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Effects of low salt concentration on structural organization and template activity of chromatin in chicken erythrocyte nuclei
Experimental Cell Research 65 (1971) 61-72
EFFECTS OF LOW SALT CONCENTRATION ON STRUCTURAL ORGANIZATION AND TEMPLATE ACTIVITY OF CHROMATIN IN CHICKEN ERYTHROCYTE NUCLEI K. BRASCH, V. L. SELIGY and G. SETTERFIELD
Department of Biology, Carleton University, and The Biochemistry Laboratory, National Research Council, Ottawa, Canada
SUMMARY Isolated chicken erythrocyte nuclei were suspended in saline, water, or saline following water, and studied both microscopically and for capacity to support R N A synthesis in vitro. Treatment in water caused complete dispersal of condensed chromatin and a reduction in width of individual fibrils from ca 200 ~ to ca 75 A; both changes were reversed by returning nuclei to saline. Studies of individual nuclei with Nomarski interference-contrast optics demonstrated that the gross configuration of condensed chromatin which reformed in saline after water dispersal was structurally identical with that present before dispersal. Nuclei in saline had very restricted capacity for R N A synthesis and no major change took place when chromatin was dispersed by water. The small amount of R N A synthesized by condensed and dispersed nuclei appeared qualitatively identical by gel electrophoresis and presaturation hybridization with erythrocyte DNA. It is concluded that the chromatin of these interphase nuclei has a specific, ordered structure which is probably determined both by the chromatin composition and interaction between chromatin and nuclear membrane. Ion-mediated condensation and dispersal of chromatin does not appear to directly regulate the capacity of the D N A to support R N A synthesis.
Eukaryotic interphase nuclei contain varying amounts of condensed and dispersed chromatin. The distribution of chromatin between the two states varies in different species [1] and in different nuclei within the organs of the same species [2]. Several studies have indicated that in intact nuclei RNA synthesis is largely associated with dispersed chromatin [3, 4, 5] and that increased condensation is correlated with restriction of genetic transcription [2, 6]. Two questions concerning this situation are as yet incompletely answered. First: is the formation of condensed chromatin a relatively unspecific type of aggregation or does each interphase nucleus have a precise internal structure resulting from specific folding and packing of the two
chromatin phases? Second: does the process of chromatin condensation directly control gene transcription or is it merely a secondary phenomenon which proceeds in conjunction with restriction of template capability of the chromatin at the molecular level? Mature chicken erythrocyte nuclei, which have almost completely condensed chromatin [7, 8, 9] and severely restricted transcription capacity [10-12], present an opportunity to obtain evidence bearing on these questions. Several authors have shown, using both light and electron microscopes, that the state of condensation of chromatin in isolated nuclei is sensitive to salt concentration: hypotonic conditions cause swelling and Exptl Cell Res 65
62
K. Brasch et al.
dispersal
of
solutions
produce
chromatin.
chromatin
while
hypertonic
abnormally
condensed
T h e c h a n g e s a r e in p a r t r e v e r -
sible [13-18]. T h i s p a p e r p r e s e n t s t h e r e s u l t s of e x p e r i m e n t s
designed to follow changes
in b o t h the specific s t r u c t u r a l c o n f i g u r a t i o n and
t e m p l a t e a c t i v i t y of t h e c h r o m a t i n
of
e r y t h r o c y t e nuclei in t h e n o r m a l c o n d e n s e d state in p h y s i o l o g i c a l saline, c o m p l e t e l y dispersed
in dilute salt s o l u t i o n
recondensed
(water)
and
in saline a f t e r h y p o t o n i c
dis-
persal.
MATERIALS
AND METHODS
Sources Spray-dried Microcoeeus lysodeiktieus ceils were purchased from Miles Chemical Corp. Chemicals were obtained as follows: Dade Tissue Tac, Canadian Laboratory Supplies; calf thymus D N A and RNase A (type RAF), Worthington Biochemical Corp.; 5-3H-UTP and Nuclear Chicago Solubilizer (NCS), Amersham/Searle; CTP and GTP, Pabst Laboratories; ATP, protamine phosphate and spermidine phosphate, Sigma Chemical Corp.; formamide, Eastman Kodak. Nitrocellulose membrane filters were Schleicher and Schuell type B-6.
Preparation of nuclei Nuclei were prepared from blood of White Leghorn roosters (5-8 months old, Ottawa Meat Control strain) through lysis of erythrocytes with saponin as described by Neelin and co-workers [12, 19]. The isolated nuclei were freed of hemoglobin by repeated washing in saline (0.15 M NaC1). To prepare nuclei with dispersed chromatin, washed nuclei suspended in saline were sedimented at 1 000 g for 5 min and the supernatant was decanted and replaced by an equal volume of distilled water. Nuclei with recondensed chromatin were produced from these nuclei by sedimenting after 5 min water treatment and resuspending in the same volume of saline. Chicken liver nuclei were prepared by a modification of the method previously described [11], using a medium containing 0.35 M sucrose and 0.15 M NaC1 only. After purification the isolated nuclei were suspended in saline.
Electron microscopy For examination by electron microscopy samples of pelleted nuclei (1 000 g, 5 min) were fixed at 0-4~ as follows: 1.5 % glutaraldehyde, 30 min; 6 % glutaraldehyde, 2 h; three washes in saline or water overnight; 2 % OsO4, 2 h; three washes in saline or water, 15 min each. Glutaraldehyde and OsO4 were dis-
Exptl Cell Res 65
solved in the medium in which the nuclei were initially suspended, i.e. saline or water, and the pH was adjusted to 6.8 with 0.01 N NaOH. Following fixation small clumps of nuclei were dehydrated at 0-4~ in methoxyethanol and ethanol, two changes of 1 h each, and embedded in Epon-Araldite [20]. Sections were stained for 30--40 min in 25 % uranyl acetate in methanol [211.
Light microscopy The processes of chromatin dispersion and recondensation were followed in individual nuclei using a • 100 oil immersion objective and Nomarski interferencecontrast optics on a Zeiss Photomicroscope. A suspension of unfixed nuclei in saline was mounted beneath a coverslip on a slide coated with a thin film of Tissue-Tac adhesive. The adhesive prevented many nuclei from moving during subsequent manipulation. A drop of distilled water was then placed at one edge of the coverglass and drawn under with absorbent paper placed at the opposite edge. When dispersion of chromatin was observed to be complete saline was again drawn under the coverslip in the same manner. The same nucleus was continuously observed and periodically photographed during these procedures.
D NA and protein determinations Nuclei were fractionated into D N A and protein components as described previously [11]. D N A was measured quantitatively by the diphenylamine reaction using calf thymus D N A as standard [22]. Protein was determined by the method of Lowry et al. [231.
