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Evidence for an association of most nuclear RNA with chromatin
J . M d . Biol.-(1976) 102, 177-191
Evidence for an Association o f m o s t Nuclear R N A with Chromatin C~ARLES B. Kra~'MEL,STANLEY K. SESSIONS AND MIC~AV.L C. MAcLEOD t
Department of Biology University of Oregon Eugene, Ore. 97403, U.S.A. (Received 17 .February 1975, and in revisedform 30 October1975) Chromatin prepared from mouse myeloma cell nuclei under conditions of low ionic strength and with minimal shearing contains at least 80% of the nuclear RNA which is labeled during a 5 or 30-minute pulse with uridine, or during a 5-m~nute pulse followed by a 25-minute chase. The RNA remains associated with chromatin after shearing by sonication, as determined by sedimentation and equilibrium density-gradient analyses. I t is released from chromatin by procedures developed elsewhere to extract ribonucleoprotein particles from intact nuclei. These results, and the results of reconstruction experiments, suggest that this association reflects the /n rive location of nuclear RNA and is not the result of non-specific binding during preparation. The chromatin-associated RNA includes RNA species which are known to be post-transcriptional; 32 S precursor ribosomal RNA and poly(A)-cont,.inlng heterogeneous nuclear RNA. Fractions of chromatin enriched in associated RNA were obtained by two methods derceloped elsewhere, and in both ~ f these the enrichment obtained (3 to 10-fold) appears to be independent of the age of the RNA. The implications of these results for models of RNA !processing are discussed. 1. I n t r o d u c t i o n I n m a m m a l i a n cells transcription of R N A molecules occurs along restricted regions of chromatin. One class of transcriptional product, the 45 S ribosomal R N A precursor, remains in close association with the ribosomal genes in the nucleolus, where processing of the transcrip~ takes place. Transpor~ of the fine,lied 28 S a n d 18 S r R N A molecules m u s t be rapid, since t h e y cannot be detected in the nucleus (Penman, 1966). The other main class of transcriptional products, heterogeneous nuclear RNA, is thought to be processed differently. I t has been suggested (Samarina et al., 1968) t h a t the newly synthesized hnRNA:~ molecules leave the chromatin fibril in associa. tion with proteins, and enter the nucleoplasm, where post-transcriptional "processing" takes place. This includes a rapid degradation of the m a j o r p a r t of most h n R N A molecules to nucleotides (Sherrer & Marcaud, 1965), and the addition of a sequence t Present address: Biology Division, Oak Ridge National Lab.. Oak Ridge, Tenn. 87830, U.S.A. Abbreviations used: hnRNA, heterogeneous nuclear RNA; RN'P, ribonucloopretein; tmRNP, heterogeneous nuolear RIq-P. 12
177
178
C. B. K I M M E L E T A L .
of adenylate residues to some of t h e transcripts (Jellinek et al., 1973; P e r r y et al., 1974). I n this scheme, small poly(A)-containing molecules are t h o u g h t t o be transp o r t e d to the cytoplasm a n d eventually associate with ribosomes as functional messenger R N A . The evidence for the existence of nucleoplasmic h n R N P particles has a c c u m u l a t e d from a series of analyses (Samarina eta/., 1968; Pederson, 1974a,b; Martin et al., 1973) of R N P contained in nuclei, which h a v e properties expected for post-transcriptional h n R N P particles, a n d wlfieh p r e s u m a b l y contain no D N A . W e report here t h e results of experiments which suggest t h a t post-transcriptional R N A molecules, including h n R N A , r e m a i n t i g h t l y associated with c h r o m a t i n fibrils during the m a j o r p a r t of their lifetimes in the nucleus. T h e distributions of nascent a n d post-transcriptional R N A molecules a m o n g different classes of c h r o m a t i n particles are non-random, and appear to be identical.
2. M a t e r i a l s a n d M e t h o d s
(a) Cells and labeling procedures Cultures of a cloned strain of myeloma S 194 were obtained from Dr G. Waring, and grown as cell suspensions in fortified Eagle's medium (Volt & Dulbecco, 1963) containing 10~o home serum (North American Biological Supply Co.). Cells in the late exponential phase of growth (Kimmel, 1971) were treated with [2-14C]thymidine (0.01 to 0-03 ~Ci/ml, 0.23 Ci/g; New England Nuclear) for about 1 day to label cellular DNA, and continued growth during this period was routinely checked by cell counts (generation time ~ 14 h). To pulse-label RNA, the cells were centrifuged to a pellet, and concentrated up to 10-fold in fresh, warm culture meditun. After a period of 5 min at 37~ [5, 6-3H]uridine was added (10 to 40 ~Ci/ml, 185 Ci/g; New England Nuclear), and the cells incubated for various periods (0.5 to 20 rain). I n pulse-chase experiments, the cells in medium containing [5,6-3H]uridine were diluted 4-fold in warm medium containing 10-~ m-unlabeled uridine (zero time of chase). The cells were centrifuged to a pellet (500 g for 3 rain) and resuspended in warm medium containing 10 -4 ~-uridine. (b) Cell fractionation Radioactive labeling was terminated by diluting the cell suspension in a slurry of iced medium, and subsequent operations were carried out rapidly at 0 to 4~ The cells were centrifuged to a pellet (500 g for 3 min), and washed once by centrifugation in LS buffer (5 mM-NaC1, 5 mM-KC1, 1-5 mM-MgC12, 10 m~-Tris-HC1, p H 7"4). The cells were resuspended in this buffer, allowed to swell for 3 min, and gently homogenized with a Dounce homogenizer (Kontes Glass Co., B pestle). Care was taken at this stage not to lyse nuclei, and the homogenate usually contained 5 to 10% of unbroken cells, as determined by phase microscopy. The nuclei were sedimented (1000 g for 5 rain), and the pellet was resuspended with vortex mixing and gradual addition of 1 mM-Tris-HC1 (pH 7.0). This procedure lyses most nuclei, and complete lysis was achieved with gentle Dounce homogenization. I n some experiments the nuclei were lysed, and subsequent operations carried out in 1 mM or 0.2 mM-EDTA (pH 7.0). Chromatin was centrifuged to a pellet (27,000 g for 5 rain; Sorvall centrifuge, SS34 rotor; 15,000 revs/min) and resuspended, again with vigorous mixing and gradual addition of buffer (usually 1 mM-Tris.HC1, p H 7.0). (c) Shearing of chromatin "Soluble" chromatin particles (i.e. those remaining in the supernate after centrifugation at 27,000 g for 5 min) were prepared by shearing the chromatin suspension described above. Samples of 1 ml, in a 5-ml beaker in ice, were sonicated with the standard 0"5-in probe of the Branson model 185 sonifier, using three 5-s treatments at 20 V. The sonicate w~s Clarified by centrifugation (27,000 g for 5 min).
A S S O C I A T I O N OF N U C L E A R R N A AND C H R O M A T I N
179
(d) Analytical procedures (i) Ghemical assays D N A was determined b y the B u r t o n (1956) procedure, R N A b y the oreinol reaction (Schneider, 1957), and protein b y the method of Lowry et al. (1951). Calf t h y m u s I)NA, yeast soluble RNA, a n d crystalline bovine serum a l b u m i n served as standards (Sigma Chemical Co.). (ii) Precipitation with trichloroacetic acid A total of 250/~g of yeast R N A (Sigma Chemical Co.) was added to ehromatin samples, and a n equal volume of cold 10% trichloroacetic acid was rapidly mixed with the sample. Insoluble material was centrifuged to a pellet (9000 g for 1 min), washed by eentrifugation in 5 ~/o triehloroaeetm acid, a n d dissolved in 0-1 N-NaOH. (iii) Determination of 8H and 14G radioactivity Aqueous samples were diluted to between 0.2 a n d 1 ml with water, a n d 10 vol. of a scintillation fluid were added (1 part Triton X-100, 2 parts toluene containing 0"825~o PPO a n d 0-25% Me2POPOP) prior to counting. (iv) Release and analysis of nucleic acids in chromatin To chromatin samples of 0.3 ml were added 50 /~1 of 5~/o (w/v) sodium dodeeyl sulfate a n d 0.2 ml of Pronase (1 mg/ml in 0-1 M-EDTA, 0-05 M-Tris.HC1 (pH 8), and self-digested for 30 rain at 37~ Pronase was obtained from Calbioehem, and contained no measurable nuclease activity under these conditions. The samples were incubated for 0"5 h at 37~ and layered over 15% to 30~/o (w/w) linear sucrose gradients, prepared in 20 mM-sodium acetate, 1 mM-EDTA, 40 mM-Tris.HCl (pH 7.2) for sedimentation analysis as described (MacLeod, 1975b). (v) R N A isolation for poly(A) analysi s Cells were labeled with [2,8-3H]adenosine (40/zCi/ml, 32 Ci/mol; New E n g l a n d Nuclear) for 30 min. Chromatin fractions were digested with 50/~g/ml of eleetrophoretieally purified DNase I (Worthington Biochemical Co.) in the presence of 1 m~-MgC12, for 5 rain at 37~ The sample was diluted 3-fold with water, brought to p H 9 in 0.1 M-Tris.HCl, 1% (w/v) sodium dodecyl sulfate, and extracted with phenol at 0 to 4~ as described b y Brawerman et al. (1972). The final aqueous phase was adjusted to 1% {w/v) sodium dodeeyl sulfate a n d 0.1 M-NaC1, and the R N A was precipitated overnight with 2.5 vol. ethanol at -- 20~ Poly(A)-eontaining R N A was isolated b y chromatography on poly(U)Sepharose, as described b y MaeLeod (1975a). We have shown previously (MacLeod, 1975b) t h a t poly(A)-containing R N A in cytoplasmic extracts from myeloma cells binds specifically to poly(U)-Sepharose trader these conditions; in t h a t cytoplasmic marker R N A molecules lacking poly(A) (e.g. rRNA) do not bind, a n d molecules which do bind contain an RNase-resistant, adenylate-rich sequence.
(e) Density gradient analyses of chromatin (i) Sedimentation of chromatin through sucrose gradients Non-linear steep gradients (0-28 M to 2.8 M-sucrose in 1.0 mM-Tris.HCl, p H 7"0) were prepared b y the layering method described by Chance et al. (1974), a n d left for 1 day at 4~ These were overlaid with 0"3 to 0"5 ml of a sample of nuclear material, a n d centrifuged at 20,000 revs/min for 30 min at 4~ (SW50L rotor, Spineo model L-2, gay ~ 3"25 X 104). Sedimentation of sheared "soluble" chromatin was arlalyzed in gradients of 5% to 20% (w/w) linear sucrose in 1-0 m~-Tris.HCl (pH 7.0), centrifuged at 35,000 revs/min for 3 h at 4~ (SW40 rotor, gay = 1"52 • 10s). (ii) Equilibrium centrifugation of chromatin in metrizamide Samples of chromatin were mixed with 2-10 ml of 60% (w/v) metrizamide (Nyegaard, Oslo) i n 1.0 mM-Tris-HC1 (pH 7.0), to give a final volume of 3.50 ml (refractive index ---- 1.391; see Birnie et al., 1973). These preparations were overlaid with mineral oil a n d
180
C.B.
