Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

J. Mol. Riol.

(1985) 186. 743-758

Different Conformations of Ribosomal DNA in Active and Inactive Chromatin in Xenupus Zaevis Corrado Spadaforat and Patrizia Riccardi Universite de Genthe, Laboratoire d’Embryologie-Mole’culaire Ecole de Me’decine, 20, rue Ecole-de-Me’decine 1211 Geneve 4, Switzerland (Received 9 November 1984, and in revised form 24 July

1985)

The chromatin structure of the ribosomal DNA in Xenopus laevis was st’udied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BgZI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the “Barn Islands”. In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment, populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretir conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

1. Introduction

contain the regulatory sequences controlling gene expression (Stalder et al., 1980; Wu, 1980; Keene et al., 1981; Wu & Gilbert, 1981). A variet,y of such sites has been described and many of them are located near the 5’ end of the gene. However, hypersensitive sites have been found far upstream from the gene and also within the gene (Fritton et al., 1983; Parslow & Granner, 1982). In contrast, the chromatin structure of ribosomal genes, transcribed by polymerase I, remains obscure in many aspects and differs substantially from the chromatin of the genes transcribed by polymerase TI (Ness et aZ.: 1983; Davis et a.Z., 1983; Udvary et at.. 1984). \Ve have recently shown (Spadafora & Grippa, 1984) that the ribosomal chromatin in Xenopus Zaevis, particularly in its active state, is organized in a very compact structure. hardly permeable to micrococcal nuclease. Here we digest nuclei from blood, liver and embryos of X. Zaevis with micrococcal nuclease, and show restriction enzyme analysis of the extracted DNA. Our results indicate t.hat the n&ease introduces single-strand

The chromatin structure of a variety of genes has been studied extensively by the use of nuclease digestion. Significant differences have been found comparing the active versus the inactive state of many genes transcribed by polymerase II (for reviews, see Mathis et al., 1980; Igo-Kemenes et al.. 1982). Such differences in the chromatin organization have been shown t’o correlate with the differential gene expression. It is now well-established that active genes transcribed by polymerase II are, in general, more sensitive to nuclease degradation than inactive genes (Weintraub & specific sites on Groudine, 1976). Tn addition, chromatin, called “hypersensitive” are extremely sensitive to nuclease attack. These sit’es often t Author to whom all correspondence should be addressed at: Dipartimento di Biopatologia Vmana, Sezione di Biologia Cellula.re. Policlinico Umberto I, 00161 Koma, Tta.ly. 0021-2836, xl

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nicks along the ribosomal repeat, unit. These nicks can be resealed by ligation and are created not onI> by micrococcal nuclease: but also by DNase 1 and I)Nase 11 digestions. The nicks appear to modify an unusual DNA conformation present in the completely inactive ribosomal cahromatin of blood cells.

2. Materials and Methods (a) Materials Restrication enzgmea were I)urchased from ?Jw l3ngland Biolabs and Bethesda Research Laborat,ories. Ewhvrichia coli DNA polymerase, calf intestinal phos1) were obtained from phat,asr and actinomycm Borhringer-Mannheim (Srhweiz). phagr T4 DNA ligase from New England Biolabs and ribonuclease A from Il’orthington. Proteinase K was obtained from Merck. chorionir gonadotropin and agarosr type II from Sigma. and phage T4 polynucleotide kinase from PL Biochemicals. Nitrocellulose filters were from Gchleirher and Schiill (KA85). [cr-32P]deoxynucleotide triphosphate and [Y-~~P]ATP at high specific activity (2000 to 3000 Ci/mmol) were purchased from Amersham. Ribonucleasr A was boiled for IO min in 20 mM-Tris. HCl (pH 7.5). SRC is 0.15 M-NaCl. 0.015 N-sodium citrate: Denhardts solution is 0.029, (w/v) bovine serum albumin. ~02% (w/v) polyvinylpyrrolidone, 0.02:/, (v/v) Ficoll.