Measurement of template activity DNA-dependent R N A polymerase (FVI, post DEAE) was prepared from spray-dried M. lysodeikticus cells by the method of Nakamoto et al. [24]. Template activities of nuclear suspensions and erythrocyte D N A were determined as previously described [11]. Each 0.25 ml of reaction mixture contained: 50 ktg of D N A template; 6.0 units of R N A polymerase; 0.2 /~moles each of ATP, CTP, GTP and 5-3H-UTP (0.6 /~Ci/0.2 /~M UTP); 0.4 /~moles spermidine phosphate; 1.25/~moles MnCI~; 50/~moles Tris-HC1, pH 7.5. In control experiments either R N A polymerase or MnClz was omitted from the assay mixture. Incubation was carried out at 30~ for 10 min. The reaction was then stopped and the acid-insoluble precipitate collected on nitrocellulose membrane filters, dried and counted at 23~ in 15 ml of scintillation fluid (6 g, 2,5-diphenyl-oxazole/l toluene) in a Beckman LS-250 counter. A quench curve, prepared to relate counting efficiency to an external standard ratio was used to estimate disintegrations/min and to convert amounts of U M P incorporated into nmoles. After the TCA-insoluble material on each filter was counted the D N A was extracted with hot perchloric acid and its concentration determined by diphenylamine [22].
Structure and template activity of nuclei Purification of R N A synthesized in vitro For purification of labelled RNA synthesized in vitro a reaction mixture 20 times that employed for template assays was used. Synthesis was carried out for 30 min at 30~ and was stopped by addition of 10 ml of solution containing 0.02 M sodium acetate (pH 4.5), 0.15 M NaC1 and 0.1% SDS and an equal volume of aqueous-phenol. The mixture was phenol extracted three times at 23~ followed by three extractions with diethyl ether. RNA was precipitated from solution by addition of 2 vol of pre-filtered ethanol and stored at - 2 0 ~ for at least 2 h. The precipitates were collected at 30 000 g for 20 min, dissolved in 2 x SSC (0.30 M NaC1, 0.03 M sodium citrate) and dialyzed against the same for 24 h.
Electrophoretic separation of R N A RNA synthesized in vitro with nuclear suspensions was fractionated by electrophoresis using gels (0.52 cm diam. x8.5 cm) of 7% polyacrylamide crosslinked with bisacrylamide [25]. Gels were scanned at 260 nm using a Gilford spectrophotometer equipped with a Model 2410 Linear Transport. For determination of radioactivity gels were frozen on dry ice and cut into 1 mm thick slices. Slices were digested for 1 h at 23~ in 0.1 ml NCS before counting in 15 ml of scintillation fluid.
D N A - R N A hybridization ,-,,~"~'TAfrom mature erythrocytes was aikaiine denatured and immobilized on 25 mm nitrocellulose membrane filters [26]. Adsorption to filters was 89-92 % as judged from measurement of UV absorption before and after filtration. Hybridization was by the formamide technique [27, 28] in screw cap vials containing a solution of 2 x SSC, 30 % formamide and various amounts of 8H-RNA to which were added 1 or 2 filters with approx. 1.5/zg erythrocyte DNA per filter and 1 or 2 filters containing 5 #g E. eoli DNA. Final volume was 1 ml and incubation was for 30 h at 37~ For counting, the filters were washed before and after RNase treatment [26] and a 17 mm diameter center portion of each was removed with a stainless steel cutting tube.
RESULTS AND DISCUSSION
Microscopical observations T h e electron m i c r o g r a p h s , figs 1-6, show t h a t the o r g a n i z a t i o n of the c h r o m a t i n of the e r y t h r o c y t e nuclei was strikingly sensitive to salt c o n c e n t r a t i o n . The c h r o m a t i n of freshly isolated nuclei was c o m p o s e d of fibrils 150-250 A in width, m a i n l y c o n d e n s e d in large masses which were d i s t r i b u t e d t h r o u g h the nucleus with some preferential
63
a s s o c i a t i o n with the nuclear m e m b r a n e (figs 1, 2). This d i s t r i b u t i o n of c h r o m a t i n is essentially similar to t h a t f o u n d in nuclei of i n t a c t a v i a n e r y t h r o c y t e s [7, 8]. Owing to t h e thinness of the sections it c a n n o t be established if all of the c h r o m a t i n masses are a s s o c i a t e d with the m e m b r a n e . W h e n the nuclei were p l a c e d in w a t e r a m a r k e d change in b o t h gross o r g a n i z a t i o n a n d fine structure t o o k place (figs 3, 4). T h e c o n d e n s e d c h r o m a tin masses dispersed to give a m o r e o r less u n i f o r m d i s t r i b u t i o n of fibrils a n d the w i d t h of i n d i v i d u a l fibrils was s h a r p l y r e d u c e d to 50-100 A. N e a r the p e r i p h e r y of the nucleus the fibrils showed a r a d i a l o r i e n t a t i o n a n d were associated w i t h the m e m b r a n e . The n u c l e a r m e m b r a n e itself was relatively m o r e distinct a n d was often p a r t i a l l y b r o k e n (fig. 3). N u c l e a r pores were extended to a w i d t h of 2 000-3 000 A f r o m a n original 5 0 0 - I 500 A . W h e n w a t e r - t r e a t e d nuclei were r e t u r n e d to saline these changes were largely reversed (figs 5, 6); c o n d e n s e d c h r o m a t i n masses, with the s a m e general d i s t r i b u t i o n as in the original nuclei, r e a p p e a r e d a n d the d i a m e t e r of i n d i v i d u a l fibrils a g a i n fell in the 150-250 A range. These o b s e r v a t i o n s c o n f i r m previous rep o r t s [13-17] t h a t e x p o s u r e to low c a t i o n c o n c e n t r a t i o n induces dispersal of cond e n s e d c h r o m a t i n in isolated nuclei a n d that this c h a n g e is reversible. Since mere h y p o t o n i c e x p o s u r e does n o t significantly c h a n g e the m a c r o m o l e c u l a r c o m p o s i t i o n of the c h r o m a t i n (table 1) it a p p e a r s that such ionm e d i a t e d c o n f i g u r a t i o n a l changes of c h r o m a tin d e p e n d p r i m a r i l y on m o d i f i c a t i o n of charge interactions. The general c o n d e n s a tion in response to ion c o n c e n t r a t i o n does, however, have a degree of specificity which m u s t be d e t e r m i n e d b y the c o m p o s i t i o n of the c h r o m a t i n . C o m p a r i s o n of figs 1 a n d 7 clearly shows that at the s a m e salt c o n c e n t r a tion, the chicken liver nucleus has far less
Exptl CellRes 65
64
K. Brasch et aL
Figs 1-7 are electron micrographs of thin sections. Scale lines represent 1 fire. Fig. 1. Isolated chicken erythrocyte nucleus suspended and fixed in saline, x 32 000. Fig. 2. Higher magnification view of a portion of a saline-suspended nucleus showing ehromatin fibrils with a width of 150-250 A. x 72 000.
E.~:ptl Cell Res 65
Structure and template activity of nuclei 65
Fig. 3. Nucleus suspended and fixed in water. Note that the nuclear membrane has ruptured (R). x 30 000. Fig. 4. Higher magnification view of a portion of a water-suspended nucleus showing chromatin fibrils with diameter 50-100 .~. x 72 000.
5 - 711804
Exptl Cell Res 65
66
K. Brasch et al.
Fig. 5. Nucleus resuspended and fixed in saline after suspension in water, x 30 000. Fig. 6. Higher magnification view of a portion of a nucleus resuspended in saline. Note reeondensed chromatin fibrils with a width similar to that of fibrils in fig. 2. x 72 000.
