Study of a chromatin domain different from bulk chromatin in barley nuclei

286

Biochimwa et Biophv~ica Acta, 781 (1984) 286 293 Elscvier

BBA 91333

S T U D Y OF A CHROMATIN DOMAIN DIFFERENT FROM BULK CHROMATIN IN BARLEY NUCLEI GILLES MITHIEUX and BERNARD ROUX Laboratoire de Physico-Chirnie Biologique, Universitb Claude Bernard, Lyon 1, 43 Bd du 11 Novernbre 1918, 69622 Villeurbanne Cbdex, (France) (Received January 10th, 1984)

Key words: Chromatin; Nucleosorne," Histone," High-mobility-group protein; (Barley)

A method for fractionation of barley chromatin was developed. A soluble fraction, eluted from micrococcal nuclease-digested nuclei, was recovered and studied in comparison with the low ionic strength soluble bulk chromatin obtained after lysis of nuclei. The eluted fraction contained mainly mononucleosomes with less than 10% dinucleosomes. It was highly micrococcal nuclease-sensitive, totally depleted in the highest molecular mass H1 subspecies, and enriched both in the lowest M r proteins and in other acid-soluble proteins. The soluble bulk chromatin exhibited protein and DNA contents analogous to those of the low ionic strength insoluble fraction. In addition, high-mobility.group proteins purification was carried out. Four species were identified. They were almost exclusively localized in the highly micrococcal nuclease-sensitive fraction.

Introduction Experimental evidence has shown that eukaryotic DNA is packaged in a repetitive structure called the nucleosome, containing approx. 200 basepairs wrapped around a core of histones (for reviews, see Refs. 1-4). Although the length of nucleosomal DNA is variable, ranging from 154 basepairs [5] to 241 basepairs [6], all nucleosomes contain a relatively constant nuclease-resistant core of approx. 140-145 base pairs [5-9]. Different chromatin domains are well known in animal chromatins. Mg2+-soluble chromatin has been shown to be enriched in transcriptionally active nucleosomes [10]. It is now quite evident that both active and inactive regions of the eukaryotic genome are organized in nucleosomes Abbreviations: HMG, high mobility group; Mes; 4-morpholineethanesulfonic acid; E fraction, eluted chromatin fraction; L fraction, lysed chromatin fraction.

[11-17], although transcriptional domains, very susceptible to nuclease attack, presumably exhibit an open conformation [12,15,18-23]. Many authors found that active regions are depleted in histone H I [24,25], while they are enriched in high-mobility-group protein species 1 and 2 [24,26] or 14 and 17 [25,27-30]. H M G 14 and 17 are probably involved in the DNAase I sensitivity of transcriptionally competent chromatin [28,30,31]. It has also been shown that active domains of the genome are enriched in acetylated species of histone H4 [32-35], and depleted in 5-methylcytosine [25]. Others have studied newly replicated chromatin, which has also been shown to be organized into nucleosomal structures [36-38]. The repetitive length of DNA associated with replicating chromatin has been reported to increase during maturation [39-45]. A reason could be that newly replicated chromatin is not yet structured into mature nucleosomes [46-48]. Particularly, the reassociation of histone H1 has been shown to occur

287 later than core histones [49], and it seems that H1 is more weakly bound to chromatin during replication [50]. Other authors think that the shorter repetitive unit could be due to the sliding of nucleosomes close to the replication fork [51]. In contrast to animal chromatins, very little is known concerning active domains of plant chromatins. Four H M G proteins have been identified in wheat embryos [52]. More recently, Spiker et al. [53] have shown that transcriptionally active genes are preferentially DNAase I-sensitive in wheat embryo nuclei. Barley (Hordeum vulgaris) constitutes an interesting model with regard to its high heterogeneity in histone H1 and H2 content [9,54-56], which may be correlated to specific physiological states of development, or to transcriptionally active or dormant states of chromatin. In a previous work [56], we reported that barley nucleosome shows a structure slightly different from that of its animal counterpart and that its nucleosomal fiber exhibits a superstructure containing approximately six nucleosomes. In this paper, we present a convenient method for fractionation of barley chromatin in two soluble domains, exhibiting different properties. Proteins and DNA content of both domains were examined and discussed, particularly H M G proteins. Materials and Methods