S. IaerG were obtained from South African Snake Farm (Cape Town). Ovulation was induced by injection of chorionir gonadotropin (Brown & Littna, 1964). The embryos were kept in water at 20 to 24°C until the desired stage of development was reached. Embryos were staged according to Nieuwkoop & Faber (1956). (v) ,V~ucleipreparation, digestions of nuclei xith micrococcal nucleasr, DNase 1 and DBase II and DNA extraction Kuc-lei were isolated from X. /arcis liver or blood cells and from embryos at stages 9. 13 and 40 of development (Sieuwkoop & Faber, 1956) as described by Burgoyne et ~1. (1974). Nuclei were suspended in buffer A (Burgoyne el at.. 1974) at a concent.rat,ion of about I mg/ml. The concentration was determined spectrophotometrica,lly at 260 nm, dissolving 5 ~1 of the nuclear suspension in 1 ml of 1 M-Nash. Micrococcal nucleasr digestions of nuclei were performed at 37°C’ with 0.5 unit,/pg of chromatin in the presence of 1 mM-CaCi2. Samples were withdrawn at different times and the reactions were stopped by adding EDTA to a final concentration of 10 mM. Digestions with DPu’ase I were done in buffer A. with 3 mM-MgCl,. h’urlei were suspended at, a concentration of 1 pig/m! and divided into portions of 1 ml each. Increasing amounts of DEase I were added to the different porbions of nuclei and t,he samples incubated at 37°C for 5 min. Reactions were stopped by adding EDTA to a final concentration of 20 mM. DBase II digestions were done on nuclei resuspended in 10 mu-Tris. HCl (pH 6.9). 1 rnM-EDTA at a concentration of 1 mg/ml. 0.5 unit of DNase II/pg of chromatin were added and portions were withdrawn at) different times from the incubation mixture. Reactions were stopped by mixing the samples with an equal volume of Tris . HCl (pH 8.8). The DNA was extracted as desrribed (Spadafora & Crippa. 1984).

and t? Kiccclrdi

The recaombinant plasmids used S~h probrs \\ t’r<’ described by Rungper ef cti. ( 1979). The isolation of specific fragment,s of the inserted-DXA was performed using low gelling agarose gels as reported (Spadafora & Crippa. 1984).

ottd h?ybridizatiotl Sativt, agarose gel t,lrc,troI)horesis. blots antI b~~britliz;rtions were performed using eondit.ions that ha\-<, brerl described. if not otherwise indicat,etl (Spadafora A (‘rippa 1984). Sative agarosr gels always c-olrtainrd 0.5 ILK of’ ethidium bromide/ml and DKA samples \vcre Ioadrd ill a buffer containing 100 mfiI-Ka(‘l. Denatrcri~lp agaros’ gels were used essentiallv as described by Maniatis ei al. (1982) without ethidium bromide. and nic.k-trartnlat,ior~~ werp according to Rigby ut al. (I 977). In c*ontrol experiments denaturing gels containing 0.5 tug ethidium bromide/ml were used and restricted DKA samples were loaded in denaturing buffer containing 100 rniv-?;a( ‘I. This did not influence t,he results. Treatment of DX:l with Klenow enz.vme and ligase were done in the following way: 10 fig of DKJA were incubat,ed for I h at 25”(* in a final volume of 270 fil. containing the 1 deoxyribonucleotides (0.02 mM final concn), Klenow buffer (100 miv-Tris . HCl. pH 7.8. 10 mM-MgCl,. 100 pg bovine serum albumin/ml) and 15 units of Klenon enzyme. After incubation the DKA was extracted with phenol and precipitated with 3 vol. ethanol. The I)R;A was resuspended in 100 ~1 of ligase buffer (50 rn?lTris. HCl. pH 7% 10 miv-MgCl,. 20 rn~-dit,hiothreitol. 1 mM-ATP and 50 pg bovine serum albumin/ml) and incubated with 5 units of phage T4 ligase at 15,“(’ overnight. DNA restrictions were performed according to manufacturer’s recommendations, Routinely 1 pg of DKA was rrstrict,rd with 5 units of enzyme a(, 37°C’ overnight. In SOIW rxperiments the same filter \vas hybridized a stvoml time with a different probe. The first ratlioac~tivv probe was stripped b>- boiling the filt,t’r for I5 rnin ~II 0.1 XSSC’. WIO,, ( \\‘,#X -). so<1’mm dodecyl sulphat~e. Tht, iiltrr vvas then air-dried and rehvbridizrd \vith the s(v.on(l probe. omitting t,hrhi”.eh~briciizilti~~t, str.1).