K I M M E L E T AI~.
centrifuged at 33,000 revs/min for 65 h at 4~ (SW50L rotor, gay = 8'8 • 10a). Fractions (10 drops) were collected through a 20-gauge needle which pierced the bottom of the tube, and 100-/A portions were taken and diluted with 100 IA of water for determination of radioactivity. The refractive index of portions of the sample was determined with a Bausch and Lomb refractometer at 25~ (iii) Equilibrium centrifugation of chromatin in CsCl Samples of sheared, soluble chromatin in 1 nn~-EDTA (pH 7.0) were treated with 0-1 vol. 12~/o formaldehyde in 10 raM-sodium phosphate (pH 7-0), and incubated for 3 to 5 h at 0~ A known volume of this mixture was added to 1-60 ml of saturated CsCI in 0.2 m_~-EDTA (pH 7.0), and 0-2 m_~-EDTA was added to bring the final volume to 3.50 ml, in a polyallomer tube. The mixture was overlaid with mineral oil, and centrifuged at 33,000 revs/min for 65 h at 12~ (SWSOL rotor, Spinco model L-2, gay = 8"8 • 10~). The gradients were fractionated and analyzed as described above for metrizamide gradients. (f) Extraction of nuclear ribonucleoprotein Nuclei, prepared as described above, were washed once by centrifugation in LS buffer, and extracted for 20-min periods in STM buffer (0.1 ~-NaC1, 1 m~-MgC12, 10 m~t-Tris.HC1) at pH 7.0 and p H 8.0 at 0~ followed by extractions at pH 8"0 at 20~ and 37~ according to the method of Martin (Martin & McCarthy, 1972; Martin et a/., 1973). Nuclei remained intact throughout, as determined by phase microscopy, and were sedimented to a pellet after each step by centrifugation (1000 g for 5 rain). Unsheared chromatin was extracted identically, except that it was initially washed in 1 m~-Tris.HCl, and was sedlmented to a pellet after each step by centrifugation at 27,000 g for 5 rain. 3. R e s u l t s
(a) Association of nuclear R N A with chromatin Rapid lysis of nuclei in low-sMt buffers permits the isolation of a chromatin preparation which is minimally degraded through the action of endogenous nuclear enzymes, or through shear. Extraction of macromolecules from chromatLa should also be minimal at low ionic strength. The preparations so obtained are characterized b y an A26oam/A28oam ratio of 1-4 to 1.5. Analyses of three preparations yielded an average DNA/protein ratio of 0-60 and an average RNA/protein ratio of 0.25. As shown b y the data in Table 1, greater t h a n 60% of 5-minute pulse-labeled total cellular R N A is recovered in the chromatin fraction, whether it is prepared in Tris.HCl (preparation I), or E D T A (preparation II). This amount, over 8 0 ~ of the radioactive R N A in the nucleus, is also observed to be recovered in the chromatin fraction ff the 5-minute pulse is followed b y a 25-minute chase (preparation I I I ) in unlabeled uridine. Under the conditions of the chase, net incorporation of radioactivity into total cellular R N A is not detected after 13 minutes (8 rain of chase, data not shown). These findings suggest t h a t the chromatin fraction contains, in addition to nascent R N A chains, R N A molecules whose transcription has been completed. This is because transcription time is expected to be short, relative to the length of the chase (at least with respect to r R N A ; Greenberg & Penman, 1966). This was confirmed for r R N A precursors b y sedimentation analysis. Figure 1 compares the sedimentation profiles of chromatinassociated, 5-minute pulse-labeled R N A (a), and 5-minute pulse--25-minute chased R N A (b). Radioactivity is observed in the 45 S r R N A p r i m a r y transcript in b o t h preparations, b u t the 32 S post-transcriptional molecule is a prominent peak in only the pulse-chased preparation (Fig. 1 (b)). About one-half of nuclear R N A consists of h n R N A molecules (Brandhorst & lVlcConkey, 1974), and heterogeneously sedimenting R N A molecules are also present in these gradients, as expected from the
ASSOCIATION
OF NUCLEAR
RNA
AND CHROMATIN
181
TABLE 1
Distrib~tio~ of radioactively labeled R N A in 8~bcell~lar fraction~ Percentage of recovered radioactivity~ Preparation I II III
Fraction
Cytoplasm Chromatin supernate Chromatln Total recovery (% of homogenate) Tots] radioactivity in homogenate (cts/min)
26.6 12.8 60-1
7.3 10.3 82.4
24.1 2-6 73.3
103.2 7.2 x 104
75.8 2.1 x 105
75.4 1.4 x 105
Subce]]ular fractions were prepared as described in Materials a n d Methods from 1 x 1 0 ~ to 4 x 107 cells labeled for one day with [2-z4C]thymldine, a n d for short intervals with [5,6-3H] uridine (see below). A t least 80% of 14C was in D N A as indicated b y dlge~tion experiments with electrophoretically purified DNase I (Worthington Biochemical Co.). A t least 95% of ~H was in RNA, ss indicated b y digestion experiments with h e a t - t r e a t e d pancreatic RNase (Sigma Chemical Co.). The c h r o m a t i n fraction contained a b o u t 95% of recovered DNA, w i t h 1 to 3 % in the cytoplasmic fraction a n d in the c h r o m a t i n s u p e r n a t a n t fraction. These d a t a are calculated from t h e recovered trichloroacetic acid-insoluble radioactivity in 3H (labeling RNA) in three separate preparations : I, 5-min pulse in uridine, isolation in 1.0 mMTris.HC1; I I , 5-rain pulse in uridine, isolation in 1.0 mM-EDTA; III, 5-min pulse in urldlne, 25-min chase, isolation in 1.0 mz~-TrJs.HC1.
'o_ 6~I-
45s
z2s
-t--
20
0
~ssl ~22s
-
32S
c
0
I0
I0
20
Bottom
Top Fraction no. (o)
(b)
Fzo. 1. Sedimentation distribution of radioactive R N A associated with c h r o m s t i n after a 5-min pulse of [5,6-SH]uridine (a) or 5-min pulse--25-min chase in 10 -4 •-urldine (b). Labeling a n d fractionation procedures were as described in Materials a n d Methods. R N A was released from protein in portions containing c h r o m s t i n from 3 • 10 s cells, w i t h sodium dodecyl sulfate a n d Pronase as described in Materials a n d Methods, a n d layered over 15% ~o 30O/o sucrose/sodium dodecy] sulfate gradients. The gradients were centrifuged for 3 h a t 35,000 revs/min (20~ SW20 rotor), fraotionated into 0.5-ml portions, a n d total radioactivity in 3H in each fraction is shown in the Figure. Sedimentation values indicated in t h e Figure were assigned according to the migration of identifiable cytoplasmic R N A species in s gradient r u n in parallel with these.
C. B . K I M i ~ I E L E T
182
AL.
r e c o v e r y d a t a . T h e r e is no m a j o r difference in its s e d i m e n t a t i o n p a t t e r n i n t h e t w o p r e p a r a t i o n s , h n R N A molecules m a y be s y n t h e s i z e d a t r a t e s s i m i l a r t o r R N A ( P e r r y et al., 1974). P r e v i o u s w o r k h a s s h o w n t h a t s o m e h n R N A molecules are p o l y a d e n y l a t e d , a n d t h a t this occurs a f t e r t r a n s c r i p t i o n is c o m p l e t e d . W h e n cells were l a b e l e d for 30 m i n u t e s w i t h r a d i o a c t i v e adenosine, 6~ o f t h e l a b e l e d c h r o m a t i n - a s s o c i a t e d R N A was b o u n d b y p o l y ( U ) - S e p h a r o s e u n d e r c o n d i t i o n s w h i c h were p r e v i o u s l y d e m o n s t r a t e d t o be specific for p o l y ( A ) - c o n t a i n i n g R N A (MacLeod, 1975b). T h u s t w o t y p e s o f p o s t - t r a n s c r i p t i o n a l R N A molecules, 32 S p r e c u r s o r r R N A a n d p o l y ( A ) - c o n r a i n i n g h n R N A , are o b t a i n e d in t h e c h r o m a t i n f r a c t i o n p r e p a r e d b y our procedures. I t was possible t h a t R N A was n o t a s s o c i a t e d w i t h c h r o m a t i n , b u t c o n t a m i n a t e d t h e c h r o m a t i n f r a c t i o n i s o l a t e d b y differential c e n t r i f u g a t i o n . F o r e x a m p l e , t h i s f r a c t i o n c o n t a i n s n u c l e a r m e m b r a n e s , as d e t e r m i n e d b y p h a s e m i c r o s c o p y . To e x a m i n e this we s t u d i e d t h e s e d i m e n t a t i o n of n u c l e a r m a t e r i a l t h r o u g h v e r y s t e e p sucrose gradients. I n t h e e x p e r i m e n t s h o w n in F i g u r e 2(a), a n u n f r a c t i o n a t e d n u c l e a r l y s a t e was centrifuged t h r o u g h such a g r a d i e n t . T h e m a j o r p a r t s of c h r o m a t i n D N A a n d o f n u c l e a r R N A (pulse-labeled for 5 rain) are in a b a n d n e a r t h e b o t t o m o f t h e g r a d i e n t , a n d a small f r a c t i o n of e a c h n e a r t h e top. T h e t o p f r a c t i o n c o r r e s p o n d s t o t h e " c h r o m a t i n s u p e r n a t e " f r a c t i o n in T a b l e 1. T h e r e is also a l i g h t s c a t t e r i n g b a n d n e a r t h e c e n t e r of t h e g r a d i e n t , w h i c h c o n t a i n s m e m b r a n e s (phase m i c r o s c o p y ) , b u t o n l y s m a l l a m o u n t s o f l a b e l e d mmlei acids. T h e r a p i d c o s e d i m e n t a t i o n o f c h r o m a t i n
6-4-
3H / '4C
0
S-~ '0 x
l
II
I
2-
-
4
6-ii I ,
i i
x
,o o, I ,,
4_
i
:
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i
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,
~
t i i
2
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i
o o
._o
0~
~
Bottom
(a)
I0
-
20
0 =,No
Fraction no.
1
"
0
"Fop
(b)
FIo. 2. Sedimentation distribution on steep (0.28 ~ to 2.8 M) sucrose gradients of unsheared (a) and sheared soluble (b) ehromatin. (a} The cells were labeled for 2 days with [2-14C]thymidine (0.05 gCi/ml) and for 5 min with [5,6-3H]m'idine (40/~Ci/ml). Nuclei were prepared from 5.2 x 107 cells and one-haft of the nuclear lysate, in 1.0 m~-Tris.HC1, was layered over the gradient. The Figure shows radioactivity in triehloroaeetie acid-insoluble material taken from 0.1-ml portions of the gradient fractions. (b) The cells were labeled for 1 day with [2-14C]thymidine (0-02 ttCi/ml) and for 20 min with [5,6-3H]uridine (20 /xCi/ml). The nuclear lysate, in 1.0 m~Tris. HC1, was sonicated as described in Materials and Methods and a portion of the sonieate (not clarified in this experiment) containing nuclear material from 2.4 x 10e cells was layered over the gradient. The Figure shows radioactivity in triehloroacetic acid-insoluble material in the fractions. Background levels of radioactivity were obtained in the lower 3/4 of the gradient. - - O - - Q - - , aH radioactivity; - - O - - 9 14C radioactivity.
ASSOCIATION
OF NUCLEAR
RNA AND CHROI~IATIN
183
and most nuclear RNA, obtained here, shows that the RNA is associated with chromatin, and not with some unidentified very large particle. Shearing of chromatin into slowly sedimenting "soluble" particles by sonieation also quantitatively slows the sedimentation of radiolabeled associated RNA (:Fig. 2(b)). Analyses (not shown) of the sedimentation of sheared chromalia showed t h a t both RNA and DNA sediment together in a complex of particles ranging from about 20 S to 100 S, indicating t h a t the two nucleic acids m a y remain associated throughout the procedure. Association of RNA with chromatin was also tested by equilibrium densitycentrifugation analyses. Centrifugation of chromatin in metrizamide densitygradients is shown in Figure 3. In the case of sheared (Fig. 3(a)) and unsheared I ~
1"4
F--T--
1"2 ~
20
--
U;
I'0
_
",.
li6 x
jl
x
0
-
E
'1
-
0
I0
I'
20 0
u v
20
I0
Fraction no.
(a)
(b)
FIG. 3. Buoyant density of chromatin in metrizamide. Cells were labeled for l day with [2-z4C]thymidino (0.016 ~Ci/rnl) and for 20 rain with [5,6-3H]uridino (10 ~Ci/ml). Clu-omatin was prepared in 1.0 mM-Tris.HC1, and centrifuged in metrizamido as described in Materials and Methods. Each gradient hero contains chromatin prepared from 1-6 • 106 cells. (a) The chromatin was sheared by sonication before mixing with metrizamide; (b) non-sheared (insoluble) ehromatin was analyzed. - - 0 - - 0 - - , 3H radioactivity and buoyant density; - - O - - O - - , z4C radioactivity and 3H/z4C ratio as appropriate.
chromatin (Fig. 3(b)) label in RNA is found in the chromatin band (density approx. 1.26 g/cm3), and significantly shifted to its heavy side as reported by Birnie et al. (1973). The shift is more pronounced in the sheared preparation (Fig. 3(a)). Such Sheared particles were also analyzed in CsC1 gradients, after treatment with formaldehyde (Fig. 4). Here the chromatin bands at about 1.41 g/cm 3, and the pulse-labeled RNA is in the same band. (b) Exchange of added nucleic acids with chromatin It is possible t h a t some fraction of nuclear RNA is not associated with ehromatin in rive, but binds with it during isolation. To examine this possibility we attempted to create artificial complexes between RNA and chromatin. Various concentrations of labeled preparations of purified DNA, RNA, or R N P in Tris. HC1 were added to
C.B. KIMMEL ET AL.
184
1.412
1"5 F
1"4
-4
-2
3H/J'*C
Q..
i.ii
I
~I I f
~
~7 0
'
x c
-
-6
~
, i
:
i
!
i
i
~ '~
0
-
g" i
-~
p2 o
E m
_~,
1I -
o
0
~./i J ~176 1 = 2
I0
20
Jo
Fraction no.