Isolation of barley leaves nuclei Nuclei were isolated according to the method of Muller et al. [55] with slight modifications. Young leaves of 7-days-germinated seeds, grown at 25°C, were harvested. 100 g of cut leaves were immersed in 250 ml of grinding medium, stored for 10 min at 4°C, and immediately ground in a Waring blender. All operations were carried out at 4°C. The grinding medium contained 5 mM Mes (pH 6.1) (adjusted with KOH), 4 mM magnesium acetate, 250 mM sucrose, 3% (w/v) gum arabic, 0.35% ( w / v ) T r i t o n X-100, 5 m M 2mercaptoethanol and 1 mM sodium bisulfite. This buffer was centrifuged at 3500 x g for 15 min before use. The resulting suspension was filtered through a calibrated nylon screen (25 # m square mesh). After centrifugation at 1500 x g for 15 rain, the pellet was resuspended in fresh grinding

medium, layered on a 1.2 M sucrose cushion containing 5 mM Mes (pH 6.1), 4 mM magnesium acetate, 5 mM 2-mercaptoethanol, 1 mM sodium bisulfite, and centrifuged at 450 x g for 15 min. The pellet was resuspended in the grinding medium without gum arabic, but containing 1% (w/v) Triton X-100. This suspension was gently agitated at 4°C during 20 min, filtered through a calibration nylon screen (10/~m square mesh) and centrifuged through a second sucrose cushion as described previously.

Fractionation of chromatin Barley chromatin fractionation was performed according to the methods described by Worcel et al. [49] and Noll et al. [57]. 108 nuclei were suspended in 1 ml digestion buffer containing 5 mM Mes (pH 7.0), 1 mM CaC12,250 mM sucrose and 1 mM sodium bisulfite as proteinase inhibitor. The suspension was incubated at 37°C and 10 U of micrococcal nuclease (Sigma) were added. 1 min 30 s later, the reaction was stopped by addition of 100 mM EDTA to a final concentration of 5 mM, and quick-chilled in ground ice. The suspension was then incubated at 4°C for 10 min with gentle agitation. After a 5-rain centrifugation at 1500 x g, the supernatant was removed and dialysed overnight at 4°C against 1 mM sodium bisulfite and 1 mM EDTA (pH 7.5). Pelleted nuclei were lysed in 1 mM sodium bisulfite containing 1 mM EDTA (pH 7.5), with agitation at 4°C for 20 min. After a 5-min centrifugation at 1500 x g, the supernatant was recovered, as well as the pellet.

Fractionation of nucleosome oligomers Micrococcal nuclease-digested chromatin was fractionated on isokinetic sucrose gradients according to Finch et al. [58]. The chromatin solution was layered on isokinetic sucrose gradients containing 1 mM phosphate buffer (pH 7.4), 1 mM EDTA and 1 mM sodium bisulfite. The sucrose concentration increased from 5% (w/v) at the top of the gradient up to 28.2% (w/v) at the bottom. Centrifugation was performed for 19 h at 26 000 rev./min in a SW 27 rotor (Beckman) at 4°C. The fractions were collected with a density gradient fractionator ISCO, and dialysed overnight against 1 mM phosphate (pH 7.4), 1 mM EDTA and 1 mM sodium bisulfite.

288

DNA purification and analysis

H M G purification

The solutions containing soluble chromatin or digested nuclei were adjusted to a final concentration of 3 m M EDTA, 1% ( w / v ) SDS and 1 M NaCI. They were incubated overnight at 37°C in presence of 1 0 0 / x g / m l of proteinase K. The suspension was further deproteinized by four or five extractions with c h r o r o f o r m / i s o a m y l alcohol (24:1, v / v ) . The purified D N A was precipitated by addition of 2 vol. of ethanol at - 2 0 ° C and stored for 48 h at - 2 0 ° C . Precipitated D N A was collected by centrifugation at 10000 x g for 10 min, washed twice with ethanol and dried under vacuum. The analysis on agarose polyacrylamide slab gels was performed as described by Philipps and Gigot [59]. The slab gel (12 cm long) was 2% ( w / v ) polyacrylamide/0.5% ( w / v ) agarose, 0.1% ( w / v ) SDS, 40 m M Tris-HCl (pH 7.8), 20 mM sodium acetate and 1 m M EDTA. D N A was dissolved in 10 m M Tris-HC1 (pH 8.0), 1 m M E D T A and 20% ( w / v ) glycerol. Migration was for 4 h at 80 V in a buffer containing 40 m M Tris-HC1 (pH 7.8), 20 m M sodium acetate and 1 m M EDTA. Gels were stained with ethidium bromide and photographed under ultraviolet illumination using an orange filter.