Single-stranded probes were preparetl from c~lonetl DNA -snd end-labellrd as described by Jlaniatis rt trl. (1982). Strands were separated on W,, (WV) polyacrylamide gels and were recovered by elevtrot~lution.

Ribosomal DS.4 was purified essentially AS described by MacLeod & Bird (1983). DIGA was extracted from t’hr blood of an individual female and from embryos at st.aptA 40 and was diluted to a concentration of’ 1%) /Lg,irnl in a buffer containing 10 m,I-Tris . HC’I (pH 7.5). .5 rnhl-EI)TB. CsCl was added to a linal concentration of 1 g/m1 of’ solut,ion a.nd actinornycin I) to a final <,oncentration 01’ 60 pgjml. (:radients were ventrifugrd at 3H.000 reys/tnin for about -COh at %O‘(’ in a Ti .X.-2 rotor anti wt‘rv thtatl collected from the top. I’ortions (50 ~1) from racab fraction filter an<1 were denatured, spotted on a nitrovrllnlosr hybridized with nick-t.ranslat.ed ribosomal I)NA prol)tts. The frartions containing ribosomal 11X.4 were poolrtl. diluted 4 times lvitb water and thrn precipitated wit11 ethanol. This DK.1 \vas further ljurifird through a stlconcl

X. laevis

Ribosomal

WJ/actinomyrin D gradient in a Ti 50. I rotor. Fractions of about 400 ~1 were collected from the top. Fractions containing rihosomal DPU’A were detected by hybridizaCon: pooled and precipitated with ethanol. The purit,y of the ribosomal DEA was checked by restriction. Dot hybridizations were performed essentially as described by Kafatos et al. (1979), with the following modifications: the samples were spotted on a nitrocellulose filter which was then washed in 4 x SSC and heated for 2 h at 80”(~, The filt,er was then pre-hybridized as described for Southern blots. DNA samples for electron microscopy were treated and spread on grids as described by Davis it al. (1971).

3. Results (a) Micrococcal nuclease digestion of blood and liver ribosomal chromatin of X. laevis ?;uclei prepared from liver and blood cells of adult’ individuals were digested with micrococcal nuclease and portions were withdrawn at different The DNA was purified and times of digestion. restricted with BgTT restriction subsequently nuclease. DNA samples were then fractionated on agarose gels containing ethidium bromide, blott.ed ont.o nitrocellulose filters, and hybridized with DNA probes corresponding to different portions of the X. laevis ribosomal unit (see Fig. 1). Figure 2 shows the hybridization patterns using probe II (“Ham Islands”) of the DPiAs prepared from liver (Fig. 2(a)) and blood cells (Fig. 2(b)) of two different animals. The restrict,ion pat’terns of t,he two DNA controls (slot 1, (a) and slot 1. (b)) are different due to the high degree of heterogeneity occurring in the spacer region between individuals (Wellauer et al.. 1976; Botchan et al.. 1977). It is clear that micrococcal nuclease digestion has a striking effect on blood cell DNA ((b). slots 2 to 4). The fragments prepared from micrococcal nucleasetreated nuclei have a faster electrophoretic mobility t,han the fragments of the control DNA (Fig. 2(b), slot 1). This effect is present but much less pronounced in liver chromatin. To rule out the possibility that the smaller shift in liver chromatin was due t)o electrophoretic artefact,s. we repeated t’he experiment on the liver of another animal