Exptl Cell Res 65
Structure and template activity of nuclei
67
Fig. 7. Isolated chicken liver nucleus suspended and fixed in saline. Compare with fig. 1. • 20 000.
condensed chromatin than the chicken erythrocyte nucleus. The reversible change in diameter of the chromatin fibrils in response to salt concentration lends support to the view advanced by several authors [8, 29, 30] that the commonly observed cbromatin fibril with a diameter of 150-250 A is formed of a supercoiled deoxyribonucleoprotein fibril of lesser diameter. As with changes in gross configuration, transition between supercoiled and uncoiled states of individual fibrils appears to depend in part on conditions of electrostatic charge in the chromatin. Light microscope views of individual nuclei before, during and after water treatment are presented in fig. 8. The freshly prepared nuclei in saline showed complex internal structure resulting from chromatin condensation. As expected from the electron microscope observations suspension of the
nuclei in water caused swelling and a complete disappearance of internal structure. Most interestingly, however, when the nuclei were returned to saline, recondensation of the chromatin gave almost identical internal structure to that present before water treatment. In addition to the six specific examples in fig. 8, similar results were observed visually with m a n y more nuclei. Within the limits of resolution of the technique employed, this observation clearly indicates that the ion-mediated condensation Table 1. Protein/DNA ratios chicken erythrocyte nuclei
of mature
Pretreatment of nuclei
Protein/DNA (duplicate samples)
0.15 M NaC1 Distilled water 0.15 M NaCI after water
1.03 0.87 1.38
0.99 0.94 0.87 Exptl Cell Res 65
68
K. Brasch et al.
Fig. 8. Photomicrographs of six individual erythrocyte nuclei as viewed by Nomarski interference-contrast optics. Each horizontal series of three micrographs represents the same nucleus: in saline (left), in water (center); and resuspended in saline (right). Magnification in all micrographs x 5 000. Exptl Cell Res 65
Structure and template activity of nuclei of chromatin in erythrocyte nuclei is not merely random aggregation but rather, is a specific folding and packing which gives rise to an ordered interphase structure. Although the condensed masses of chromatin in these interphase nuclei do not appear the same as condensed mitotic chromosomes the two types of condensation both have a high degree of specificity and might depend on similar basic mechanisms. Several authors have commented on this possibility [8, 13, 31]. Presumably interactions between proteins and D N A in the chromatin are fundamentally responsible for the specific configurations but the association of the chromatin with the nuclear membrane seen in both condensed and dispersed conditions (figs 2, 4) may also be important in ordering the interphase structure. In this regard it is interesting to note that often in the course of the light microscope studies nuclei were seen to partially rupture as water was added. When such nuclei were recondensed in saline the original structural configuration of the chromatin was not recognizable. Davies [8] has shown that chromatin of avian erythrocyte nuclei is packed in highly ordered arrays at the nuclear membrane and Davies & Tooze [32] have argued that membrane association might be important in both interphasic and mitotic condensation of chromatin. The finding of specificity of interphasic structure in the erythrocyte nuclei is not necessarily unexpected. Striking species-specific differences in the organization of interphase chromatin are found in plants and in the so-called prochromosal plant nuclei the number and position of distinct heterochromatic bodies is quite characteristic of a given species [1]. In animal cells, regularly occurring heterochromatic structures such as the Barr body of human epithelial cells [33] and the chromocenter of Drosophila
69
Table 2. Template activity of mature erythrocyte nuclei and pure DNA using M. lysodeikticus DNA-dependent RNA polymerase Pmoles [SH]-UMPincorporated/#g DNA/6.0 units RNA polymerase Pre-treatment of nuclei 0.15 M NaC1 Distilled water 0.15 M NaC1 after water Erythrocyte DNA in 0.15 M NaC1
Pre-incubation No prefor 2 mina incubation 0.23 1.25
0.26 0.65
1.05
0.92
99.20
100.30
a Nuclei were pre-incubated with 6.0 units RNA polymerase for 2 min at 30~ before the addition of reaction mixture and MnC12respectively.
salivary gland nuclei [34] reflect specific structural organization within interphase nuclei. Comings [35] has reviewed the evidence concerning ordered arrangement of chromosomes in interphase nuclei and concludes that such ordered arrangements probably occur generally and result in part from specific attachment of chromatin to nuclear membranes. Our observations seem to lend direct support to these suggestions. Thus, although it is not yet possible to examine directly the large scale organization of most interphase nuclei it seems quite possible that all nuclei might have a specifically ordered arrangement of condensed and dispersed chromatin derived from different chromosomes. The chromatin might then be viewed as analagous to a protein molecule with tertiary and quaternary structure. Specific folding and packing of chromatin fibrils might bring genes spaced in different parts of the genome into close physical proximity to allow for correlated function, just as folding of polypeptides allows widely spaced amino acids to function together in active centers.
Exptl CellRes 65
70
K. Brasch et al. 4s
IO O "
I\ 1.2,
80'
60--
O.8'
40' 0.4
20"
O
,~
'
3g
'
5g
' 7~
30
50
70
I 90
lO Fig. 9. Abscissa: distance in mm; ordinate: A~60. Polyacrylamide gel electrophoresis of RNA synthesized in vitro from nuclei suspended in saline (---), water ( ) and saline following water (-.-). Fig. 10. Abscissa: % labelled RNA removed from filters; ordinate: incubation temperature. Stability of DNA-RNA hybrids formed by incubating filters with bound DNA in ~H-RNA synthesized in vitro from mature erythrocyte nuclei (9 and purified DNA (e). Filters were washed in 2 • SSC and RNase treated before heating in 0.5 ml of 2 • SSC. Both filters and washes were counted.
tion was measured (table 2). The recondensed nuclei also showed low activity. In addition to the lack of major quantitative response there was also no measurable difference in the qualitative characteristics of the small a m o u n t of R N A which was synthesized by the condensed and dispersed nuclei. W h e n R N A synthesis was allowed to proceed for long intervals (ca 30 rain) approximately the same a m o u n t of polyribonucleotides with the same specific activities were synthesized. These polymers were similar in molecular weight distribution as indicated by polyacrylamide gel electrophoresis (fig. 9) and readily formed stable R N A - D N A hybrids which exhibited thermal dissociation profiles similar to that obtained for R N A synthesized f r o m deproteinized erythrocyte D N A (fig. 10). W h e n presaturation hybridization was carried out as outlined in table 3, where D N A filters were first incubated with unlabelled R N A synthesized f r o m fresh
R N A synthesis Freshly prepared nuclei in saline incubated with heterologous D N A - d e p e n d e n t R N A polymerase showed m a r k e d restriction in template activity in comparison to deproteinized erythrocyte D N A (table 2). This result is very similar to that obtained using isolated chromatin rather than intact nuclei as template [11, 12] and no d o u b t reflects the lack of R N A synthesis in mature nucleated erythrocytes [2, 6, 10]. C h r o m a t i n f r o m chicken liver and kidney nuclei, i.e. nuclei which have an in vivo capability for R N A synthesis, shows considerably greater template activity than erythrocyte c h r o m a t i n in an in vitro system similar to that used here [11]. Despite the marked structural changes in the chromatin which t o o k place when the nuclei were suspended in water (above) there was no correlated m a j o r change in template activity; only a small increase in transcrip-
Exptl Cell Res 65
Table 3. Comparison of R N A synthesized in vitro from saline- and water-treated erythrocyte nuclei by pre-saturation hybridization
Expt no.