FIG. 4. Buoyant density profile of formaldehyde-treated chromatin in CsC1. A total of 1.6 • 107 cells were labeled for 1 day with [2-14C]thymidine (0.016 ~Cilml) followed by 10 rain with [5,6-3H]uridine (10 ~Cilml). ChromaLin was prepared and sheared in 1.0 m~-EDTA, and a portion treated with formaldehyde for 3 h as described in Materials and Method~. A portion representing 1]4 of the total preparation was mixed with CsC1 for the gradient analysis. In control experiments, no change in sedimentation of chromatin could be detected after formaldehyde treatment, nor were artificial complexes created between ehromatin particles and ribosomal subunits, indicating that neither aggregation nor non-specific cross-linking was occurring (data not shown). - - Q - - Q - - , 3H radioactivity and buoyant density; - - C ) - - O - - , 14C radioactivity and 3H/14C ratio as appropriate. nuclear pellets, a n d the nuclei lysed in t h e presence of these molecules; after differential centrffugation, radioactivity associated with the c h r o m a t i n was determined. The d a t a from several experiments are summarized in Table 2. W e found, first of all, significant binding of D N A to c h r o m a t i n (Table 2, experiments 1 a n d 2). Binding was as high as 22% of a d d e d material, a n d the m a x i m a l level was a p p r o x i m a t e l y 12~/o of t h e D N A calculated to be present in the ehromatin preparation. However, binding of R N A to chromatin was at b a c k g r o u n d levels (<3~/0 of a d d e d material over a range of i n p u t concentrations). This was true for purified nuclear R N A (experiments 2 a n d 3), R N P extracted from nuclei b y the m e t h o d of Martin et al. (1973) (experiments 3 a n d 4), and ribosomes (experiment 5). These d a t a t h u s provide no evidence for a rapid exchange occurring between R N P a n d ehromatin during isolation. (e) Extraction of ribonucleoprotein from chromatin I f h n l ~ N P particles were associated with c h r o m a t i n in vivo, we reasoned t h a t t h e same washing procedure in which t h e y are released f r o m intact nuclei (the usual m e t h o d of obtaining these particles) would also effect their release f r o m c h r o m a t i n preparations. W e h a v e c o m p a r e d R N A extraction f r o m these two sources, following t h e m e t h o d of Martin et al. (1973) developed for aseites t u m o r cells.
ASSOCIATION
OF NUCLEAR
RNA
AND
185
CHROMATIN
TABLE 2 Bicdi~
o f added n u c ~ i c acids a n d n c d e o p r o t e i ~ to chromalin Amount
Experiment
Addition a
No. of nuclei b
added
(/~g) 1
2 3
4 5
DNA o DNA DNA DNA DNA ~ RNA d RNA s RNA RNP e RNP RNP t Rib 9 Rib Rib
5 • l0 s 5 • l0 s 5 • l0 s 5 • l0 s 1 • 10 v 1 • 107 1.2 • l0 s 1.2 • l 0 s 1-2 • l0 s 1-2 • l0 s 5-3 • l 0 s 2.9 • l0 s 2.9 • l0 s 2.9 • l 0 s
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Binding (%)
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(/,g)
a Radioactive nucleic acids or nucleoproteins were added in 1-0 mM-Tris.HC1 (pH 7.0) to pc/lets containing nuclei, which were t h e n lysed as usual. Chromatin was sedimented (27,000 g for 5 rain) a n d radioactivity in t h e c h r o m a t i n pellet a n d supernate determined. Recoveries of added radioactivity were greater t h a n 90%. b Usually of the order of 2 Ace0 units of c h r o m a t l n arc obtained from 107 cells, corresponding to approx. 80 ~g of DNA. a Uniformly l~C-labeled D N A was prepared b y e x t r a c t i o n with phenol/chloroform (Penman, 1966) of sodium dodecyl sulfate a n d Pronase-treated chromatin. The D N A (A28o/A2so ~ 1.86) contained 1.3 • 103 ets/min per pg. a 20-min pulse-labeled R N A [5,6-3H]uridine was prepared from c h r o m a t i n b y t r e a t i n g it w i t h DNase I (25/~g/ml for 10 rain a t 37~ in 1.0 mM-MgC12, 1.0 m ~ - T r i s . H C l , p H 7.0) followed b y t r e a t m e n t with sodium dodecyl sulfate a n d Pronase (see Materials a n d Methods) a n d extraction w i t h phenol/chloroform (Penman, 1966). The R N A (A28o/A2so ~ 1.96) contained 4.7X104 cts/min per pg. e R N P particles were extracted from t h e nuclei of cells pulse-labeled for 20 mln w i t h [5,6-aH]uridine as described in Materials a n d Methods, a n d dialyzed vers~ 1-0 mM-Tris.HC1 (pH 7.0). The R N P (A26o/Asso ~ 1.43) contained R N A which sedimented a t approx. 6 S (obtained also b y Martin et al., 1973), which contained 3.45 • 103 cts/min per ~g. f Another p r e p a r a t i o n of 20-min labeled R N P particles, extracted as above, was tested. The R N A conta!ned 1.15 • 104 cts/min per /zg. Ribosomes were sedimented (40,000 revs/min for 90 rain a t 4~ in t h e SW50L rotor) from t h e cytoplasmic fraction of S194 cells which h a d been t r e a t e d with 3 /~Ci [5,6-3H]uridine]ml, 20 h. They were re3uspended in 1-0 mM-Tris.HC1 (pH 7.0). The ribosomes (Asso/A28o ~ 1.73) cont a i n e d R N A labeled a t 9.7 • 10~ cts/mln per pg.
"Figure 5(a) shows the cumulative release of 10-minute pulse-labeled RNA and uniformly labeled DNA from whole nuclei, according to Martin's washing schedule. T h e r e is s p e c i f i c e x t r a c t i o n o f R N A (less t h a n 1~/c o f D N A is s o l u b i l i z e d ) , a n d i n a g r e e m e n t w i t h t h e f i n d i n g s o f M a r t i n et al. (1973) R N A e x t r a c t i o n is m o s t e f f i c i e n t at piE/8, and at e]evated temperature. Chromatin, prepared from a portion of the s a m e cells, w a s e x t r a c t e d a c c o r d i n g t o t h e s a m e s c h e d u l e a n d , a s s h o w n i n l ~ i g u r e 5(b), the specific extraction of RNA occurs nearly identically to that from whole n u c l e i . A s i m i l a r r e l e a s e o f R N A o c c u r s a t 20~ a n d 37~ i n 1.0 m M - T r i s . H C 1 ( p H 7.0) b u t , a s s h o w n i n F i g u r e 5(c), s u b s t a n t i a l l y m o r e D N A is a l s o s o l u b i l i z e d , a m o u n t i n g t o m o r e t h a n 10~/o o f t h e t o t a l .
186
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(a) (b) (c) FIG. 5. Extraction of RNP particles from nuclei (a) and chromatin (b) and (c). A total of 3.4 • 107 cells were treated with [2-14C]thymidine (20 h, 0.16/zCi/ml) followed by [5,6-3H]uridine (10 min, 20/~Ci/rnl). The cells were homogenized, and 1/3 of the nuclei washed once in LS buffer (see Materials and Methods), and used for the extractions shown in (a). Chromatin was prepared from the remaining 2/3, washed once in 1.0 mM-Tris (pH 7.0) and extracted with the buffers as described by Martin etal. (1973) (b), or with 1.0 m~-Tris-HC1 (c). The extraction procedure is described in Materials and Methods, and the Figure shows the cumulative release of radioactivity in 3H ( - - O - - O - - ) and xdC ( - - O - - 9 as a percentage of the total in portions from each supernate. These experiments demonstrate t h a t R N A , p r e s u m a b l y in association with proteins (A26o/A28o = 1.4), can readily be released from a complex with chromatin, in the same procedure where h n R N P particles are obtained from nuclei. (d) Distribution of R N A in fl'actionated chromatin M a n y workers h a v e shown t h a t chromatin preparations m a y be fractionated to obtain classes of particles enriched for associated pulse-labeled R N A molecules. I n this section we show t h a t in two m e t h o d s t h e associated R N A is n o t fractionated according t o its age. A slowly sedimenting minor fraction of ctu'omatin enriched for associated R N A can be obtained from mildly sheared preparations (Chalkley & Jonson, 1968 ; M u r p h y etal., 1973; M c C a r t h y etal., 1973; Chance etal., 1974). Figure 6 shows the sedimentation patterns obtained from chromatin in a nuclear lysate (Fig. 6(a)), compare with Fig. 2(a)), a n d f r o m chromatin sheared b y sedimentation to a pellet with v o r t e x resuspension (Fig. 6(b)). I n the latter a b o u t 3 % of the D N A a n d 3 0 % of the R N A is slowly sedimenting. Continued association of the D N A a n d R N A in this minor fraction was confirmed b y sedimentation analysis in sucrose gradients a n d equilibrium density analysis in metrizamide gradients (data n o t shown). W e examined the distribution of R N A of different ages in chromatin fractionated b y this m e t h o d in double-label experiments. I n the experiment shown in Figure 7, R N A was labeled for 20 minutes with [2-zdC]uridine, a n d [5,6-3H]uridine was a d d e d for an additional 4 minutes. A similar proportion of molecules of each age is slowly sedimenting. W h e n deproteinized R N A from this fraction was examined b y sucrose gradient centrifugation b o t h isotopes were f o u n d to label 45 S precursor r R N A a n d h n R N A .
ASSOCIATIOI~
OF NUCLEAR
RNA AND CHROMATIN
187
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Fro. 6. Fl'aetionation of ehromatin in steep (0-28 M to 2.8 M) sucrose gradients. Cells were labeled f r 1 day with [2-x4C]thymidine (0-01 /~Ci/ml) and for 10 rain with [5,6-aH]uridino (20 /~Ci]ml). Nuclei were prepared from 3 x l0 T cells and lysed in 0.9 ml 0.2 mM-EDTA, and 0.2 ml of the lysate used for the gradient shown in (a). Chromatin in the rest of the lysate was centrifuged to a pellet and resusponded with vortex mixing. Half of this preparation was used for the gradient shown in (b). The Figure shows triehloroaeetic acid-insoluble radioactivity in 100-/~1 portions from the gradient fractions. - - 0 - - 0 - - , 3H radioactivity; - - O - - O - - , 140 radioactivity.
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:FIG. 7. Gradient fraetionation of R N A in slightly sheared chromatin. Cells were labeled with [2-x4C]uridino (0"5 ~Ci/ml) for 20 rain, and [5,6-3H]uridine (40 ~Ci/ml) for an additional 4 rain. A chromatin suspension was prepared in 0-2 mM-EDTA, centrifuged through a 0.28 • to 2.8 sucrose gradient, and fractionated. The Figure shows the distribution of trichloroaeeti0 acidinsoluble radioactivity of each isotope ( 0 , 3H; O, 14C), and the ~H]:4C ratio.