The procedure was based on the method of Gordon et al. [61] as modified by Spiker et al. [52] for wheat embryo H M G proteins. The suspensions containing nucleoproteins or nuclei were adjusted to a final concentration of 0.35 M NaC1 by adding the appropriate volume of 2 M NaCI. The suspension was agitated at 4°C during 15 min. After a 10 rain centrifugation at 2000 X g, the supernatant was removed and the extraction procedure of the pellet repeated once under the same conditions. Both supernatants were combined and clarified at 100000 x g for 2 h. The supernatant was brought to 5% ( w / v ) trichloroacetic acid with a 50% ( w / v ) solution. The acid-insoluble proteins were sedimented by a 10-min centrifugation at 10000 x g, after 5 rain under mild agitation at 4°C. The resulting supernatant was removed and brought to 20% ( w / v ) trichloroacetic acid. After mild agitation for 5 min at 4°C, it was centrifuged as before. The pelleted H M G proteins were sequentially washed with acetone-HC1 (6:1, v / v ) , twice with acetone and once with diethyloxide, and dried under vacuum.

Histone purification The solutions containing nucleoproteins were adjusted to A260nm ---5 with distilled water and dialysed overnight against 0.4 N H2SO 4. After centrifugation at 20 000 x g for 20 min, the supernatant was removed and dialysed for 48 h against ethanol. The precipitate was collected by centrifugation at 20 000 x g for 20 rain, washed twice with ethanol and dried under vacuum. Histones were extracted from insoluble chromatin using the method of Panyim et al. [60], slightly modified. Pelleted lysed nuclei were suspended in distilled water with agitation at 4°C for 15 min. The solution was further adjusted t o A 2 6 0 n m --- 5 and made 0.4 N HzSO 4, then placed overnight at 4°C with gentle agitation. After a 20 min centrifugation at 20000 x g, the supernatant was removed and dialysed for 48 h against ethanol. Histone were recovered by centrifugation at 20000 x g for 20 min, washed twice with ethanol and dried under vacuum.

Protein analysis Proteins were analysed on polyacrylamide slab gel as described by Laemmli [62]. Stacking gel (1 cm long) was 5% ( w / v ) polyacrylamide, 125 mM Tris-HCl (pH 6.8) and 0.1% ( w / v ) SDS. Resolving gel (18 cm long) was 16% ( w / v ) polyacrylamide, 375 mM Tris-HC1 (pH 8.8) and 0.1% ( w / v ) SDS. Proteins were dissolved in 62.5 m M Tris-HC1 (pH 6.8), 2% ( w / v ) SDS, 5% ( w / v ) 2-mercaptoethanol, 10% ( w / v ) glycerol and heated for 3 min at 100°C before depositing. Migration was for 15 h at 40 mA in buffer containing 25 mM Tris-HCl (pH 8.3), 192 m M glycin and 0.1% ( w / v ) SDS. Gels were stained with Coomassie brilliant blue and destained by diffusion. Scanning gels and integrating peaks were performed using a Vernon PHI 3 photometric densitometer recorder. Results

Fractionation of barley chromatin The method used allowed us to obtain approx. nuclei per kg of fresh young leaves. The mild micrococcal nuclease digestion of them led to two 10 9