Input 3H-RNA from water (/~g/ml) nuclei a
3H-RNA hybridized (dpm//~gDNA) Pre-incubated with unlabeled RNA from saline nuclei b
1
10.5
--
2 3 4 5
26.3 43.7 94.5 140.0
0 2 4 2
Controlc 12 28 50 72 156
a Spec. act. 12 400 dpm//~g. b Hybridization conditions as described in Methods. DNA filters were preincubated with 150/~g/ml unlabelled RNA synthesized from saline-suspended nuclei for 30 h before washing with hybridization medium and a further 30 h incubation with labelled RNA synthesized from water-suspended nuclei. e DNA filters were preincubated with hybridization medium only for the first 30 h and then incubated with labelled RNA synthesized from water-suspended nuclei.
Structure and template activity of nuclei 71 nuclei in saline and then incubated with labelled R N A synthesized from nuclei in water, little or no labelled hybrid was detected. Furthermore, the R N A synthesized from all three types of nuclei (normalcondensed, water-dispersed and recondensed) hybridized with less than 2 % of the D N A while R N A synthesized from purified D N A hybridized with approx. 20 % of the DNA. These results suggest that a very large proportion, if not all of the quantitatively small amount of RNA transcribed from the condensed and dispersed nuclei was qualitatively similar. The small increase in transcription activity which was observed with water-dispersed and recondensed nuclei (table 2) might be best explained through reduction in interference by nuclear and ghost cytoplasmic membranes with access of the RNA-polymerase to the chromatin. As noted in the structural studies above, water treatment often caused rupturing of the nuclear membrane and release of chromatin. Such chromatin might be more available to the polymerase in both the water-dispersed and recondensed preparations. In order to minimize the effect of chromatin condensation which might result from the higher ionic strength of the reaction mixture used to measure template activity the nuclei were preincubated with RNA-polymerase prior to adding the reaction mixture. This procedure had no major effect on transcription but gave a small increase which might again be explained by better accessibility of the little functional template which was available to RNA-polymerase. The main significance of these in vitro transcriptional studies is to indicate that mere loss of structural condensation, in the mature erythrocyte system at least, need not have a major effect on the restricted template properties of the chromatin. This conclusion
is consistent with an earlier study [11] which showed that differences in such physical properties as solubility and viscosity could not account for differences in template activity of chicken liver, kidney and erythrocyte chromatin. This in turn suggests the possibility that both the progressive condensation of chromatin observed in vivo during erythrocyte maturation [2, 6] and the decondensation which occurs in heterokaryons [36] may be secondary processes which are associated with, but not directly responsible for, depression or stimulation of R N A synthesis. The fact that R N A synthesis in maturing nucleated red blood cells ceases before full condensation of the chromatin takes place [2, 6] is a natural expectation from such a situation. Presumably other processes, which may or may not be causally related to chromatin condensation, and which involve molecular changes within the chromatin to make D N A available for template purposes, must be involved in regulating gene transcription. It has been shown that the restriction in template activity of intact erythrocyte chromatin can be eliminated in a stepwise fashion by extraction of the nuclei with either acid [12] or salt [7]. Of particular interest is the marked increase in template activity that occurs after removal of the erythrocyte specific "serine rich" histone by low p H [12]. Parallel electron microscopic studies indicate that removal of this histone fraction causes a loosening of the condensed chromatin fibrils [38]. In at least some cell types, however, gene activity in vivo is not associated with major changes in the qualitative or quantitative content of histone proteins in the chromosomes [39, 40]. Presumably, therefore, normal derepression and repression of D N A in living cells must involve as yet unresolved changes in DNA-protein interactions within the chromatin complex. Exptl Cell Res 65
72
K . B r a s c h e t al.
We thank Dr A. T. Matheson, Dr J. M. Neelin and Dr K. R. Shelton for technical assistance and advice. K. Brasch held a Province of Ontario Graduate Fellowship and V. L. Seligy an N.R.C. Post-Doctoral Fellowship. This research was supported in part by Operating and Capital grants awarded by the National Research Council of Canada. REFERENCES I. Lafontaine, J G, The nucleus (ed A J Dalton & F Haguenau) vol. 3, p. 152. Academic Press, New York (1968). 2. Grasso, J A & Woodard, J W, J cell biol 31 (1966) 279. 3. Frenster, J H, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 50 (1963) 1026. 4. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc nat/acad sci US 52 (1964) 93. 5. Allfrey, V G, Aspects of protein biosynthesis (ed C Anfinsen jr), vol 1, p. 247. Academic Press, New York (1970). 6. Cameron, I L & Prescott, D M, Exptl cell res 30 (1963) 609. 7. Davies, H G, J biophys biochem cytol 9 (1961) 671. 8. - - J cell sci 3 (1968) 129. 9. Zentgraf, H, Deumling, B & Franke, W W, Exptl cell res 56 (1969) 333. 10. Dingman, C W & Sporn, M B, J biol chem 239 (1964) 3483. 11. Seligy, V L & Miyagi, M, Exptl cell res 58 (1969) 27. 12. Seligy, V L & Neelin, J M, Biochim biophys acta 213 (1970) 380. 13. Ris, H & Mirsky, A E, J gen physiol 32 (1949) 489. 14. Anderson, N G & Wilbur, K M, J gen physiol 35 (1952) 781. 15. Barnicott, N A & Huxley, H E, Quart j microscop sci 106 (1965) 197.
Exptl Cell Res 65
16. Robbins, E, Pederson, T & Klein, P, J cell biol 44 (1970) 400. 17. Crawley, J C W & Harris, H, Exptl cell res 31 (1963) 70. 18. Lezzi, M & Gilbert, L I, J cell sci 6 (1970) 615. 19. Purkayastha, R & Neelin, J M, Biochim biophys acta 117 (1966) 468. 20. Mollenhauer, H H, Stain technol 39 (1964) 111. 21. Stempak, J & Ward, R, J cell biol 22 (1964) 697. 22. Burton, K, Biochem j 62 (1956) 315. 23. Lowry, O H, Rosebrough, N J, Farr, N A L & Randall, R J, J biol chem 193 (1951) 265. 24. Nakamoto, T, Fox, C F & Weiss, S B, J biol chem 239 (1964) 167. 25. Birnboim, H C, Anal biochem 29 (1969) 498. 26. Gillespie, D & Spiegelman, S, J tool biol 12 (1965) 829. 27. Bonner, J, Knng, G & Bekhor, I, Biochemistry 6 (1967) 3650. 28. Tan, C H & Miyagi, M, J mol biol 50 (1970) 641. 29. DuPraw, E J, Cell and molecular biology. Academic Press, New York (1969). 30. Pardon, J F, Wilkins, M H F & Richards, B M, Nature 215 (1967) 508. 31. Tooze, J & Davies, H G, J cell biol 16 (1963) 501. 32. Davies, H G & Tooze, J, J cell sci 1 (1966) 331. 33. Mittwoch, U, J reed genet 1 (1964) 50. 34. Swanson, C P, Merz, T & Young, W J, Cytogenetics. Prentice-Hall, Englewood Cliffs, N.J. (1967). 35. Comings, D E, A m j human genet 20 (1968) 440. 36. Harris, H, Nucleus and cytoplasm. Oxford University Press, Oxford (1968). 37. Scligy, V L & Miyagi, M. In preparation. 38. Brasch, K, Setterfield, G & Neelin, J M. In preparation. 39. Gorovsky, M A & Woodard, J, J cell bio133 (1967) 723. 40. Comings, D E, J cell biol 35 (1967) 699.