188
C. B . K I M M E L
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T h e 32 S p r e c u r s o r r R N A p o s t - t r a n s c r i p t i o n a l molecule was o b s e r v e d o n l y i n t h e p o p u l a t i o n l a b e l e d for 24 m i n u t e s ( d a t a n o t shown). W e p r e p a r e d t h e slowly sedim e n t i n g e h i ' o m a t i n f r a c t i o n f r o m cells l a b e l e d for 30 m i n u t e s w i t h [2,8-3H]adenosine, a n d f o u n d t h a t 6~ o f t h e r a d i o a c t i v i t y was o b t a i n e d in p o l y ( A ) - e o n t a i n i n g R N A molecules. This was t h e s a m e p r o p o r t i o n as f o u n d p r e v i o u s l y (see a b o v e ) for t o t a l e h r o m a t i n . T h e size d i s t r i b u t i o n s o f p o l y ( A ) - c o n t a i n i n g R N A f r o m f r a c t i o n a t e d a n d u n f r a c t i o n a t e d c h r o m a t i n were i d e n t i c a l ( d a t a n o t shown). T h u s f r a c t i o n a t i o n o f s l i g h t l y s h e a r e d c h r o m a t i n does n o t f r a e t i o n a t e t h e a s s o c i a t e d R N A a c c o r d i n g t o its age, n o r does i t select for or a g a i n s t p o s t - t r a n s c r i p t i o n a l R N A molecules. E x t e n s i v e l y s h e a r e d c h r o m a t i n m a y b e f r a c t i o n a t e d b y differential p r e c i p i t a t i o n w i t h MgC12 (Bonner et al., 1973; G o t t e s f e l d et al., 1974). I n a g r e e m e n t w i t h t h e findings of B o n n e r ' s group, we o b s e r v e d t h a t c h r o m a t i n p a r t i c l e s e n r i c h e d in a s s o c i a t e d p u l s e - l a b e l e d R N A a r e r e l a t i v e l y r e s i s t a n t t o Mg 2+ p r e c i p i t a t i o n . T h i s is s h o w n in F i g u r e 8(a), w h e r e t h e s o l u b i l i t y o f c h r o m a t i n associated, 5 - m i n u t e l a b e l e d R N A (ordinate) is p l o t t e d as a f u n c t i o n o f c h r o m a t i n I ) N A s o l u b i l i t y (abscissa), in a p r e p a r a t i o n t r e a t e d w i t h 0 t o 2 mM-MgCI2. W e find, a t a c o n c e n t r a t i o n of 1.0 mM-MgC12, 5 t o 10~/o o f e h r o m a t i n D N A , a n d 35 t o 4 5 % o f a s s o c i a t e d R N A , r e m a i n i n t h e s u p e r n a t a n t f r a c t i o n a f t e r c e n t r i f u g a t i o n a t 27,000 g for 5 m i n u t e s . S e d i m e n t a t i o n a n a l y s i s of t h i s m a t e r i a l in sucrose g r a d i e n t s d e m o n s t r a t e d t h a t t h i s
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Fzo. 8. Fraetionation of ehromatin by MgOl2 precipitation. (a) Sheared "soluble" chromatin particles were prepared from cells labeled for 1 day with [2-14C]thymidine (0.005 FCi/m]) and for 5 rain with [5,6-3H]uridine (40 FCi/ml). Samples of 50 ~1, at an absorbanee (260 nm) of 2.0 were mi~ed with 50 pl of 0 to 4 m•-MgC12 and centrifuged at 27,000 g for 5 rain as described in Materials and Methods. The supernate was assayed for radioactivity in 3H and 14C. In the Figure, 100% corresponds to 216 cts/mln in 14C and 2529 cts/min in 3H. The Figure shows RNA solubility as a function of chromatin DNA solubility. (b) Both isotopes are in RNA. A total of 5 • 107 cells in 5 ml of culture medium were labeled with [2-14C]uridine (2 ~Ci/ml, 57 Ci/mol; New England Nuclear) for 22 rain, and [5,6-3H]uridine (40/LCi/ml) for an additional 3 rain. Sheared chromatin was prepared and 100-pl portions at an initial absorbance (260 nm) of 2-0 treated with 100 pl of 0 to 4 m~-MgC12 as described in Materials and Methods. After centrifugation, radioactivity was measured in 100-pl portions of the supernate. The open and closed circles show the data from 2 separate preparations, which were treated identically throughout. In one of these (open circles) 100% in the Figure corresponds to 1882 ots/min in 8H and 1125 ot~/min in 14C. In the other (closed circles) 100% corresponds to 2142 cts/min in 3H and 1525 e~s/min in 14C.
ASSOCIATION
OF NUCLEAR
RNA AND CHROMATIN
]89
technique does not select for a particular size class of RNA-rich particles, and equilibrium density analysis in metrizamide gradients revealed no apparent density fractionation of chromatin by this method (data not shown). RNP particles, extracted from nuclei by the method of Martin et al. (1973), and dialyzed versus 1.0 mM-Tris-HC1 (pH 7.0) were found to be quantitatively soluble when treated with 2.0 m~-MgCl2. The age-distribution of RNA molecules in Mg2+-fractionated sheared chromatin was followed in double-label experiments. Two are shown in Figure 8(b), where in each case RNA was labeled for 3 minutes and 25 minutes. Here it is seen that the proportions of RNA molecules marked by each isotope remaining soluble in various concentrations of MgC12 (0 to 2.0 raM) are the same; the points cluster closely about the diagonal. Thus fractionation of ehromatin by Mg2+ does not fractionate the chromatin-assoeiated RNA according to its age.
4. Discussion We have shown that when chromatin is prepared rapidly, and in low ionic strength buffers, the major part (at least 80~/o) of nuclear RNA is obtained in the chromatin preparation. The cosedimentation of RNA with chromatin in sucrose gradients, and the similar isopycnic positions in metrizamide gradients argue that the RNA is associated with chromatin, and not simply present as a contaminant. Furthermore, shearing of the chromatin by sonication does not appear to release the associated RNA, as determined by the same criteria. It has been reported (de Pomerai et al., 1974) that different procedures for the isolation of chromatin yield preparations which differ considerably with respect to composition and biolo~cal activity. In particular, extensively purified chromatin contains less RNA than we obtained with our relatively simple procedure. We found that both DNA and RNA are solubilized if ct~romatin is repeatedly extracted at low ionic strength (Fig. 5), which is the expected result of degradation. We tested directly for binding of exogenous RNA or RNP to our preparation and could find none (Table 2). These observations suggest that the RNA is functionally associated with some fraction of chromatin in vivo. Evidence was obtained that both known major classes of nuclear RNA, the rRNA precursors and hnRNA, remain associated with chromatin after transcription is completed. The primary rRNA transcript, 45 S RNA, was identified after a very short pulse of label, while the major transcriptional intermediate, 32 S precursor, rRNA could only be observed after a more extended period (Fig. 1). Processing of precursor rRNA is known to occur in the nucleolns (Perry, 1962; Penman et al., 1966), which is associated with the nucleolar organizer region of chromatin (Steele, 1968; Miller & Beatty, 1969). Also present in the chromatin were rapidly labeling heterodisperse molecules which contained, significantly, poly(A) sequences. The addition of poly(A) to some hnRNA molecules is thought to occur as a post-transcriptional step (Perry et al., 1974; Jellinek et al., 1973). These data are thus not in accord with the hypothesis (Samarina et al., 1968) that post-transcriptional processing of hnRNA occurs in relatively simple nucleoplasmic organelles (hnRNP particles), and we consider it likely that such particles are a part of the chromatin matrix in vivo. This view is supported by the observation that RNP possessing several properties of hnl~NP could be detached from chromatin (Ishil~awa e~ a/., 1974), and we showed here (Fig. 5) that RNA could be extracted from chromatin by
190
C. B . K I M M E L
ET AL.
the same procedure used in another system (Martin et al., 1973) to prepare h n R N P from whole nuclei. A detailed analysis of the release of chroma~in R N P is made difficult b y the fact that sheared chromatin sediments heterogeneously in the same rauge as reported for h n R N P particles (Samarina el al., 1968; Pederson, 1974a), and the buoyant density of h n R N P and sheared chromatin in CsC1 are similar (~-~I.4 g/ca3). The buoyant density of h n R N P in metrizamide has not been reported. In this solvent, chromatin and its associated RNA band at 1.2 to 1.3 g/cm 3 (Birnie et al., 1973; and Fig. 3). Recently Scott & Sommerville (1974) showed b y immunofluorescent methods t h a t h n ~ N P proteins are associated specifically with the lateral loops of giant lampbrush chromosomes in the newt ooeyte. In accord with these results, we found t h a t a large part (30 to 40 ~/o) of chromatin RNA could be shown to be contained in a fraction containing 3 to 10% of chromatin DNA. In particular, we established that, in two methods to obtain RNA-enriched chromatin classes, the RNA is not fractionated according to its age. For one of the methods we showed that post-transcriptional molecules, identified as above, were present in the same proportion as in total chromatin. The basis of separation is thought to be different in each method; the slowly sedimenting fraction prepared from large particles having a special " e x t e n d e d " conformation (Chalkley & Jenson, 1968), and the Mg 2§ soluble fraction prepared from small chromatin particles containing reduced amounts of histone proteins (Gottesfeld et al., 1974). We have not tested directly whether the same minor class of chromatin is obtained in each isolation. Both have been shown, in the above-cited studies but not repeated here, to yield chromatin especially active in in vitro transcription assays. We found that neither method separates a class of chromatin of special buoyant density, and Rickwood et al. (1974) obtained no purification of template-active chromatin b y metrizamide density-fraetionation. The minor fractions are thought to contain, as is the case for lampbrush loops, regions active in in vivo RNA synthesis. I t is possible, therefore, that events in the post-transcriptional history of hnRNA, as for precursor rRNA, occur while molecules remain complexed with active genes in chromatin. This research was supported by National Science Foundation grant no. GB-37495. REFERENCES
Birnie, G. D., Rickwood, D. & Hell, A. (1973). Biochim. Biophys. Acta, 331, 283-294. Bonnet, J., Garrard, W. T., Gottesfeld, J., Holmes, D. S., Seval, J. S. & Wilkes, M. (1973). Cold Spring Harbor Syrup. Quan$. Biol. 38, 303-310. Brandhorst, B. P. & McConkey, E. H. (1974). J. Mol. Biol. 85, 451-463. Brawerman, G., Mendecld, J. & Less, S. Y. {1972). Biochemistry, l l , 637-641. Burton, K. (1956). Biochem. J. 62, 315-323. Chalkley, R. & Jensen, R. H. (1968). Biochemistry, 7, 4380-4388. Chance, H., Kadohoma, N. & Anderson, K. M. (1974). Biochcm. Biophys. Res. Commun. 58, 66-73. de Pomerai, D. I., Chesterton, C. J. & Betterworth, P. H. W. (1974). Eur. J. Biochem. 46, 461-471. Gottesfeld, J. M., Garrard, W. T., Bagi, G., Wilson, R. F. & Bonner, J. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 2193-2197. Greenberg, H. & Penman, S. (1966}. J. Mol. Biol. 21, 527-535. Ishikawa, K., Sato, T., Sato, S. & Ogata, K. (1974). Biochim. Biophys. Acta, 353, 420-437. Jellinek, W., Molloy, G., Salditt, M., Wall, 1%., Scheiness, D. & Darnell, J. E., Jr {1973). Cold Spring Harbor Syrap. Quant. Biol. 38, 891-898.
A S S O C I A T I O N OF N U C L E A R RNA AND CHROMATIN
191
Kimmel, C. B. (1971). Expt. Cell R ~ . 65, 202-208. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 198, 265-275. MacLeod, M. C. (1975a). A w l . Biochem. 68, 299-310. MaeLeod, M. C. (19755). Biochem~try, 14, 4011-4018. Martin, T. E. & McCarthy, B. J. (1972). Biochim. Biophys. Acta, 277, 354-367. Martin, T., Billings, P., Levey, A., Ozarslan, S., Quinlin, T., Swift, H. & Urbas, L. (1973). Cold Spring Harbor Syrup. Quant. Biol. 38, 921-932. McCarthy, B. J., Nisiura, J. T., Doenecke, D., Nasser, D. S. & Johnson, C. B. (1973). Cold Spring Harbor Syrup. Quant. Biol. 38, 763-771. Miller, O. L., Jr & Beatty, B. R. (1969). Science, 164, 955-957. Murphy, E. C., Jr, Hall, S. H., Shepherd, J. H. & Weiser, R. S. (1973). Biochemistry, 12, 3843-3853. Pederson, T. (1974a). J. Mol. Biol. 83, 163-183. Pedorson, T. (1974b). Prec. Nat. Acad. Sci., U.S.A. 71, 617-621. Penman, S. (1966). J. Mol. Biol. 17, 117-130. Penman, S., Smith, I. & Holtzman, E. (1966). Science, 154, 786-789. Perry, R. P. (1962). Prec. Nat. Acad. Sci., U.S.A. 48, 2179-2186. Perry, R. P., Kelley, I). E. & LaTorre, J. (1974). J. Mol. Biol. 82, 315-351. Rickwood, D., Hell, A., Malcolm, S., Birnie, G. D., Macgillivray, A. J. & Paul, J. (1974). Biochim. Biophys. Acta, 353, 353-361. Samarina, O. P., Lukanidin, E. M., Molnar, J. & Georgiev, G. P. (1968). J. Mol. Biol. S3, 251-263. Schneider, W. C. (1957). In Methods in Enzymology (Colowick, S. P. & Kaplan, N. O., eds), vol. 3, p. 680, Academic Press, New York. Scott, S. E. M. & Sommerville, J. (1974). Nature (London), 250, 680-682. Sherrer, K. & Maraud, L. (1965). Bull. Soc. Ghim. Biol. 47, 1697-1713. Steele, W. J. (1968). J. Biol. Chem. 243, 3333-3341. Vogt, M. & Dulbecco, R. (1963). Prec. Nat. Acad. Sci., U.S.A. 49, 171-179.