289

soluble chromatin fractions. One was eluted from the digested nuclei during a 10-min incubation in ground ice with gentle agitation, in a medium containing 5 mM Mes (pH 7.0), 1 mM CaC12,250 mM sucrose, 1 mM sodium bisulfite and 5 mM EDTA. We called it E (for elution). The other, containing bulk chromatin, was extracted from the lysed nuclei and was soluble at low ionic strength: 1 mM sodium bisulfite and 1 mM EDTA (pH 7.5). We called it L (for lysis). A third fraction remained in the lysis pellet and consisted of the low ionic strength insoluble chromatin. The yield was approx. 5 units of A260n m per 108 nuclei for the E fraction (after dialysis) and 10 units for the L. L oligonucleosomes were fractioned on isokinetic sucrose gradients (Fig. 1). Under these conditions, only three nucleosomal oligomers could be separated from each other: monomer, dimer and trimer. Monomer and dimer fractions were recovered and studied in order to compare them to E and L chromatin. Absorption spectra recording and A260/A280 ratios calculation showed the first difference between the barley chromatin fractions. A26o/A28o ratio was 1.30 + 0.05 for E fraction, 1.45 + 0.05 for L soluble chromatin and 1.60 + 0.05 for L monomer and dimer. The increase i n A26o/A28 o

A 260 nm i

I

I

BOTTOM

ratio from E fraction up to L monomer constitutes a good indication that E chromatin contained a higher proportion of associated proteins than total L chromatin and than L monomer and dimer. D N A analysis Micrococcal nuclease-digested DNA fragments were extracted from E and L fractions, from L monomer and from insoluble chromatin remaining in the lysed nuclei pellet (Fig. 2). L and insoluble chromatin showed approximately the same electrophoretic pattern, indicating that barley nuclei exhibited a typical nucleosomal organization. This proved that micrococcal nuclease was able to digest practically all barley chromatin domains, although insoluble chromatin contained some oligonucleosomes of higher order than those in L. Insoluble chromatin resulted than from an aggregation phenomenon. E fraction contained only a monomer species with less than 10% of dimer. But the most important result concerned the D N A length of both. The comparison to HaelII digested ~X174 DNA allowed us to calculate the mean length of DNA associated to E and L oligonucleosomes (table I). E monomer and dimer DNA length was significantly lower than for the L counterparts: 10 basepairs less for monomer and 40 basepairs less for dimer. This proved that E chromatin was much more micrococcal nuclease-sensitive than L, particularly to the exonucleolytic activity.

a

b

c

d

1.5

TOP 0.5 ~L ~E

I

I

I

I

0

20

40

60

FRACTION NUMBER

Fig. 1. Sedimentation profile of soluble bulk chromatin after micrococcal nuclease digestion.

dlmer dimer

~Lmonomer ~1~, monomer

Fig. 2 . Comparative electrophoretic patterns of DNA fragments extracted from: (a) insoluble chromatin; (b) isolated L monomer; (c) E chromatin; (d) L chromatin.

290 TABLE I

H3

MEAN LENGTHS OF DNA FRAGMENTS ASSOCIATED TO E, L AND INSOLUBLE CHROMATIN

MIGRATION

Values were determined by comparison with Haelll-digested ~pXI74 DNA. Data represent means to within 5 basepairs for three determinations, and are expressed as basepairs.

H4

H1 c

H1B H1A ' START

DNAassociated to

Monomer Dimer Trimer Tetramer Pentamer Hexamer Heptamer

E chromatin

L chromatin

Insoluble chromatin

160 335 -

170 375 570 765 960 1155 1 340

170 380 565 770 960 1150 1350

H2

J.

Protein analysis H i s t o n e were extracted from all fractions of barley chromatin. I n s o l u b l e c h r o m a t i n , L m o n o mer, L dimer a n d total L c h r o m a t i n exhibited exactly the same electrophoretic p a t t e r n (Fig. 3a). Particularly, histone H1 appeared as three subspecies that we called A, B a n d C from the highest to the lowest molecular mass. E c h r o m a t i n (Fig. 3b) was totally depleted in H1A, enriched in H1C, whilst H I B a n d the total percentage of H1 class r e m a i n e d c o n s t a n t (Table II). Variations could not be detected for other histone classes (Table III). F u r t h e r m o r e , a certain n u m b e r of c o n t a m i n a n t acid-soluble proteins were present, c o n s t i t u t i n g approx. 10% of total histones, H M G p r o t e i n extraction was performed as described previously. It allowed us to identify four H M G species: H M G I, H M G II, H M G III and H M G IV. A p p r o x i m a t e relative molecular masses have been d e t e r m i n e d by c o m p a r i s o n with SDS molecular weight markers of Sigma a n d calf t h y m u s histones [63]. The values o b t a i n e d were 22600 for H M G I, 22200 for H M G II, 19800 for H M G III a n d 19 500 for H M G IV. W e noted that our procedure led to H M G proteins c o n t a m i n a t e d by histones H2 a n d H3. By c o m p a r i n g panels b a n d c of Fig. 3 one can see that some acid-soluble proteins which c o n t a m i n a t e d E histone p a t t e r n were p r o b a b l y H M G species. E c h r o m a t i n was very enriched in H M G proteins, particularly H M G III a n d H M G IV (Fig. 3c). As L c h r o m a t i n con-