Received August 24, 1970
EFFECTS OF LOW SALT CONCENTRATION ON STRUCTURAL ORGANIZATION AND TEMPLATE ACTIVITY OF CHROMATIN IN CHICKEN ERYTHROCYTE NUCLEI K. BRASCH, V. L. SELIGY and G. SETTERFIELD
Department of Biology, Carleton University, and The Biochemistry Laboratory, National Research Council, Ottawa, Canada
SUMMARY Isolated chicken erythrocyte nuclei were suspended in saline, water, or saline following water, and studied both microscopically and for capacity to support R N A synthesis in vitro. Treatment in water caused complete dispersal of condensed chromatin and a reduction in width of individual fibrils from ca 200 ~ to ca 75 A; both changes were reversed by returning nuclei to saline. Studies of individual nuclei with Nomarski interference-contrast optics demonstrated that the gross configuration of condensed chromatin which reformed in saline after water dispersal was structurally identical with that present before dispersal. Nuclei in saline had very restricted capacity for R N A synthesis and no major change took place when chromatin was dispersed by water. The small amount of R N A synthesized by condensed and dispersed nuclei appeared qualitatively identical by gel electrophoresis and presaturation hybridization with erythrocyte DNA. It is concluded that the chromatin of these interphase nuclei has a specific, ordered structure which is probably determined both by the chromatin composition and interaction between chromatin and nuclear membrane. Ion-mediated condensation and dispersal of chromatin does not appear to directly regulate the capacity of the D N A to support R N A synthesis.
Eukaryotic interphase nuclei contain varying amounts of condensed and dispersed chromatin. The distribution of chromatin between the two states varies in different species [1] and in different nuclei within the organs of the same species [2]. Several studies have indicated that in intact nuclei RNA synthesis is largely associated with dispersed chromatin [3, 4, 5] and that increased condensation is correlated with restriction of genetic transcription [2, 6]. Two questions concerning this situation are as yet incompletely answered. First: is the formation of condensed chromatin a relatively unspecific type of aggregation or does each interphase nucleus have a precise internal structure resulting from specific folding and packing of the two
chromatin phases? Second: does the process of chromatin condensation directly control gene transcription or is it merely a secondary phenomenon which proceeds in conjunction with restriction of template capability of the chromatin at the molecular level? Mature chicken erythrocyte nuclei, which have almost completely condensed chromatin [7, 8, 9] and severely restricted transcription capacity [10-12], present an opportunity to obtain evidence bearing on these questions. Several authors have shown, using both light and electron microscopes, that the state of condensation of chromatin in isolated nuclei is sensitive to salt concentration: hypotonic conditions cause swelling and Exptl Cell Res 65
62
K. Brasch et al.
dispersal
of
solutions
produce
chromatin.
chromatin
while
hypertonic
abnormally
condensed
T h e c h a n g e s a r e in p a r t r e v e r -
sible [13-18]. T h i s p a p e r p r e s e n t s t h e r e s u l t s of e x p e r i m e n t s
designed to follow changes
in b o t h the specific s t r u c t u r a l c o n f i g u r a t i o n and
t e m p l a t e a c t i v i t y of t h e c h r o m a t i n
of
e r y t h r o c y t e nuclei in t h e n o r m a l c o n d e n s e d state in p h y s i o l o g i c a l saline, c o m p l e t e l y dispersed
in dilute salt s o l u t i o n
recondensed
(water)
and
in saline a f t e r h y p o t o n i c
dis-
persal.
MATERIALS
AND METHODS
Sources Spray-dried Microcoeeus lysodeiktieus ceils were purchased from Miles Chemical Corp. Chemicals were obtained as follows: Dade Tissue Tac, Canadian Laboratory Supplies; calf thymus D N A and RNase A (type RAF), Worthington Biochemical Corp.; 5-3H-UTP and Nuclear Chicago Solubilizer (NCS), Amersham/Searle; CTP and GTP, Pabst Laboratories; ATP, protamine phosphate and spermidine phosphate, Sigma Chemical Corp.; formamide, Eastman Kodak. Nitrocellulose membrane filters were Schleicher and Schuell type B-6.
Preparation of nuclei Nuclei were prepared from blood of White Leghorn roosters (5-8 months old, Ottawa Meat Control strain) through lysis of erythrocytes with saponin as described by Neelin and co-workers [12, 19]. The isolated nuclei were freed of hemoglobin by repeated washing in saline (0.15 M NaC1). To prepare nuclei with dispersed chromatin, washed nuclei suspended in saline were sedimented at 1 000 g for 5 min and the supernatant was decanted and replaced by an equal volume of distilled water. Nuclei with recondensed chromatin were produced from these nuclei by sedimenting after 5 min water treatment and resuspending in the same volume of saline. Chicken liver nuclei were prepared by a modification of the method previously described [11], using a medium containing 0.35 M sucrose and 0.15 M NaC1 only. After purification the isolated nuclei were suspended in saline.
Electron microscopy For examination by electron microscopy samples of pelleted nuclei (1 000 g, 5 min) were fixed at 0-4~ as follows: 1.5 % glutaraldehyde, 30 min; 6 % glutaraldehyde, 2 h; three washes in saline or water overnight; 2 % OsO4, 2 h; three washes in saline or water, 15 min each. Glutaraldehyde and OsO4 were dis-
Exptl Cell Res 65
solved in the medium in which the nuclei were initially suspended, i.e. saline or water, and the pH was adjusted to 6.8 with 0.01 N NaOH. Following fixation small clumps of nuclei were dehydrated at 0-4~ in methoxyethanol and ethanol, two changes of 1 h each, and embedded in Epon-Araldite [20]. Sections were stained for 30--40 min in 25 % uranyl acetate in methanol [211.
Light microscopy The processes of chromatin dispersion and recondensation were followed in individual nuclei using a • 100 oil immersion objective and Nomarski interferencecontrast optics on a Zeiss Photomicroscope. A suspension of unfixed nuclei in saline was mounted beneath a coverslip on a slide coated with a thin film of Tissue-Tac adhesive. The adhesive prevented many nuclei from moving during subsequent manipulation. A drop of distilled water was then placed at one edge of the coverglass and drawn under with absorbent paper placed at the opposite edge. When dispersion of chromatin was observed to be complete saline was again drawn under the coverslip in the same manner. The same nucleus was continuously observed and periodically photographed during these procedures.
D NA and protein determinations Nuclei were fractionated into D N A and protein components as described previously [11]. D N A was measured quantitatively by the diphenylamine reaction using calf thymus D N A as standard [22]. Protein was determined by the method of Lowry et al. [231.
Measurement of template activity DNA-dependent R N A polymerase (FVI, post DEAE) was prepared from spray-dried M. lysodeikticus cells by the method of Nakamoto et al. [24]. Template activities of nuclear suspensions and erythrocyte D N A were determined as previously described [11]. Each 0.25 ml of reaction mixture contained: 50 ktg of D N A template; 6.0 units of R N A polymerase; 0.2 /~moles each of ATP, CTP, GTP and 5-3H-UTP (0.6 /~Ci/0.2 /~M UTP); 0.4 /~moles spermidine phosphate; 1.25/~moles MnCI~; 50/~moles Tris-HC1, pH 7.5. In control experiments either R N A polymerase or MnClz was omitted from the assay mixture. Incubation was carried out at 30~ for 10 min. The reaction was then stopped and the acid-insoluble precipitate collected on nitrocellulose membrane filters, dried and counted at 23~ in 15 ml of scintillation fluid (6 g, 2,5-diphenyl-oxazole/l toluene) in a Beckman LS-250 counter. A quench curve, prepared to relate counting efficiency to an external standard ratio was used to estimate disintegrations/min and to convert amounts of U M P incorporated into nmoles. After the TCA-insoluble material on each filter was counted the D N A was extracted with hot perchloric acid and its concentration determined by diphenylamine [22].