Evidence for an Association o f m o s t Nuclear R N A with Chromatin C~ARLES B. Kra~'MEL,STANLEY K. SESSIONS AND MIC~AV.L C. MAcLEOD t
Department of Biology University of Oregon Eugene, Ore. 97403, U.S.A. (Received 17 .February 1975, and in revisedform 30 October1975) Chromatin prepared from mouse myeloma cell nuclei under conditions of low ionic strength and with minimal shearing contains at least 80% of the nuclear RNA which is labeled during a 5 or 30-minute pulse with uridine, or during a 5-m~nute pulse followed by a 25-minute chase. The RNA remains associated with chromatin after shearing by sonication, as determined by sedimentation and equilibrium density-gradient analyses. I t is released from chromatin by procedures developed elsewhere to extract ribonucleoprotein particles from intact nuclei. These results, and the results of reconstruction experiments, suggest that this association reflects the /n rive location of nuclear RNA and is not the result of non-specific binding during preparation. The chromatin-associated RNA includes RNA species which are known to be post-transcriptional; 32 S precursor ribosomal RNA and poly(A)-cont,.inlng heterogeneous nuclear RNA. Fractions of chromatin enriched in associated RNA were obtained by two methods derceloped elsewhere, and in both ~ f these the enrichment obtained (3 to 10-fold) appears to be independent of the age of the RNA. The implications of these results for models of RNA !processing are discussed. 1. I n t r o d u c t i o n I n m a m m a l i a n cells transcription of R N A molecules occurs along restricted regions of chromatin. One class of transcriptional product, the 45 S ribosomal R N A precursor, remains in close association with the ribosomal genes in the nucleolus, where processing of the transcrip~ takes place. Transpor~ of the fine,lied 28 S a n d 18 S r R N A molecules m u s t be rapid, since t h e y cannot be detected in the nucleus (Penman, 1966). The other main class of transcriptional products, heterogeneous nuclear RNA, is thought to be processed differently. I t has been suggested (Samarina et al., 1968) t h a t the newly synthesized hnRNA:~ molecules leave the chromatin fibril in associa. tion with proteins, and enter the nucleoplasm, where post-transcriptional "processing" takes place. This includes a rapid degradation of the m a j o r p a r t of most h n R N A molecules to nucleotides (Sherrer & Marcaud, 1965), and the addition of a sequence t Present address: Biology Division, Oak Ridge National Lab.. Oak Ridge, Tenn. 87830, U.S.A. Abbreviations used: hnRNA, heterogeneous nuclear RNA; RN'P, ribonucloopretein; tmRNP, heterogeneous nuolear RIq-P. 12
177
178
C. B. K I M M E L E T A L .
of adenylate residues to some of t h e transcripts (Jellinek et al., 1973; P e r r y et al., 1974). I n this scheme, small poly(A)-containing molecules are t h o u g h t t o be transp o r t e d to the cytoplasm a n d eventually associate with ribosomes as functional messenger R N A . The evidence for the existence of nucleoplasmic h n R N P particles has a c c u m u l a t e d from a series of analyses (Samarina eta/., 1968; Pederson, 1974a,b; Martin et al., 1973) of R N P contained in nuclei, which h a v e properties expected for post-transcriptional h n R N P particles, a n d wlfieh p r e s u m a b l y contain no D N A . W e report here t h e results of experiments which suggest t h a t post-transcriptional R N A molecules, including h n R N A , r e m a i n t i g h t l y associated with c h r o m a t i n fibrils during the m a j o r p a r t of their lifetimes in the nucleus. T h e distributions of nascent a n d post-transcriptional R N A molecules a m o n g different classes of c h r o m a t i n particles are non-random, and appear to be identical.
2. M a t e r i a l s a n d M e t h o d s
(a) Cells and labeling procedures Cultures of a cloned strain of myeloma S 194 were obtained from Dr G. Waring, and grown as cell suspensions in fortified Eagle's medium (Volt & Dulbecco, 1963) containing 10~o home serum (North American Biological Supply Co.). Cells in the late exponential phase of growth (Kimmel, 1971) were treated with [2-14C]thymidine (0.01 to 0-03 ~Ci/ml, 0.23 Ci/g; New England Nuclear) for about 1 day to label cellular DNA, and continued growth during this period was routinely checked by cell counts (generation time ~ 14 h). To pulse-label RNA, the cells were centrifuged to a pellet, and concentrated up to 10-fold in fresh, warm culture meditun. After a period of 5 min at 37~ [5, 6-3H]uridine was added (10 to 40 ~Ci/ml, 185 Ci/g; New England Nuclear), and the cells incubated for various periods (0.5 to 20 rain). I n pulse-chase experiments, the cells in medium containing [5,6-3H]uridine were diluted 4-fold in warm medium containing 10-~ m-unlabeled uridine (zero time of chase). The cells were centrifuged to a pellet (500 g for 3 rain) and resuspended in warm medium containing 10 -4 ~-uridine. (b) Cell fractionation Radioactive labeling was terminated by diluting the cell suspension in a slurry of iced medium, and subsequent operations were carried out rapidly at 0 to 4~ The cells were centrifuged to a pellet (500 g for 3 min), and washed once by centrifugation in LS buffer (5 mM-NaC1, 5 mM-KC1, 1-5 mM-MgC12, 10 m~-Tris-HC1, p H 7"4). The cells were resuspended in this buffer, allowed to swell for 3 min, and gently homogenized with a Dounce homogenizer (Kontes Glass Co., B pestle). Care was taken at this stage not to lyse nuclei, and the homogenate usually contained 5 to 10% of unbroken cells, as determined by phase microscopy. The nuclei were sedimented (1000 g for 5 rain), and the pellet was resuspended with vortex mixing and gradual addition of 1 mM-Tris-HC1 (pH 7.0). This procedure lyses most nuclei, and complete lysis was achieved with gentle Dounce homogenization. I n some experiments the nuclei were lysed, and subsequent operations carried out in 1 mM or 0.2 mM-EDTA (pH 7.0). Chromatin was centrifuged to a pellet (27,000 g for 5 rain; Sorvall centrifuge, SS34 rotor; 15,000 revs/min) and resuspended, again with vigorous mixing and gradual addition of buffer (usually 1 mM-Tris.HC1, p H 7.0). (c) Shearing of chromatin "Soluble" chromatin particles (i.e. those remaining in the supernate after centrifugation at 27,000 g for 5 min) were prepared by shearing the chromatin suspension described above. Samples of 1 ml, in a 5-ml beaker in ice, were sonicated with the standard 0"5-in probe of the Branson model 185 sonifier, using three 5-s treatments at 20 V. The sonicate w~s Clarified by centrifugation (27,000 g for 5 min).
A S S O C I A T I O N OF N U C L E A R R N A AND C H R O M A T I N
179
(d) Analytical procedures (i) Ghemical assays D N A was determined b y the B u r t o n (1956) procedure, R N A b y the oreinol reaction (Schneider, 1957), and protein b y the method of Lowry et al. (1951). Calf t h y m u s I)NA, yeast soluble RNA, a n d crystalline bovine serum a l b u m i n served as standards (Sigma Chemical Co.). (ii) Precipitation with trichloroacetic acid A total of 250/~g of yeast R N A (Sigma Chemical Co.) was added to ehromatin samples, and a n equal volume of cold 10% trichloroacetic acid was rapidly mixed with the sample. Insoluble material was centrifuged to a pellet (9000 g for 1 min), washed by eentrifugation in 5 ~/o triehloroaeetm acid, a n d dissolved in 0-1 N-NaOH. (iii) Determination of 8H and 14G radioactivity Aqueous samples were diluted to between 0.2 a n d 1 ml with water, a n d 10 vol. of a scintillation fluid were added (1 part Triton X-100, 2 parts toluene containing 0"825~o PPO a n d 0-25% Me2POPOP) prior to counting. (iv) Release and analysis of nucleic acids in chromatin To chromatin samples of 0.3 ml were added 50 /~1 of 5~/o (w/v) sodium dodeeyl sulfate a n d 0.2 ml of Pronase (1 mg/ml in 0-1 M-EDTA, 0-05 M-Tris.HC1 (pH 8), and self-digested for 30 rain at 37~ Pronase was obtained from Calbioehem, and contained no measurable nuclease activity under these conditions. The samples were incubated for 0"5 h at 37~ and layered over 15% to 30~/o (w/w) linear sucrose gradients, prepared in 20 mM-sodium acetate, 1 mM-EDTA, 40 mM-Tris.HCl (pH 7.2) for sedimentation analysis as described (MacLeod, 1975b). (v) R N A isolation for poly(A) analysi s Cells were labeled with [2,8-3H]adenosine (40/zCi/ml, 32 Ci/mol; New E n g l a n d Nuclear) for 30 min. Chromatin fractions were digested with 50/~g/ml of eleetrophoretieally purified DNase I (Worthington Biochemical Co.) in the presence of 1 m~-MgC12, for 5 rain at 37~ The sample was diluted 3-fold with water, brought to p H 9 in 0.1 M-Tris.HCl, 1% (w/v) sodium dodecyl sulfate, and extracted with phenol at 0 to 4~ as described b y Brawerman et al. (1972). The final aqueous phase was adjusted to 1% {w/v) sodium dodeeyl sulfate a n d 0.1 M-NaC1, and the R N A was precipitated overnight with 2.5 vol. ethanol at -- 20~ Poly(A)-eontaining R N A was isolated b y chromatography on poly(U)Sepharose, as described b y MaeLeod (1975a). We have shown previously (MacLeod, 1975b) t h a t poly(A)-containing R N A in cytoplasmic extracts from myeloma cells binds specifically to poly(U)-Sepharose trader these conditions; in t h a t cytoplasmic marker R N A molecules lacking poly(A) (e.g. rRNA) do not bind, a n d molecules which do bind contain an RNase-resistant, adenylate-rich sequence.
(e) Density gradient analyses of chromatin (i) Sedimentation of chromatin through sucrose gradients Non-linear steep gradients (0-28 M to 2.8 M-sucrose in 1.0 mM-Tris.HCl, p H 7"0) were prepared b y the layering method described by Chance et al. (1974), a n d left for 1 day at 4~ These were overlaid with 0"3 to 0"5 ml of a sample of nuclear material, a n d centrifuged at 20,000 revs/min for 30 min at 4~ (SW50L rotor, Spineo model L-2, gay ~ 3"25 X 104). Sedimentation of sheared "soluble" chromatin was arlalyzed in gradients of 5% to 20% (w/w) linear sucrose in 1-0 m~-Tris.HCl (pH 7.0), centrifuged at 35,000 revs/min for 3 h at 4~ (SW40 rotor, gay = 1"52 • 10s). (ii) Equilibrium centrifugation of chromatin in metrizamide Samples of chromatin were mixed with 2-10 ml of 60% (w/v) metrizamide (Nyegaard, Oslo) i n 1.0 mM-Tris-HC1 (pH 7.0), to give a final volume of 3.50 ml (refractive index ---- 1.391; see Birnie et al., 1973). These preparations were overlaid with mineral oil a n d
180
C.B.