@

i

HMGIv

HMG]]I

Fig. 3. Comparative electrophoretic scans of proteins associated to different barley chromatin fractions: (a) histones extracted from soluble bulk chromatin; (b) histones extracted from E chromatin; (c) HMG proteins extracted from E chromatin; (d) HMG proteins extracted from barley nuclei depleted in E chromatin. In the case of scans (c) and (d), the entirety of extracted HMG proteins was suspended in 100 FI of suspension buffer and 10/~1 were deposited on the gel.

tained only a very small a m o u n t of H M G , we extracted them from the whole digested nuclei depleted in E fraction (Fig. 3d). F r o m the whole

291

T A B L E II E S T I M A T E D P E R C E N T A G E S OF HISTONE HI SUBF R A C T I O N S C O M P A R E D W I T H T O T A L HISTONES IN E, L A N D I N S O L U B L E C H R O M A T I N The estimation was performed by scanning gels and integrating peaks using a Vernon PH1 3 photometric densitometer. Data are expressed as percent. Chromatin fraction E L Insoluble

HI A

HI B

H1C

4.5 5

6 6 6.5

10.5 5.5 5.5

molecular H M G proteins, 70% of H M G III and H M G IV and 50% of H M G I and H M G II were concentrated in E chromatin, all H M G subspecies being present in approximately the same amounts in the barley nucleus. Three other acid-soluble proteins, whose relative molecular masses ranged from 20 000 to 21000, were also associated with E chromatin (indicated by small arrows in Fig. 3b). Nevertheless, they represented no more than 4% of the total histones. Discussion

The method described in this paper allowed the fractionation of barley chromatin into three different domains: E, L and insoluble chromatin. The E fraction was extensively micrococcal nuclease-digested, and eluted from nuclei, eWe have shown that the length of DNA associated to E monomer and dimer was less than that of DNA associated to counterparts in other fractions. It has been shown

T A B L E II1 E S T I M A T E D P E R C E N T A G E S OF HISTONE CLASSES C O M P A R E D W I T H T O T A L HISTONES IN E, L A N D INSOLUBLE CHROMATIN The estimation was performed by scanning gels and integrating peaks using a Vernon PHI 3 photometric densitometer. Data are expressed as percent. Chromatin fraction

HI

H2

H3

H4

E L Insoluble

16.5 16 17

31.5 32.5 31

27 27 28

25 24.5 24

that 160 basepairs of barley nucleosomal DNA were effectively protected against micrococcal nuclease digestion in the presence of histone H1 [55]. It thus seems reasonable to suppose that the whole linker DNA of E monomers has been digested by the enzyme (160 basepairs is the mean length that we calculated for E monomer DNA). The same assertion can be made concerning the E dimer-associated DNA, since we have found a mean length of 335 basepairs, much shorter than that of L dinucleosome. Nevertheless, this very low value could be due to the sliding of nucleosomes along the chromatin fiber, which has been shown to occur after micrococcal nuclease digestion [64]. This result strongly suggests that E chromatin exhibits a very open conformation which makes the associated D N A highly micrococcal nucleasesensitive. It is worth noting that E chromatin was totally depleted in the highest molecular mass histone H1 and enriched in the lowest. This suggests that H I A may have a negative control role in micrococcal nuclease accessibility, and therefore H1C would have a permissive role. Another interesting property is that E chromatin was highly enriched in H M G proteins, particularly in III and IV species. This fact has often been reported in relation to the high nuclease sensitivity of active chromatin [25,28,30,31]. E nucleosomes were also enriched to a small extent with other acid-soluble proteins. Such a characteristic has already been reported in calf thymus transcriptionally competent chromatin[26]. Though they represent only a small proportion compared to histones, they may play a significant role in E chromatin because they are concentrated in this fraction. In fact, they can be detected in non-digested nuclei histone patterns when overloading the gel (data not shown). It is then out of question that they are degradation products, related to the 37°C incubation. It seems then clear that some associated proteins, particularly H M G species and H1C subspecies, play an important role in the E chromatin nucleosomal structure, presumably in fixing an open nuclease-sensitive conformation. In contrast, H1A would be a constriction factor of the nucleosomal chain, and therefore would play a negative control role in nuclease accessibility.