Structure and template activity of nuclei Purification of R N A synthesized in vitro For purification of labelled RNA synthesized in vitro a reaction mixture 20 times that employed for template assays was used. Synthesis was carried out for 30 min at 30~ and was stopped by addition of 10 ml of solution containing 0.02 M sodium acetate (pH 4.5), 0.15 M NaC1 and 0.1% SDS and an equal volume of aqueous-phenol. The mixture was phenol extracted three times at 23~ followed by three extractions with diethyl ether. RNA was precipitated from solution by addition of 2 vol of pre-filtered ethanol and stored at - 2 0 ~ for at least 2 h. The precipitates were collected at 30 000 g for 20 min, dissolved in 2 x SSC (0.30 M NaC1, 0.03 M sodium citrate) and dialyzed against the same for 24 h.
Electrophoretic separation of R N A RNA synthesized in vitro with nuclear suspensions was fractionated by electrophoresis using gels (0.52 cm diam. x8.5 cm) of 7% polyacrylamide crosslinked with bisacrylamide [25]. Gels were scanned at 260 nm using a Gilford spectrophotometer equipped with a Model 2410 Linear Transport. For determination of radioactivity gels were frozen on dry ice and cut into 1 mm thick slices. Slices were digested for 1 h at 23~ in 0.1 ml NCS before counting in 15 ml of scintillation fluid.
D N A - R N A hybridization ,-,,~"~'TAfrom mature erythrocytes was aikaiine denatured and immobilized on 25 mm nitrocellulose membrane filters [26]. Adsorption to filters was 89-92 % as judged from measurement of UV absorption before and after filtration. Hybridization was by the formamide technique [27, 28] in screw cap vials containing a solution of 2 x SSC, 30 % formamide and various amounts of 8H-RNA to which were added 1 or 2 filters with approx. 1.5/zg erythrocyte DNA per filter and 1 or 2 filters containing 5 #g E. eoli DNA. Final volume was 1 ml and incubation was for 30 h at 37~ For counting, the filters were washed before and after RNase treatment [26] and a 17 mm diameter center portion of each was removed with a stainless steel cutting tube.
RESULTS AND DISCUSSION
Microscopical observations T h e electron m i c r o g r a p h s , figs 1-6, show t h a t the o r g a n i z a t i o n of the c h r o m a t i n of the e r y t h r o c y t e nuclei was strikingly sensitive to salt c o n c e n t r a t i o n . The c h r o m a t i n of freshly isolated nuclei was c o m p o s e d of fibrils 150-250 A in width, m a i n l y c o n d e n s e d in large masses which were d i s t r i b u t e d t h r o u g h the nucleus with some preferential
63
a s s o c i a t i o n with the nuclear m e m b r a n e (figs 1, 2). This d i s t r i b u t i o n of c h r o m a t i n is essentially similar to t h a t f o u n d in nuclei of i n t a c t a v i a n e r y t h r o c y t e s [7, 8]. Owing to t h e thinness of the sections it c a n n o t be established if all of the c h r o m a t i n masses are a s s o c i a t e d with the m e m b r a n e . W h e n the nuclei were p l a c e d in w a t e r a m a r k e d change in b o t h gross o r g a n i z a t i o n a n d fine structure t o o k place (figs 3, 4). T h e c o n d e n s e d c h r o m a tin masses dispersed to give a m o r e o r less u n i f o r m d i s t r i b u t i o n of fibrils a n d the w i d t h of i n d i v i d u a l fibrils was s h a r p l y r e d u c e d to 50-100 A. N e a r the p e r i p h e r y of the nucleus the fibrils showed a r a d i a l o r i e n t a t i o n a n d were associated w i t h the m e m b r a n e . The n u c l e a r m e m b r a n e itself was relatively m o r e distinct a n d was often p a r t i a l l y b r o k e n (fig. 3). N u c l e a r pores were extended to a w i d t h of 2 000-3 000 A f r o m a n original 5 0 0 - I 500 A . W h e n w a t e r - t r e a t e d nuclei were r e t u r n e d to saline these changes were largely reversed (figs 5, 6); c o n d e n s e d c h r o m a t i n masses, with the s a m e general d i s t r i b u t i o n as in the original nuclei, r e a p p e a r e d a n d the d i a m e t e r of i n d i v i d u a l fibrils a g a i n fell in the 150-250 A range. These o b s e r v a t i o n s c o n f i r m previous rep o r t s [13-17] t h a t e x p o s u r e to low c a t i o n c o n c e n t r a t i o n induces dispersal of cond e n s e d c h r o m a t i n in isolated nuclei a n d that this c h a n g e is reversible. Since mere h y p o t o n i c e x p o s u r e does n o t significantly c h a n g e the m a c r o m o l e c u l a r c o m p o s i t i o n of the c h r o m a t i n (table 1) it a p p e a r s that such ionm e d i a t e d c o n f i g u r a t i o n a l changes of c h r o m a tin d e p e n d p r i m a r i l y on m o d i f i c a t i o n of charge interactions. The general c o n d e n s a tion in response to ion c o n c e n t r a t i o n does, however, have a degree of specificity which m u s t be d e t e r m i n e d b y the c o m p o s i t i o n of the c h r o m a t i n . C o m p a r i s o n of figs 1 a n d 7 clearly shows that at the s a m e salt c o n c e n t r a tion, the chicken liver nucleus has far less
Exptl CellRes 65
64
K. Brasch et aL
Figs 1-7 are electron micrographs of thin sections. Scale lines represent 1 fire. Fig. 1. Isolated chicken erythrocyte nucleus suspended and fixed in saline, x 32 000. Fig. 2. Higher magnification view of a portion of a saline-suspended nucleus showing ehromatin fibrils with a width of 150-250 A. x 72 000.
E.~:ptl Cell Res 65
Structure and template activity of nuclei 65
Fig. 3. Nucleus suspended and fixed in water. Note that the nuclear membrane has ruptured (R). x 30 000. Fig. 4. Higher magnification view of a portion of a water-suspended nucleus showing chromatin fibrils with diameter 50-100 .~. x 72 000.
5 - 711804
Exptl Cell Res 65
66
K. Brasch et al.
Fig. 5. Nucleus resuspended and fixed in saline after suspension in water, x 30 000. Fig. 6. Higher magnification view of a portion of a nucleus resuspended in saline. Note reeondensed chromatin fibrils with a width similar to that of fibrils in fig. 2. x 72 000.