K I M M E L E T AI~.
centrifuged at 33,000 revs/min for 65 h at 4~ (SW50L rotor, gay = 8'8 • 10a). Fractions (10 drops) were collected through a 20-gauge needle which pierced the bottom of the tube, and 100-/A portions were taken and diluted with 100 IA of water for determination of radioactivity. The refractive index of portions of the sample was determined with a Bausch and Lomb refractometer at 25~ (iii) Equilibrium centrifugation of chromatin in CsCl Samples of sheared, soluble chromatin in 1 nn~-EDTA (pH 7.0) were treated with 0-1 vol. 12~/o formaldehyde in 10 raM-sodium phosphate (pH 7-0), and incubated for 3 to 5 h at 0~ A known volume of this mixture was added to 1-60 ml of saturated CsCI in 0.2 m_~-EDTA (pH 7.0), and 0-2 m_~-EDTA was added to bring the final volume to 3.50 ml, in a polyallomer tube. The mixture was overlaid with mineral oil, and centrifuged at 33,000 revs/min for 65 h at 12~ (SWSOL rotor, Spinco model L-2, gay = 8"8 • 10~). The gradients were fractionated and analyzed as described above for metrizamide gradients. (f) Extraction of nuclear ribonucleoprotein Nuclei, prepared as described above, were washed once by centrifugation in LS buffer, and extracted for 20-min periods in STM buffer (0.1 ~-NaC1, 1 m~-MgC12, 10 m~t-Tris.HC1) at pH 7.0 and p H 8.0 at 0~ followed by extractions at pH 8"0 at 20~ and 37~ according to the method of Martin (Martin & McCarthy, 1972; Martin et a/., 1973). Nuclei remained intact throughout, as determined by phase microscopy, and were sedimented to a pellet after each step by centrifugation (1000 g for 5 rain). Unsheared chromatin was extracted identically, except that it was initially washed in 1 m~-Tris.HCl, and was sedlmented to a pellet after each step by centrifugation at 27,000 g for 5 rain. 3. R e s u l t s
(a) Association of nuclear R N A with chromatin Rapid lysis of nuclei in low-sMt buffers permits the isolation of a chromatin preparation which is minimally degraded through the action of endogenous nuclear enzymes, or through shear. Extraction of macromolecules from chromatLa should also be minimal at low ionic strength. The preparations so obtained are characterized b y an A26oam/A28oam ratio of 1-4 to 1.5. Analyses of three preparations yielded an average DNA/protein ratio of 0-60 and an average RNA/protein ratio of 0.25. As shown b y the data in Table 1, greater t h a n 60% of 5-minute pulse-labeled total cellular R N A is recovered in the chromatin fraction, whether it is prepared in Tris.HCl (preparation I), or E D T A (preparation II). This amount, over 8 0 ~ of the radioactive R N A in the nucleus, is also observed to be recovered in the chromatin fraction ff the 5-minute pulse is followed b y a 25-minute chase (preparation I I I ) in unlabeled uridine. Under the conditions of the chase, net incorporation of radioactivity into total cellular R N A is not detected after 13 minutes (8 rain of chase, data not shown). These findings suggest t h a t the chromatin fraction contains, in addition to nascent R N A chains, R N A molecules whose transcription has been completed. This is because transcription time is expected to be short, relative to the length of the chase (at least with respect to r R N A ; Greenberg & Penman, 1966). This was confirmed for r R N A precursors b y sedimentation analysis. Figure 1 compares the sedimentation profiles of chromatinassociated, 5-minute pulse-labeled R N A (a), and 5-minute pulse--25-minute chased R N A (b). Radioactivity is observed in the 45 S r R N A p r i m a r y transcript in b o t h preparations, b u t the 32 S post-transcriptional molecule is a prominent peak in only the pulse-chased preparation (Fig. 1 (b)). About one-half of nuclear R N A consists of h n R N A molecules (Brandhorst & lVlcConkey, 1974), and heterogeneously sedimenting R N A molecules are also present in these gradients, as expected from the
ASSOCIATION
OF NUCLEAR
RNA
AND CHROMATIN
181
TABLE 1
Distrib~tio~ of radioactively labeled R N A in 8~bcell~lar fraction~ Percentage of recovered radioactivity~ Preparation I II III
Fraction
Cytoplasm Chromatin supernate Chromatln Total recovery (% of homogenate) Tots] radioactivity in homogenate (cts/min)
26.6 12.8 60-1
7.3 10.3 82.4
24.1 2-6 73.3
103.2 7.2 x 104
75.8 2.1 x 105
75.4 1.4 x 105
Subce]]ular fractions were prepared as described in Materials a n d Methods from 1 x 1 0 ~ to 4 x 107 cells labeled for one day with [2-z4C]thymldine, a n d for short intervals with [5,6-3H] uridine (see below). A t least 80% of 14C was in D N A as indicated b y dlge~tion experiments with electrophoretically purified DNase I (Worthington Biochemical Co.). A t least 95% of ~H was in RNA, ss indicated b y digestion experiments with h e a t - t r e a t e d pancreatic RNase (Sigma Chemical Co.). The c h r o m a t i n fraction contained a b o u t 95% of recovered DNA, w i t h 1 to 3 % in the cytoplasmic fraction a n d in the c h r o m a t i n s u p e r n a t a n t fraction. These d a t a are calculated from t h e recovered trichloroacetic acid-insoluble radioactivity in 3H (labeling RNA) in three separate preparations : I, 5-min pulse in uridine, isolation in 1.0 mMTris.HC1; I I , 5-rain pulse in uridine, isolation in 1.0 mM-EDTA; III, 5-min pulse in urldlne, 25-min chase, isolation in 1.0 mz~-TrJs.HC1.
'o_ 6~I-
45s
z2s
-t--
20
0
~ssl ~22s
-
32S
c
0
I0
I0
20
Bottom
Top Fraction no. (o)
(b)
Fzo. 1. Sedimentation distribution of radioactive R N A associated with c h r o m s t i n after a 5-min pulse of [5,6-SH]uridine (a) or 5-min pulse--25-min chase in 10 -4 •-urldine (b). Labeling a n d fractionation procedures were as described in Materials a n d Methods. R N A was released from protein in portions containing c h r o m s t i n from 3 • 10 s cells, w i t h sodium dodecyl sulfate a n d Pronase as described in Materials a n d Methods, a n d layered over 15% ~o 30O/o sucrose/sodium dodecy] sulfate gradients. The gradients were centrifuged for 3 h a t 35,000 revs/min (20~ SW20 rotor), fraotionated into 0.5-ml portions, a n d total radioactivity in 3H in each fraction is shown in the Figure. Sedimentation values indicated in t h e Figure were assigned according to the migration of identifiable cytoplasmic R N A species in s gradient r u n in parallel with these.
C. B . K I M i ~ I E L E T
182
AL.
r e c o v e r y d a t a . T h e r e is no m a j o r difference in its s e d i m e n t a t i o n p a t t e r n i n t h e t w o p r e p a r a t i o n s , h n R N A molecules m a y be s y n t h e s i z e d a t r a t e s s i m i l a r t o r R N A ( P e r r y et al., 1974). P r e v i o u s w o r k h a s s h o w n t h a t s o m e h n R N A molecules are p o l y a d e n y l a t e d , a n d t h a t this occurs a f t e r t r a n s c r i p t i o n is c o m p l e t e d . W h e n cells were l a b e l e d for 30 m i n u t e s w i t h r a d i o a c t i v e adenosine, 6~ o f t h e l a b e l e d c h r o m a t i n - a s s o c i a t e d R N A was b o u n d b y p o l y ( U ) - S e p h a r o s e u n d e r c o n d i t i o n s w h i c h were p r e v i o u s l y d e m o n s t r a t e d t o be specific for p o l y ( A ) - c o n t a i n i n g R N A (MacLeod, 1975b). T h u s t w o t y p e s o f p o s t - t r a n s c r i p t i o n a l R N A molecules, 32 S p r e c u r s o r r R N A a n d p o l y ( A ) - c o n r a i n i n g h n R N A , are o b t a i n e d in t h e c h r o m a t i n f r a c t i o n p r e p a r e d b y our procedures. I t was possible t h a t R N A was n o t a s s o c i a t e d w i t h c h r o m a t i n , b u t c o n t a m i n a t e d t h e c h r o m a t i n f r a c t i o n i s o l a t e d b y differential c e n t r i f u g a t i o n . F o r e x a m p l e , t h i s f r a c t i o n c o n t a i n s n u c l e a r m e m b r a n e s , as d e t e r m i n e d b y p h a s e m i c r o s c o p y . To e x a m i n e this we s t u d i e d t h e s e d i m e n t a t i o n of n u c l e a r m a t e r i a l t h r o u g h v e r y s t e e p sucrose gradients. I n t h e e x p e r i m e n t s h o w n in F i g u r e 2(a), a n u n f r a c t i o n a t e d n u c l e a r l y s a t e was centrifuged t h r o u g h such a g r a d i e n t . T h e m a j o r p a r t s of c h r o m a t i n D N A a n d o f n u c l e a r R N A (pulse-labeled for 5 rain) are in a b a n d n e a r t h e b o t t o m o f t h e g r a d i e n t , a n d a small f r a c t i o n of e a c h n e a r t h e top. T h e t o p f r a c t i o n c o r r e s p o n d s t o t h e " c h r o m a t i n s u p e r n a t e " f r a c t i o n in T a b l e 1. T h e r e is also a l i g h t s c a t t e r i n g b a n d n e a r t h e c e n t e r of t h e g r a d i e n t , w h i c h c o n t a i n s m e m b r a n e s (phase m i c r o s c o p y ) , b u t o n l y s m a l l a m o u n t s o f l a b e l e d mmlei acids. T h e r a p i d c o s e d i m e n t a t i o n o f c h r o m a t i n
6-4-
3H / '4C
0
S-~ '0 x
l
II
I
2-
-
4
6-ii I ,
i i
x
,o o, I ,,
4_
i
:
E i-
_
i
.~
,
~
t i i
2
E
i
o o
._o
0~
~
Bottom
(a)
I0
-
20
0 =,No
Fraction no.
1
"
0
"Fop
(b)
FIo. 2. Sedimentation distribution on steep (0.28 ~ to 2.8 M) sucrose gradients of unsheared (a) and sheared soluble (b) ehromatin. (a} The cells were labeled for 2 days with [2-14C]thymidine (0.05 gCi/ml) and for 5 min with [5,6-3H]m'idine (40/~Ci/ml). Nuclei were prepared from 5.2 x 107 cells and one-haft of the nuclear lysate, in 1.0 m~-Tris.HC1, was layered over the gradient. The Figure shows radioactivity in triehloroaeetie acid-insoluble material taken from 0.1-ml portions of the gradient fractions. (b) The cells were labeled for 1 day with [2-14C]thymidine (0-02 ttCi/ml) and for 20 min with [5,6-3H]uridine (20 /xCi/ml). The nuclear lysate, in 1.0 m~Tris. HC1, was sonicated as described in Materials and Methods and a portion of the sonieate (not clarified in this experiment) containing nuclear material from 2.4 x 10e cells was layered over the gradient. The Figure shows radioactivity in triehloroacetic acid-insoluble material in the fractions. Background levels of radioactivity were obtained in the lower 3/4 of the gradient. - - O - - Q - - , aH radioactivity; - - O - - 9 14C radioactivity.
ASSOCIATION
OF NUCLEAR
RNA AND CHROI~IATIN
183
and most nuclear RNA, obtained here, shows that the RNA is associated with chromatin, and not with some unidentified very large particle. Shearing of chromatin into slowly sedimenting "soluble" particles by sonieation also quantitatively slows the sedimentation of radiolabeled associated RNA (:Fig. 2(b)). Analyses (not shown) of the sedimentation of sheared chromalia showed t h a t both RNA and DNA sediment together in a complex of particles ranging from about 20 S to 100 S, indicating t h a t the two nucleic acids m a y remain associated throughout the procedure. Association of RNA with chromatin was also tested by equilibrium densitycentrifugation analyses. Centrifugation of chromatin in metrizamide densitygradients is shown in Figure 3. In the case of sheared (Fig. 3(a)) and unsheared I ~
1"4
F--T--
1"2 ~
20
--
U;
I'0
_
",.
li6 x
jl
x
0
-
E
'1
-
0
I0
I'
20 0
u v
20
I0
Fraction no.
(a)
(b)
FIG. 3. Buoyant density of chromatin in metrizamide. Cells were labeled for l day with [2-z4C]thymidino (0.016 ~Ci/rnl) and for 20 rain with [5,6-3H]uridino (10 ~Ci/ml). Clu-omatin was prepared in 1.0 mM-Tris.HC1, and centrifuged in metrizamido as described in Materials and Methods. Each gradient hero contains chromatin prepared from 1-6 • 106 cells. (a) The chromatin was sheared by sonication before mixing with metrizamide; (b) non-sheared (insoluble) ehromatin was analyzed. - - 0 - - 0 - - , 3H radioactivity and buoyant density; - - O - - O - - , z4C radioactivity and 3H/z4C ratio as appropriate.
chromatin (Fig. 3(b)) label in RNA is found in the chromatin band (density approx. 1.26 g/cm3), and significantly shifted to its heavy side as reported by Birnie et al. (1973). The shift is more pronounced in the sheared preparation (Fig. 3(a)). Such Sheared particles were also analyzed in CsC1 gradients, after treatment with formaldehyde (Fig. 4). Here the chromatin bands at about 1.41 g/cm 3, and the pulse-labeled RNA is in the same band. (b) Exchange of added nucleic acids with chromatin It is possible t h a t some fraction of nuclear RNA is not associated with ehromatin in rive, but binds with it during isolation. To examine this possibility we attempted to create artificial complexes between RNA and chromatin. Various concentrations of labeled preparations of purified DNA, RNA, or R N P in Tris. HC1 were added to
C.B. KIMMEL ET AL.
184
1.412
1"5 F
1"4
-4
-2
3H/J'*C
Q..
i.ii
I
~I I f
~
~7 0
'
x c
-
-6
~
, i
:
i
!
i
i
~ '~
0
-
g" i
-~
p2 o
E m
_~,
1I -
o
0
~./i J ~176 1 = 2
I0
20
Jo
Fraction no.