292

It is interesting to note that E chromatin properties look like those of active domains of animal chromatins. Further investigations may reveal whether E chromatin is enriched in newly replicated nucleosomes or in transcriptionally competent regions. The other barley chromatin fractions, low ionic strength soluble oligonucleosomes and insoluble chromatin, exhibited the same content in nucleosomal DNA, and significant differences in protein contents could not be shown. Factors of insolubility must be investigated in more external nonhistone proteins, in connection with the nucleoskeleton and with nuclear matrix proteins. Relevant works are in process in the laboratory.

Acknowledgments We are very grateful to Professor R. Got for providing facilities, and to Dr. M.J. Quentin for critical reading of the manuscript. References 1 Chambon, P. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 1209-1234 2 Kornberg, R. (1977) Annu. Rev. Biochem. 40, 931-954 3 Thomas, J.D. (1977) in International Review of Biochemistry, Vol. 17 (Clark, B.F.C., ed.), University Park Press, Baltimore, MD 4 Cartwright, I.L., Abmayr, S.M., Fleischmann, G., Lowenhaupt, K., Elgin, S.C.R,, Keene, M.A. and Howard, G.C. (1982) CRC Crit. Rev. Biochem. 13, 1-86 5 Morris, N.R. (1976) Cell 8, 357-363 6 Noll, M. (1974) Nature (Lond,) 251, 249-251 7 Noll, M. (1976) Cell 8, 349-355 8 Spadafora, C., Bellard, M., Compton, J.L. and Chambon, P. (1976) FEBS Lett. 69, 281-285 9 Muller, A. (1979) Thesis, University Louis Pasteur, Strasbourg, France 10 Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F. and Bonner, J. (1974) Proc. Natl. Acad. Sci. USA 71, 2193-2197 11 Lacy, E. and Axel, R. (1975) Proc. Natl. Acad. Sci. USA 72, 3978-3982 12 Garel, A. and Axel, R. (1976) Proc. Natl, Acad. Sci. USA 73, 3966-3970 13 Reeves, R. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 709-722 14 Flint, S.L. and Weintraub, H.M. (1977) Cell 12, 783-794 15 Gottesfeld, J.M. and Butler, P.J.G. (1977) Nucleic Acids Res. 4, 3155-3173 16 Jackson, J.B., Pollock, J.M. Jr. and Rill, R.L. (1979) Biochemistry 18, 3739-3748