Exptl Cell Res 65
Structure and template activity of nuclei
67
Fig. 7. Isolated chicken liver nucleus suspended and fixed in saline. Compare with fig. 1. • 20 000.
condensed chromatin than the chicken erythrocyte nucleus. The reversible change in diameter of the chromatin fibrils in response to salt concentration lends support to the view advanced by several authors [8, 29, 30] that the commonly observed cbromatin fibril with a diameter of 150-250 A is formed of a supercoiled deoxyribonucleoprotein fibril of lesser diameter. As with changes in gross configuration, transition between supercoiled and uncoiled states of individual fibrils appears to depend in part on conditions of electrostatic charge in the chromatin. Light microscope views of individual nuclei before, during and after water treatment are presented in fig. 8. The freshly prepared nuclei in saline showed complex internal structure resulting from chromatin condensation. As expected from the electron microscope observations suspension of the
nuclei in water caused swelling and a complete disappearance of internal structure. Most interestingly, however, when the nuclei were returned to saline, recondensation of the chromatin gave almost identical internal structure to that present before water treatment. In addition to the six specific examples in fig. 8, similar results were observed visually with m a n y more nuclei. Within the limits of resolution of the technique employed, this observation clearly indicates that the ion-mediated condensation Table 1. Protein/DNA ratios chicken erythrocyte nuclei
of mature
Pretreatment of nuclei
Protein/DNA (duplicate samples)
0.15 M NaC1 Distilled water 0.15 M NaCI after water
1.03 0.87 1.38
0.99 0.94 0.87 Exptl Cell Res 65
68
K. Brasch et al.
Fig. 8. Photomicrographs of six individual erythrocyte nuclei as viewed by Nomarski interference-contrast optics. Each horizontal series of three micrographs represents the same nucleus: in saline (left), in water (center); and resuspended in saline (right). Magnification in all micrographs x 5 000. Exptl Cell Res 65
Structure and template activity of nuclei of chromatin in erythrocyte nuclei is not merely random aggregation but rather, is a specific folding and packing which gives rise to an ordered interphase structure. Although the condensed masses of chromatin in these interphase nuclei do not appear the same as condensed mitotic chromosomes the two types of condensation both have a high degree of specificity and might depend on similar basic mechanisms. Several authors have commented on this possibility [8, 13, 31]. Presumably interactions between proteins and D N A in the chromatin are fundamentally responsible for the specific configurations but the association of the chromatin with the nuclear membrane seen in both condensed and dispersed conditions (figs 2, 4) may also be important in ordering the interphase structure. In this regard it is interesting to note that often in the course of the light microscope studies nuclei were seen to partially rupture as water was added. When such nuclei were recondensed in saline the original structural configuration of the chromatin was not recognizable. Davies [8] has shown that chromatin of avian erythrocyte nuclei is packed in highly ordered arrays at the nuclear membrane and Davies & Tooze [32] have argued that membrane association might be important in both interphasic and mitotic condensation of chromatin. The finding of specificity of interphasic structure in the erythrocyte nuclei is not necessarily unexpected. Striking species-specific differences in the organization of interphase chromatin are found in plants and in the so-called prochromosal plant nuclei the number and position of distinct heterochromatic bodies is quite characteristic of a given species [1]. In animal cells, regularly occurring heterochromatic structures such as the Barr body of human epithelial cells [33] and the chromocenter of Drosophila
69
Table 2. Template activity of mature erythrocyte nuclei and pure DNA using M. lysodeikticus DNA-dependent RNA polymerase Pmoles [SH]-UMPincorporated/#g DNA/6.0 units RNA polymerase Pre-treatment of nuclei 0.15 M NaC1 Distilled water 0.15 M NaC1 after water Erythrocyte DNA in 0.15 M NaC1
Pre-incubation No prefor 2 mina incubation 0.23 1.25
0.26 0.65
1.05
0.92
99.20
100.30
a Nuclei were pre-incubated with 6.0 units RNA polymerase for 2 min at 30~ before the addition of reaction mixture and MnC12respectively.
salivary gland nuclei [34] reflect specific structural organization within interphase nuclei. Comings [35] has reviewed the evidence concerning ordered arrangement of chromosomes in interphase nuclei and concludes that such ordered arrangements probably occur generally and result in part from specific attachment of chromatin to nuclear membranes. Our observations seem to lend direct support to these suggestions. Thus, although it is not yet possible to examine directly the large scale organization of most interphase nuclei it seems quite possible that all nuclei might have a specifically ordered arrangement of condensed and dispersed chromatin derived from different chromosomes. The chromatin might then be viewed as analagous to a protein molecule with tertiary and quaternary structure. Specific folding and packing of chromatin fibrils might bring genes spaced in different parts of the genome into close physical proximity to allow for correlated function, just as folding of polypeptides allows widely spaced amino acids to function together in active centers.
Exptl CellRes 65
70
K. Brasch et al. 4s
IO O "
I\ 1.2,
80'
60--
O.8'
40' 0.4
20"
O
,~
'
3g
'
5g
' 7~
30
50
70
I 90
lO Fig. 9. Abscissa: distance in mm; ordinate: A~60. Polyacrylamide gel electrophoresis of RNA synthesized in vitro from nuclei suspended in saline (---), water ( ) and saline following water (-.-). Fig. 10. Abscissa: % labelled RNA removed from filters; ordinate: incubation temperature. Stability of DNA-RNA hybrids formed by incubating filters with bound DNA in ~H-RNA synthesized in vitro from mature erythrocyte nuclei (9 and purified DNA (e). Filters were washed in 2 • SSC and RNase treated before heating in 0.5 ml of 2 • SSC. Both filters and washes were counted.
tion was measured (table 2). The recondensed nuclei also showed low activity. In addition to the lack of major quantitative response there was also no measurable difference in the qualitative characteristics of the small a m o u n t of R N A which was synthesized by the condensed and dispersed nuclei. W h e n R N A synthesis was allowed to proceed for long intervals (ca 30 rain) approximately the same a m o u n t of polyribonucleotides with the same specific activities were synthesized. These polymers were similar in molecular weight distribution as indicated by polyacrylamide gel electrophoresis (fig. 9) and readily formed stable R N A - D N A hybrids which exhibited thermal dissociation profiles similar to that obtained for R N A synthesized f r o m deproteinized erythrocyte D N A (fig. 10). W h e n presaturation hybridization was carried out as outlined in table 3, where D N A filters were first incubated with unlabelled R N A synthesized f r o m fresh
R N A synthesis Freshly prepared nuclei in saline incubated with heterologous D N A - d e p e n d e n t R N A polymerase showed m a r k e d restriction in template activity in comparison to deproteinized erythrocyte D N A (table 2). This result is very similar to that obtained using isolated chromatin rather than intact nuclei as template [11, 12] and no d o u b t reflects the lack of R N A synthesis in mature nucleated erythrocytes [2, 6, 10]. C h r o m a t i n f r o m chicken liver and kidney nuclei, i.e. nuclei which have an in vivo capability for R N A synthesis, shows considerably greater template activity than erythrocyte c h r o m a t i n in an in vitro system similar to that used here [11]. Despite the marked structural changes in the chromatin which t o o k place when the nuclei were suspended in water (above) there was no correlated m a j o r change in template activity; only a small increase in transcrip-
Exptl Cell Res 65
Table 3. Comparison of R N A synthesized in vitro from saline- and water-treated erythrocyte nuclei by pre-saturation hybridization
Expt no.