FIG. 4. Buoyant density profile of formaldehyde-treated chromatin in CsC1. A total of 1.6 • 107 cells were labeled for 1 day with [2-14C]thymidine (0.016 ~Cilml) followed by 10 rain with [5,6-3H]uridine (10 ~Cilml). ChromaLin was prepared and sheared in 1.0 m~-EDTA, and a portion treated with formaldehyde for 3 h as described in Materials and Method~. A portion representing 1]4 of the total preparation was mixed with CsC1 for the gradient analysis. In control experiments, no change in sedimentation of chromatin could be detected after formaldehyde treatment, nor were artificial complexes created between ehromatin particles and ribosomal subunits, indicating that neither aggregation nor non-specific cross-linking was occurring (data not shown). - - Q - - Q - - , 3H radioactivity and buoyant density; - - C ) - - O - - , 14C radioactivity and 3H/14C ratio as appropriate. nuclear pellets, a n d the nuclei lysed in t h e presence of these molecules; after differential centrffugation, radioactivity associated with the c h r o m a t i n was determined. The d a t a from several experiments are summarized in Table 2. W e found, first of all, significant binding of D N A to c h r o m a t i n (Table 2, experiments 1 a n d 2). Binding was as high as 22% of a d d e d material, a n d the m a x i m a l level was a p p r o x i m a t e l y 12~/o of t h e D N A calculated to be present in the ehromatin preparation. However, binding of R N A to chromatin was at b a c k g r o u n d levels (<3~/0 of a d d e d material over a range of i n p u t concentrations). This was true for purified nuclear R N A (experiments 2 a n d 3), R N P extracted from nuclei b y the m e t h o d of Martin et al. (1973) (experiments 3 a n d 4), and ribosomes (experiment 5). These d a t a t h u s provide no evidence for a rapid exchange occurring between R N P a n d ehromatin during isolation. (e) Extraction of ribonucleoprotein from chromatin I f h n l ~ N P particles were associated with c h r o m a t i n in vivo, we reasoned t h a t t h e same washing procedure in which t h e y are released f r o m intact nuclei (the usual m e t h o d of obtaining these particles) would also effect their release f r o m c h r o m a t i n preparations. W e h a v e c o m p a r e d R N A extraction f r o m these two sources, following t h e m e t h o d of Martin et al. (1973) developed for aseites t u m o r cells.
ASSOCIATION
OF NUCLEAR
RNA
AND
185
CHROMATIN
TABLE 2 Bicdi~
o f added n u c ~ i c acids a n d n c d e o p r o t e i ~ to chromalin Amount
Experiment
Addition a
No. of nuclei b
added
(/~g) 1
2 3
4 5
DNA o DNA DNA DNA DNA ~ RNA d RNA s RNA RNP e RNP RNP t Rib 9 Rib Rib
5 • l0 s 5 • l0 s 5 • l0 s 5 • l0 s 1 • 10 v 1 • 107 1.2 • l0 s 1.2 • l 0 s 1-2 • l0 s 1-2 • l0 s 5-3 • l 0 s 2.9 • l0 s 2.9 • l0 s 2.9 • l 0 s
6.8 13.7 27.5 55 55 11.7 5.9 29 0.4 2.0 9.0 10-8 3.6 2.9
Amount in chromatin pellet
Binding (%)
1.5 2.3 3.8 4.8 8.0 0-36 0.04 0.09 0.005 0.016 0-28 0.07 0-03 0.03
22.0 16.8 13.8 8.7 14.6 3-1 0.7 0.3 1.2 0.8 3.1 0.6 0.9 1.1
(/,g)
a Radioactive nucleic acids or nucleoproteins were added in 1-0 mM-Tris.HC1 (pH 7.0) to pc/lets containing nuclei, which were t h e n lysed as usual. Chromatin was sedimented (27,000 g for 5 rain) a n d radioactivity in t h e c h r o m a t i n pellet a n d supernate determined. Recoveries of added radioactivity were greater t h a n 90%. b Usually of the order of 2 Ace0 units of c h r o m a t l n arc obtained from 107 cells, corresponding to approx. 80 ~g of DNA. a Uniformly l~C-labeled D N A was prepared b y e x t r a c t i o n with phenol/chloroform (Penman, 1966) of sodium dodecyl sulfate a n d Pronase-treated chromatin. The D N A (A28o/A2so ~ 1.86) contained 1.3 • 103 ets/min per pg. a 20-min pulse-labeled R N A [5,6-3H]uridine was prepared from c h r o m a t i n b y t r e a t i n g it w i t h DNase I (25/~g/ml for 10 rain a t 37~ in 1.0 mM-MgC12, 1.0 m ~ - T r i s . H C l , p H 7.0) followed b y t r e a t m e n t with sodium dodecyl sulfate a n d Pronase (see Materials a n d Methods) a n d extraction w i t h phenol/chloroform (Penman, 1966). The R N A (A28o/A2so ~ 1.96) contained 4.7X104 cts/min per pg. e R N P particles were extracted from t h e nuclei of cells pulse-labeled for 20 mln w i t h [5,6-aH]uridine as described in Materials a n d Methods, a n d dialyzed vers~ 1-0 mM-Tris.HC1 (pH 7.0). The R N P (A26o/Asso ~ 1.43) contained R N A which sedimented a t approx. 6 S (obtained also b y Martin et al., 1973), which contained 3.45 • 103 cts/min per ~g. f Another p r e p a r a t i o n of 20-min labeled R N P particles, extracted as above, was tested. The R N A conta!ned 1.15 • 104 cts/min per /zg. Ribosomes were sedimented (40,000 revs/min for 90 rain a t 4~ in t h e SW50L rotor) from t h e cytoplasmic fraction of S194 cells which h a d been t r e a t e d with 3 /~Ci [5,6-3H]uridine]ml, 20 h. They were re3uspended in 1-0 mM-Tris.HC1 (pH 7.0). The ribosomes (Asso/A28o ~ 1.73) cont a i n e d R N A labeled a t 9.7 • 10~ cts/mln per pg.
"Figure 5(a) shows the cumulative release of 10-minute pulse-labeled RNA and uniformly labeled DNA from whole nuclei, according to Martin's washing schedule. T h e r e is s p e c i f i c e x t r a c t i o n o f R N A (less t h a n 1~/c o f D N A is s o l u b i l i z e d ) , a n d i n a g r e e m e n t w i t h t h e f i n d i n g s o f M a r t i n et al. (1973) R N A e x t r a c t i o n is m o s t e f f i c i e n t at piE/8, and at e]evated temperature. Chromatin, prepared from a portion of the s a m e cells, w a s e x t r a c t e d a c c o r d i n g t o t h e s a m e s c h e d u l e a n d , a s s h o w n i n l ~ i g u r e 5(b), the specific extraction of RNA occurs nearly identically to that from whole n u c l e i . A s i m i l a r r e l e a s e o f R N A o c c u r s a t 20~ a n d 37~ i n 1.0 m M - T r i s . H C 1 ( p H 7.0) b u t , a s s h o w n i n F i g u r e 5(c), s u b s t a n t i a l l y m o r e D N A is a l s o s o l u b i l i z e d , a m o u n t i n g t o m o r e t h a n 10~/o o f t h e t o t a l .
186
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(a) (b) (c) FIG. 5. Extraction of RNP particles from nuclei (a) and chromatin (b) and (c). A total of 3.4 • 107 cells were treated with [2-14C]thymidine (20 h, 0.16/zCi/ml) followed by [5,6-3H]uridine (10 min, 20/~Ci/rnl). The cells were homogenized, and 1/3 of the nuclei washed once in LS buffer (see Materials and Methods), and used for the extractions shown in (a). Chromatin was prepared from the remaining 2/3, washed once in 1.0 mM-Tris (pH 7.0) and extracted with the buffers as described by Martin etal. (1973) (b), or with 1.0 m~-Tris-HC1 (c). The extraction procedure is described in Materials and Methods, and the Figure shows the cumulative release of radioactivity in 3H ( - - O - - O - - ) and xdC ( - - O - - 9 as a percentage of the total in portions from each supernate. These experiments demonstrate t h a t R N A , p r e s u m a b l y in association with proteins (A26o/A28o = 1.4), can readily be released from a complex with chromatin, in the same procedure where h n R N P particles are obtained from nuclei. (d) Distribution of R N A in fl'actionated chromatin M a n y workers h a v e shown t h a t chromatin preparations m a y be fractionated to obtain classes of particles enriched for associated pulse-labeled R N A molecules. I n this section we show t h a t in two m e t h o d s t h e associated R N A is n o t fractionated according t o its age. A slowly sedimenting minor fraction of ctu'omatin enriched for associated R N A can be obtained from mildly sheared preparations (Chalkley & Jonson, 1968 ; M u r p h y etal., 1973; M c C a r t h y etal., 1973; Chance etal., 1974). Figure 6 shows the sedimentation patterns obtained from chromatin in a nuclear lysate (Fig. 6(a)), compare with Fig. 2(a)), a n d f r o m chromatin sheared b y sedimentation to a pellet with v o r t e x resuspension (Fig. 6(b)). I n the latter a b o u t 3 % of the D N A a n d 3 0 % of the R N A is slowly sedimenting. Continued association of the D N A a n d R N A in this minor fraction was confirmed b y sedimentation analysis in sucrose gradients a n d equilibrium density analysis in metrizamide gradients (data n o t shown). W e examined the distribution of R N A of different ages in chromatin fractionated b y this m e t h o d in double-label experiments. I n the experiment shown in Figure 7, R N A was labeled for 20 minutes with [2-zdC]uridine, a n d [5,6-3H]uridine was a d d e d for an additional 4 minutes. A similar proportion of molecules of each age is slowly sedimenting. W h e n deproteinized R N A from this fraction was examined b y sucrose gradient centrifugation b o t h isotopes were f o u n d to label 45 S precursor r R N A a n d h n R N A .
ASSOCIATIOI~
OF NUCLEAR
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187
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Fro. 6. Fl'aetionation of ehromatin in steep (0-28 M to 2.8 M) sucrose gradients. Cells were labeled f r 1 day with [2-x4C]thymidine (0-01 /~Ci/ml) and for 10 rain with [5,6-aH]uridino (20 /~Ci]ml). Nuclei were prepared from 3 x l0 T cells and lysed in 0.9 ml 0.2 mM-EDTA, and 0.2 ml of the lysate used for the gradient shown in (a). Chromatin in the rest of the lysate was centrifuged to a pellet and resusponded with vortex mixing. Half of this preparation was used for the gradient shown in (b). The Figure shows triehloroaeetic acid-insoluble radioactivity in 100-/~1 portions from the gradient fractions. - - 0 - - 0 - - , 3H radioactivity; - - O - - O - - , 140 radioactivity.
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:FIG. 7. Gradient fraetionation of R N A in slightly sheared chromatin. Cells were labeled with [2-x4C]uridino (0"5 ~Ci/ml) for 20 rain, and [5,6-3H]uridine (40 ~Ci/ml) for an additional 4 rain. A chromatin suspension was prepared in 0-2 mM-EDTA, centrifuged through a 0.28 • to 2.8 sucrose gradient, and fractionated. The Figure shows the distribution of trichloroaeeti0 acidinsoluble radioactivity of each isotope ( 0 , 3H; O, 14C), and the ~H]:4C ratio.