17 Levy, W.B., Connor, W. and Dixon, G.H. (1979) J. Biol. Chem. 254, 609-620 18 Weintraub, H. and Groudine, M. (1976) Science 193, 848-856 19 Weintraub, H., Worcel, A. and Alberts, B. (1976) Cell 9, 409-417 20 Garel, A., Zolan, M. and Axel, R. (1977) Proc. Natl. Acad. Sci. USA 74, 4867-4871 21 Bellard, M., Gannon, F. and Chambon, P. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 779-791 22 Bloom, K.S. and Anderson, J.N. (1978) Cell 15, 141 150 23 Wood, W.I. and Felsenfeld, G. (1982) J. Biol. Chem. 257, 7730-7736 24 Jackson, J.B. and Rill, R i . (1981) Biochemistry 20, 1042-1046 25 Davie, J.R. and Saunders, C.A. (1981) J. Biol. Chem. 256, 12574-12580 26 Georgevia, E.I., Pashev, I.G. and Tsanev, R.G. (1981) Biophys. Biochim. Acta 652, 240-244 27 Sandeen, G., Wood, W.I. and Felsenfeld, G. (1980) Nucleic Acids Res. 8, 3757-3778 28 Weisbrod, S. and Weintraub, H. (1979) Proc. Natl. Acad. Sci. USA 76, 630-634 29 Weisbrod, S. and Weintraub, H. (1981) Cell 23, 391-400 30 Gazit, B., Panet, A. and Cedar, H. (1980) Proc. Natl. Acad. Sci. USA 77, 1787-1790 31 Senear, A.W. and Palmiter, R.D. (1981) J. Biol. Chem. 256, 1191-1198 32 Kuel, L., Lyness, T., Dixon, G.H. and Levy-Wilson, B. (1980) J. Biol. Chem. 255, 1090-1095 33 Nelson, D.A., Perry, W.M. and Chalkley, R. (1978) Biochem. Biophys. Res. Commun. 82, 356-363 34 Davie, J.R. and Candido, E.P.M. (1978) Proc. Natl. Acad. Sci. USA 75, 3574-3577 35 Scaly, L. and Chalkley, R. (1978) Nucleic Acids Res. 5, 1863-1876 36 MacKnight, S.L., Bustin, M. and Miller, O.L., Jr. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 741-754 37 Riley, D. and Weintraub, H. (1979) Proc. Natl. Acad. Sci. USA 76, 328-332 38 Busby, S. and Bakken, A.H. (1980) Chromosoma 79, 85-104 39 Munroe, S.H. and Latt, S.A. (1977) Exp. Cell. Res. 110, 299-313 40 Scale, R.L. (1978) Proc. Natl. Acad. Sci. USA 75, 2717-2721 41 Levy, A. and Jakob, K.M. (1978) Cell 14, 259-267 42 Murphy, R.F., Wallace, R.B. and Bonner, R.B. (1980) Proc. Natl. Acad. Sci. USA 77, 3336-3340 43 Yakura, K. and' Tanifuji, S. (1980) Biochim. Biophys. Acta 609, 448-455 44 Galili, G., Levy, A. and Jakob, K.M. (1981) Nucleic Acids Res. 9, 3991-4005. 45 Jackson, D.A., MacReady, S.J. and Cook, P.R. (1981) Nature (Lond.) 292, 552-555 46 Cusik, M.E., Herman, R.M., De Pamphilis, M.L. and Wasserman, P.M. (1981) Biochemistry 20, 6648-6658 47 Annunziato, A.T., Schindler, R.K., Thomas, C.A. and Scale, R.L. (1981) J. Biol. Chem. 256, 11880-11886 48 Annunziato, A.T., Schindler, R.K., Thomas, C.A. and Scale, R.L. (1982) J. Biol. Chem. 257, 8507-8515

293 49 Worcel, A., Hart, S. and Wong, M.L. (1978) Cell 15,969-977 50 Schlaeger, E.J. (1982) Biochemistry 21, 3167-3174 51 Vaury, C,, Gilly, C., Alix, D. and Lawrence, J.J. (1983) Biochem. Biophys. Res. Commun. 110, 811-818 5"~ Spiker, S., Mardian, J.K.W. and Isenberg, I. (1978) Biochem. Biophys. Res. Commun. 82, 129-135 53 Spiker, S., Murray, M.G. and Thompson, W.F. (1983) Proc. Natl. Acad. Sci. USA 80, 815-819 54 Liberati-Langenbuch, J., Muller, A., Philipps, G., Robert, M.J., Schmitt, C. and Gigot, C. (1980) Physiol. Veg. 2, 357-372 55 Muller, A., Philipps, G. and Gigot, C. (1980) Planta 149, 69-77 56 Mithieux, G. and Roux, B. (1983) Eur. J. Biochem. 135, 443-447

57 NoU, M., Thomas, J.O. and Kornberg, R. (1975) Science 187, 1203-1206 58 Finch, J.T., Noll, M. and Kornberg, R.D. (1975) Proc. Natl. Acad. Sci. USA 72, 3320-3322 59 Philipps, G. and Gigot, C. (1977) Nucleic Acaids Res. 4, 3617-3626 60 Panyim, S., Bilek, D. and Chalkley, R. (1971) J. Biol. Chem. 246, 4206-4215 61 Gordon, J.S., Rosenfeld, B.I., Kaufman, R. and Williams, D.L. (1980) Biochemistry 19, 4395-4402 62 Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685 63 De Lange, R.J., Fambrough, D.M., Smith, E.L. and Bonner, J. (1969) J. Biol. Chem. 244, 5669-5679 64 Spadafora, C., Oudet, P. and Chambon, P. (1979) Eur. J. Biochem. 100, 225-235