Input 3H-RNA from water (/~g/ml) nuclei a
3H-RNA hybridized (dpm//~gDNA) Pre-incubated with unlabeled RNA from saline nuclei b
1
10.5
--
2 3 4 5
26.3 43.7 94.5 140.0
0 2 4 2
Controlc 12 28 50 72 156
a Spec. act. 12 400 dpm//~g. b Hybridization conditions as described in Methods. DNA filters were preincubated with 150/~g/ml unlabelled RNA synthesized from saline-suspended nuclei for 30 h before washing with hybridization medium and a further 30 h incubation with labelled RNA synthesized from water-suspended nuclei. e DNA filters were preincubated with hybridization medium only for the first 30 h and then incubated with labelled RNA synthesized from water-suspended nuclei.
Structure and template activity of nuclei 71 nuclei in saline and then incubated with labelled R N A synthesized from nuclei in water, little or no labelled hybrid was detected. Furthermore, the R N A synthesized from all three types of nuclei (normalcondensed, water-dispersed and recondensed) hybridized with less than 2 % of the D N A while R N A synthesized from purified D N A hybridized with approx. 20 % of the DNA. These results suggest that a very large proportion, if not all of the quantitatively small amount of RNA transcribed from the condensed and dispersed nuclei was qualitatively similar. The small increase in transcription activity which was observed with water-dispersed and recondensed nuclei (table 2) might be best explained through reduction in interference by nuclear and ghost cytoplasmic membranes with access of the RNA-polymerase to the chromatin. As noted in the structural studies above, water treatment often caused rupturing of the nuclear membrane and release of chromatin. Such chromatin might be more available to the polymerase in both the water-dispersed and recondensed preparations. In order to minimize the effect of chromatin condensation which might result from the higher ionic strength of the reaction mixture used to measure template activity the nuclei were preincubated with RNA-polymerase prior to adding the reaction mixture. This procedure had no major effect on transcription but gave a small increase which might again be explained by better accessibility of the little functional template which was available to RNA-polymerase. The main significance of these in vitro transcriptional studies is to indicate that mere loss of structural condensation, in the mature erythrocyte system at least, need not have a major effect on the restricted template properties of the chromatin. This conclusion
is consistent with an earlier study [11] which showed that differences in such physical properties as solubility and viscosity could not account for differences in template activity of chicken liver, kidney and erythrocyte chromatin. This in turn suggests the possibility that both the progressive condensation of chromatin observed in vivo during erythrocyte maturation [2, 6] and the decondensation which occurs in heterokaryons [36] may be secondary processes which are associated with, but not directly responsible for, depression or stimulation of R N A synthesis. The fact that R N A synthesis in maturing nucleated red blood cells ceases before full condensation of the chromatin takes place [2, 6] is a natural expectation from such a situation. Presumably other processes, which may or may not be causally related to chromatin condensation, and which involve molecular changes within the chromatin to make D N A available for template purposes, must be involved in regulating gene transcription. It has been shown that the restriction in template activity of intact erythrocyte chromatin can be eliminated in a stepwise fashion by extraction of the nuclei with either acid [12] or salt [7]. Of particular interest is the marked increase in template activity that occurs after removal of the erythrocyte specific "serine rich" histone by low p H [12]. Parallel electron microscopic studies indicate that removal of this histone fraction causes a loosening of the condensed chromatin fibrils [38]. In at least some cell types, however, gene activity in vivo is not associated with major changes in the qualitative or quantitative content of histone proteins in the chromosomes [39, 40]. Presumably, therefore, normal derepression and repression of D N A in living cells must involve as yet unresolved changes in DNA-protein interactions within the chromatin complex. Exptl Cell Res 65
72
K . B r a s c h e t al.
We thank Dr A. T. Matheson, Dr J. M. Neelin and Dr K. R. Shelton for technical assistance and advice. K. Brasch held a Province of Ontario Graduate Fellowship and V. L. Seligy an N.R.C. Post-Doctoral Fellowship. This research was supported in part by Operating and Capital grants awarded by the National Research Council of Canada. REFERENCES I. Lafontaine, J G, The nucleus (ed A J Dalton & F Haguenau) vol. 3, p. 152. Academic Press, New York (1968). 2. Grasso, J A & Woodard, J W, J cell biol 31 (1966) 279. 3. Frenster, J H, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 50 (1963) 1026. 4. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc nat/acad sci US 52 (1964) 93. 5. Allfrey, V G, Aspects of protein biosynthesis (ed C Anfinsen jr), vol 1, p. 247. Academic Press, New York (1970). 6. Cameron, I L & Prescott, D M, Exptl cell res 30 (1963) 609. 7. Davies, H G, J biophys biochem cytol 9 (1961) 671. 8. - - J cell sci 3 (1968) 129. 9. Zentgraf, H, Deumling, B & Franke, W W, Exptl cell res 56 (1969) 333. 10. Dingman, C W & Sporn, M B, J biol chem 239 (1964) 3483. 11. Seligy, V L & Miyagi, M, Exptl cell res 58 (1969) 27. 12. Seligy, V L & Neelin, J M, Biochim biophys acta 213 (1970) 380. 13. Ris, H & Mirsky, A E, J gen physiol 32 (1949) 489. 14. Anderson, N G & Wilbur, K M, J gen physiol 35 (1952) 781. 15. Barnicott, N A & Huxley, H E, Quart j microscop sci 106 (1965) 197.
Exptl Cell Res 65
16. Robbins, E, Pederson, T & Klein, P, J cell biol 44 (1970) 400. 17. Crawley, J C W & Harris, H, Exptl cell res 31 (1963) 70. 18. Lezzi, M & Gilbert, L I, J cell sci 6 (1970) 615. 19. Purkayastha, R & Neelin, J M, Biochim biophys acta 117 (1966) 468. 20. Mollenhauer, H H, Stain technol 39 (1964) 111. 21. Stempak, J & Ward, R, J cell biol 22 (1964) 697. 22. Burton, K, Biochem j 62 (1956) 315. 23. Lowry, O H, Rosebrough, N J, Farr, N A L & Randall, R J, J biol chem 193 (1951) 265. 24. Nakamoto, T, Fox, C F & Weiss, S B, J biol chem 239 (1964) 167. 25. Birnboim, H C, Anal biochem 29 (1969) 498. 26. Gillespie, D & Spiegelman, S, J tool biol 12 (1965) 829. 27. Bonner, J, Knng, G & Bekhor, I, Biochemistry 6 (1967) 3650. 28. Tan, C H & Miyagi, M, J mol biol 50 (1970) 641. 29. DuPraw, E J, Cell and molecular biology. Academic Press, New York (1969). 30. Pardon, J F, Wilkins, M H F & Richards, B M, Nature 215 (1967) 508. 31. Tooze, J & Davies, H G, J cell biol 16 (1963) 501. 32. Davies, H G & Tooze, J, J cell sci 1 (1966) 331. 33. Mittwoch, U, J reed genet 1 (1964) 50. 34. Swanson, C P, Merz, T & Young, W J, Cytogenetics. Prentice-Hall, Englewood Cliffs, N.J. (1967). 35. Comings, D E, A m j human genet 20 (1968) 440. 36. Harris, H, Nucleus and cytoplasm. Oxford University Press, Oxford (1968). 37. Scligy, V L & Miyagi, M. In preparation. 38. Brasch, K, Setterfield, G & Neelin, J M. In preparation. 39. Gorovsky, M A & Woodard, J, J cell bio133 (1967) 723. 40. Comings, D E, J cell biol 35 (1967) 699.
Received August 24, 1970