoplasm as seen by phase microscopy, and yield of DNA is good. However, without a sucrose cushion and additional centrifugation, considerable cell debris is present. This procedure can also be employed to isolate nuclei from tissues other than liver. If this is done, the density of nuclei from other tissues is often lower than those of rat liver nuclei. For example, we have previously observed that good yields of nuclei can be obtained from chick intestine by employing a 1.6 M sucrose underlay in the centrifugation step (19). By lowering the molarity of the sucrose underlay, higher nuclear yields can be obtained although often times cytoplasmic contamination is greater. When optimizing nuclear yield in a new tissue, we often vary the molarity of the underlay in separate tubes of the same homogenate during ultracentrifugation step. The yield and purity of nuclei are then assessed by phase microscopy and the most acceptable combination of these parameters adopted for standard use. Despite the usefulness of the procedure developed, it is still apparent that the endogenous endonuclease is still a problem when divalent metal ion(s) are added for in vitro transcription. For rat liver nuclei, the loss in linearity ??UMPncorporation correlates with the rapid reduction in DNA size (Figs. 3A, 38). This short time for linearity of -in vitro transcription has also been observed in nuclei isolated from cells in culture (data not shown).
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I t has been reported that poly (ADP-ribose) attachment to the Ca+2 -Mg+2 dependent endonuclease may i n h i b i t this enzyme (20, 21). We have, however, found that incubation of these nuclei with NAD+ under conditions where poly (ADP-ribose) is formed does not prevent the breakdown of DNA in the presence of divalent cations (Duffy J . J . , and L i n d e l l , T . J . , unpublished). One major problem in many nuclear isolation procedures is the well documented loss of nuclear enzymes including RNA polymerases (9,22-27). Since most nuclear isolation procedures u t i l i z e divalent cations, which results in the activation of the endogenous endonuclease, i t is possible that the loss of nuclear enzymes is due to this endonuclease action. We have also observed that nuclei isolated by the procedure described have less "free" RNA polymerase (28) than nuclei isolated by other traditional procedures. Two additional observations appear to r e l a t e to the amount of endogenous endonuclease action in nuclei. The f i r s t is that nuclei isolated by the procedure described are not good for the i s o l a t i o n of nucleoli by sonication. Second, we have observed that i t is d i f f i c u l t to s o l u b i l i z e nuclear protein from them by sonication in the presence of (NHa)pSOa(17). We suspect that these nuclei could be b r i e f l y treated 6i~h ~ divalent cation, l i k e Mgt~prior to the preparation of nucleoli or s o l u b i l i z a t i o n of nuclear protein. Treatment of isolated nuclei from c e l l s in culture with DNase has previously been used as a method to i s o l a t e nucleoli (29). In addition, Beebee (30) has described a similar procedure for the preparation of nuclear RNA polymerases~ We are, t h e r e f o r e , l e f t with a highly desirable, rapid nuclear i s o l a t i o n procedure which yields i n t a c t DNA. These nuclei are p o t e n t i a l l y suitable for the study of this isolated organelle. However, p r i o r to characterization of nuclear enzymology in s i t u , i t w i l l be important to find a way to i n h i b i t this endogenous endonuclease. We are, however, impressed enough with the nuclear i s o l a t i o n system developed to begin the search for the putative modulator or i n h i b i t o r of the endogenous endonuclease which w i l l allow us the opportunity to study t r a n s c r i p t i o n for extended periods of time without the degradation of endogenous template.
Acknowledgements We thank Dr. Eugene Gerner and Mr. David Holms for the Chinese hamster ovary c e l l s and Dr. C. Ward Kischer for the electron microscopy. We acknowledge the expert technical assistance of Mr. Russell Beaver, Mr. Bernard Byrnes, Ms. Lois O'Brien and Ms. Susan Bovair. Supported by GM-22897, AG-01289, and CA-27502.
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References I. 2. 3. 4. 5. 6. 7. 8. 9. lO.. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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