188
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T h e 32 S p r e c u r s o r r R N A p o s t - t r a n s c r i p t i o n a l molecule was o b s e r v e d o n l y i n t h e p o p u l a t i o n l a b e l e d for 24 m i n u t e s ( d a t a n o t shown). W e p r e p a r e d t h e slowly sedim e n t i n g e h i ' o m a t i n f r a c t i o n f r o m cells l a b e l e d for 30 m i n u t e s w i t h [2,8-3H]adenosine, a n d f o u n d t h a t 6~ o f t h e r a d i o a c t i v i t y was o b t a i n e d in p o l y ( A ) - e o n t a i n i n g R N A molecules. This was t h e s a m e p r o p o r t i o n as f o u n d p r e v i o u s l y (see a b o v e ) for t o t a l e h r o m a t i n . T h e size d i s t r i b u t i o n s o f p o l y ( A ) - c o n t a i n i n g R N A f r o m f r a c t i o n a t e d a n d u n f r a c t i o n a t e d c h r o m a t i n were i d e n t i c a l ( d a t a n o t shown). T h u s f r a c t i o n a t i o n o f s l i g h t l y s h e a r e d c h r o m a t i n does n o t f r a e t i o n a t e t h e a s s o c i a t e d R N A a c c o r d i n g t o its age, n o r does i t select for or a g a i n s t p o s t - t r a n s c r i p t i o n a l R N A molecules. E x t e n s i v e l y s h e a r e d c h r o m a t i n m a y b e f r a c t i o n a t e d b y differential p r e c i p i t a t i o n w i t h MgC12 (Bonner et al., 1973; G o t t e s f e l d et al., 1974). I n a g r e e m e n t w i t h t h e findings of B o n n e r ' s group, we o b s e r v e d t h a t c h r o m a t i n p a r t i c l e s e n r i c h e d in a s s o c i a t e d p u l s e - l a b e l e d R N A a r e r e l a t i v e l y r e s i s t a n t t o Mg 2+ p r e c i p i t a t i o n . T h i s is s h o w n in F i g u r e 8(a), w h e r e t h e s o l u b i l i t y o f c h r o m a t i n associated, 5 - m i n u t e l a b e l e d R N A (ordinate) is p l o t t e d as a f u n c t i o n o f c h r o m a t i n I ) N A s o l u b i l i t y (abscissa), in a p r e p a r a t i o n t r e a t e d w i t h 0 t o 2 mM-MgCI2. W e find, a t a c o n c e n t r a t i o n of 1.0 mM-MgC12, 5 t o 10~/o o f e h r o m a t i n D N A , a n d 35 t o 4 5 % o f a s s o c i a t e d R N A , r e m a i n i n t h e s u p e r n a t a n t f r a c t i o n a f t e r c e n t r i f u g a t i o n a t 27,000 g for 5 m i n u t e s . S e d i m e n t a t i o n a n a l y s i s of t h i s m a t e r i a l in sucrose g r a d i e n t s d e m o n s t r a t e d t h a t t h i s
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Fzo. 8. Fraetionation of ehromatin by MgOl2 precipitation. (a) Sheared "soluble" chromatin particles were prepared from cells labeled for 1 day with [2-14C]thymidine (0.005 FCi/m]) and for 5 rain with [5,6-3H]uridine (40 FCi/ml). Samples of 50 ~1, at an absorbanee (260 nm) of 2.0 were mi~ed with 50 pl of 0 to 4 m•-MgC12 and centrifuged at 27,000 g for 5 rain as described in Materials and Methods. The supernate was assayed for radioactivity in 3H and 14C. In the Figure, 100% corresponds to 216 cts/mln in 14C and 2529 cts/min in 3H. The Figure shows RNA solubility as a function of chromatin DNA solubility. (b) Both isotopes are in RNA. A total of 5 • 107 cells in 5 ml of culture medium were labeled with [2-14C]uridine (2 ~Ci/ml, 57 Ci/mol; New England Nuclear) for 22 rain, and [5,6-3H]uridine (40/LCi/ml) for an additional 3 rain. Sheared chromatin was prepared and 100-pl portions at an initial absorbance (260 nm) of 2-0 treated with 100 pl of 0 to 4 m~-MgC12 as described in Materials and Methods. After centrifugation, radioactivity was measured in 100-pl portions of the supernate. The open and closed circles show the data from 2 separate preparations, which were treated identically throughout. In one of these (open circles) 100% in the Figure corresponds to 1882 ots/min in 8H and 1125 ot~/min in 14C. In the other (closed circles) 100% corresponds to 2142 cts/min in 3H and 1525 e~s/min in 14C.
ASSOCIATION
OF NUCLEAR
RNA AND CHROMATIN
]89
technique does not select for a particular size class of RNA-rich particles, and equilibrium density analysis in metrizamide gradients revealed no apparent density fractionation of chromatin by this method (data not shown). RNP particles, extracted from nuclei by the method of Martin et al. (1973), and dialyzed versus 1.0 mM-Tris-HC1 (pH 7.0) were found to be quantitatively soluble when treated with 2.0 m~-MgCl2. The age-distribution of RNA molecules in Mg2+-fractionated sheared chromatin was followed in double-label experiments. Two are shown in Figure 8(b), where in each case RNA was labeled for 3 minutes and 25 minutes. Here it is seen that the proportions of RNA molecules marked by each isotope remaining soluble in various concentrations of MgC12 (0 to 2.0 raM) are the same; the points cluster closely about the diagonal. Thus fractionation of ehromatin by Mg2+ does not fractionate the chromatin-assoeiated RNA according to its age.
4. Discussion We have shown that when chromatin is prepared rapidly, and in low ionic strength buffers, the major part (at least 80~/o) of nuclear RNA is obtained in the chromatin preparation. The cosedimentation of RNA with chromatin in sucrose gradients, and the similar isopycnic positions in metrizamide gradients argue that the RNA is associated with chromatin, and not simply present as a contaminant. Furthermore, shearing of the chromatin by sonication does not appear to release the associated RNA, as determined by the same criteria. It has been reported (de Pomerai et al., 1974) that different procedures for the isolation of chromatin yield preparations which differ considerably with respect to composition and biolo~cal activity. In particular, extensively purified chromatin contains less RNA than we obtained with our relatively simple procedure. We found that both DNA and RNA are solubilized if ct~romatin is repeatedly extracted at low ionic strength (Fig. 5), which is the expected result of degradation. We tested directly for binding of exogenous RNA or RNP to our preparation and could find none (Table 2). These observations suggest that the RNA is functionally associated with some fraction of chromatin in vivo. Evidence was obtained that both known major classes of nuclear RNA, the rRNA precursors and hnRNA, remain associated with chromatin after transcription is completed. The primary rRNA transcript, 45 S RNA, was identified after a very short pulse of label, while the major transcriptional intermediate, 32 S precursor, rRNA could only be observed after a more extended period (Fig. 1). Processing of precursor rRNA is known to occur in the nucleolns (Perry, 1962; Penman et al., 1966), which is associated with the nucleolar organizer region of chromatin (Steele, 1968; Miller & Beatty, 1969). Also present in the chromatin were rapidly labeling heterodisperse molecules which contained, significantly, poly(A) sequences. The addition of poly(A) to some hnRNA molecules is thought to occur as a post-transcriptional step (Perry et al., 1974; Jellinek et al., 1973). These data are thus not in accord with the hypothesis (Samarina et al., 1968) that post-transcriptional processing of hnRNA occurs in relatively simple nucleoplasmic organelles (hnRNP particles), and we consider it likely that such particles are a part of the chromatin matrix in vivo. This view is supported by the observation that RNP possessing several properties of hnl~NP could be detached from chromatin (Ishil~awa e~ a/., 1974), and we showed here (Fig. 5) that RNA could be extracted from chromatin by
190
C. B . K I M M E L
ET AL.
the same procedure used in another system (Martin et al., 1973) to prepare h n R N P from whole nuclei. A detailed analysis of the release of chroma~in R N P is made difficult b y the fact that sheared chromatin sediments heterogeneously in the same rauge as reported for h n R N P particles (Samarina el al., 1968; Pederson, 1974a), and the buoyant density of h n R N P and sheared chromatin in CsC1 are similar (~-~I.4 g/ca3). The buoyant density of h n R N P in metrizamide has not been reported. In this solvent, chromatin and its associated RNA band at 1.2 to 1.3 g/cm 3 (Birnie et al., 1973; and Fig. 3). Recently Scott & Sommerville (1974) showed b y immunofluorescent methods t h a t h n ~ N P proteins are associated specifically with the lateral loops of giant lampbrush chromosomes in the newt ooeyte. In accord with these results, we found t h a t a large part (30 to 40 ~/o) of chromatin RNA could be shown to be contained in a fraction containing 3 to 10% of chromatin DNA. In particular, we established that, in two methods to obtain RNA-enriched chromatin classes, the RNA is not fractionated according to its age. For one of the methods we showed that post-transcriptional molecules, identified as above, were present in the same proportion as in total chromatin. The basis of separation is thought to be different in each method; the slowly sedimenting fraction prepared from large particles having a special " e x t e n d e d " conformation (Chalkley & Jenson, 1968), and the Mg 2§ soluble fraction prepared from small chromatin particles containing reduced amounts of histone proteins (Gottesfeld et al., 1974). We have not tested directly whether the same minor class of chromatin is obtained in each isolation. Both have been shown, in the above-cited studies but not repeated here, to yield chromatin especially active in in vitro transcription assays. We found that neither method separates a class of chromatin of special buoyant density, and Rickwood et al. (1974) obtained no purification of template-active chromatin b y metrizamide density-fraetionation. The minor fractions are thought to contain, as is the case for lampbrush loops, regions active in in vivo RNA synthesis. I t is possible, therefore, that events in the post-transcriptional history of hnRNA, as for precursor rRNA, occur while molecules remain complexed with active genes in chromatin. This research was supported by National Science Foundation grant no. GB-37495. REFERENCES
Birnie, G. D., Rickwood, D. & Hell, A. (1973). Biochim. Biophys. Acta, 331, 283-294. Bonnet, J., Garrard, W. T., Gottesfeld, J., Holmes, D. S., Seval, J. S. & Wilkes, M. (1973). Cold Spring Harbor Syrup. Quan$. Biol. 38, 303-310. Brandhorst, B. P. & McConkey, E. H. (1974). J. Mol. Biol. 85, 451-463. Brawerman, G., Mendecld, J. & Less, S. Y. {1972). Biochemistry, l l , 637-641. Burton, K. (1956). Biochem. J. 62, 315-323. Chalkley, R. & Jensen, R. H. (1968). Biochemistry, 7, 4380-4388. Chance, H., Kadohoma, N. & Anderson, K. M. (1974). Biochcm. Biophys. Res. Commun. 58, 66-73. de Pomerai, D. I., Chesterton, C. J. & Betterworth, P. H. W. (1974). Eur. J. Biochem. 46, 461-471. Gottesfeld, J. M., Garrard, W. T., Bagi, G., Wilson, R. F. & Bonner, J. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 2193-2197. Greenberg, H. & Penman, S. (1966}. J. Mol. Biol. 21, 527-535. Ishikawa, K., Sato, T., Sato, S. & Ogata, K. (1974). Biochim. Biophys. Acta, 353, 420-437. Jellinek, W., Molloy, G., Salditt, M., Wall, 1%., Scheiness, D. & Darnell, J. E., Jr {1973). Cold Spring Harbor Syrap. Quant. Biol. 38, 891-898.
A S S O C I A T I O N OF N U C L E A R RNA AND CHROMATIN
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Kimmel, C. B. (1971). Expt. Cell R ~ . 65, 202-208. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 198, 265-275. MacLeod, M. C. (1975a). A w l . Biochem. 68, 299-310. MaeLeod, M. C. (19755). Biochem~try, 14, 4011-4018. Martin, T. E. & McCarthy, B. J. (1972). Biochim. Biophys. Acta, 277, 354-367. Martin, T., Billings, P., Levey, A., Ozarslan, S., Quinlin, T., Swift, H. & Urbas, L. (1973). Cold Spring Harbor Syrup. Quant. Biol. 38, 921-932. McCarthy, B. J., Nisiura, J. T., Doenecke, D., Nasser, D. S. & Johnson, C. B. (1973). Cold Spring Harbor Syrup. Quant. Biol. 38, 763-771. Miller, O. L., Jr & Beatty, B. R. (1969). Science, 164, 955-957. Murphy, E. C., Jr, Hall, S. H., Shepherd, J. H. & Weiser, R. S. (1973). Biochemistry, 12, 3843-3853. Pederson, T. (1974a). J. Mol. Biol. 83, 163-183. Pedorson, T. (1974b). Prec. Nat. Acad. Sci., U.S.A. 71, 617-621. Penman, S. (1966). J. Mol. Biol. 17, 117-130. Penman, S., Smith, I. & Holtzman, E. (1966). Science, 154, 786-789. Perry, R. P. (1962). Prec. Nat. Acad. Sci., U.S.A. 48, 2179-2186. Perry, R. P., Kelley, I). E. & LaTorre, J. (1974). J. Mol. Biol. 82, 315-351. Rickwood, D., Hell, A., Malcolm, S., Birnie, G. D., Macgillivray, A. J. & Paul, J. (1974). Biochim. Biophys. Acta, 353, 353-361. Samarina, O. P., Lukanidin, E. M., Molnar, J. & Georgiev, G. P. (1968). J. Mol. Biol. S3, 251-263. Schneider, W. C. (1957). In Methods in Enzymology (Colowick, S. P. & Kaplan, N. O., eds), vol. 3, p. 680, Academic Press, New York. Scott, S. E. M. & Sommerville, J. (1974). Nature (London), 250, 680-682. Sherrer, K. & Maraud, L. (1965). Bull. Soc. Ghim. Biol. 47, 1697-1713. Steele, W. J. (1968). J. Biol. Chem. 243, 3333-3341. Vogt, M. & Dulbecco, R. (1963). Prec. Nat. Acad. Sci., U.S.A. 49, 171-179.