28s

NTS

ETS

(Fig. 2(e)). Again, the bands derived from micrococcal nuclease-treated chromatin (slots 2 and 3) show a slightly faster mobility than the corresponding DNA bands prepared from control chromatin (slot 1). Micrococcal nuclease digestion does not generate new DNA fragments in either Gssue, with the exception of some DNA smearing in the low molecular weight range. Nutleosomal repeat patterns, as expected (Spadafora & (Irippa, 1984), can be detected only by overexposing the films. The same filter shown in Figure 2(a) and (1)). was rehybridized with probe I (28 S 3’ end) after being stripped of the first’ radioactive probe (Fig. 3). The DNA restriction pattern derived from nucleasetreated blood cell nuclei (Fig. 3(b), slots 2 to 5) shows that two DNA fragment)s are shifted (labelled II and III in Fig. 3) when compared to the control pattern (slot 1). The mobility of the largest fragment (fragment I) corresponding to the part of the spacer upstream from the first Barn Island, remains unchanged. We have fractionated the same DSA samples on a lower concent.ration agarose gel (O-X t.o I(?&) and we have not found any obvious shift, occaurring in that fragment (not shown). However. it c~annot be excluded that small differences in the electrophoretic migration may be present and are not resolved in our electrophoretic system. In contrast no shift in mobility appears during the digestion of liver chromatin (Fig. 3(a)) and the position of the bands remains unaltered even after extensive digestion. Other experiments performed on several individuals confirmed these results showing that wit,h probe I (28 S 3’ end) the shift, was observed in blood chromatin but not, in liver chromatin. DNA samples of t.he same preparation were used t,o investigate the chromatin structure of another region of the repeat unit containing the terminal part of the 18 S gene and the internal transcribed spacer (probe TTT). Figure 4 (slots 4 to 6) shows that in t’his part of the repeat unit micrococcal nuclease digestion also produced a striking effect on mobility of t’he blood cell D?;A, while no effects. ot,her than a general degradation, are observed in l>h’A from

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Figure 1. X. laeuis ribosomal RXA gene probes. The horizontal bars symbolize the hybridization probes. Probe I, 496 base-pair &oRI/HindIII restriction fragment; probe II, 1060 base-pan BumHI restriction fragment (Ram Islands); probe III, 1120 base-pair EeoRI/BumHI restriction fragment; probe IV. 2500 base-pair BamHI/EcoRI restriction fragment. E, EcoRI; R. BarnHI. NTS, non-transcribed spacer: ETS, external transcribed spacer: TTS. internal transcribed spacer.

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(b)

(c)

Figure 2. Electrophoretic separat,ion of DK’A restriction fragments obtained from S. /aP,‘i.s livrl. alld blood nucltxi digested wit,h microcorcal nuclease, and Southern blot analysis using ribosomal 11X.1 probe II. (a) Hybridization patterns of the DNA restriction fragments obtained from micrococcal nuclease digestions of‘ liver rlllc.ki f’or dif%wnt lengths of t,ime: slot 1. 0 s: slot 2, 30 s: slot, 3. I min: slot 4. 2 min; slot 5. 5 min; slot 6. IO min: slot 7. 20 min: slot 8 60 min. (b) Hybridization patterns of the DK’A rest,rict,ion patterns obtained from micrococ~cal nu&asr digestion of pattrrlls of blood nuclei for different lengths of time: slot 1, 0 s; slot 2. 30 s: slot 3. 1 min; slot 1. 5 min. (c) Hybridization the I)iXA rrstriction fragments obt,ained from micrococral nuclease digestion of liver nuclei for dlfferrnt lengths of timts. slot I. OS: slot 2, I min: slot, 3, 5 min. Nurlei used in this experiment were preparrd from R different animal tllen thr nuclei used in (a). Digestions with micrococcal nuclease were r)erformrd at 37°C using 0.K unit of rtlzymcA:pg ot vhromntin. I)I$A was ext,ra&d and restrict,ed wit,h RgII. Fractionation of the l)K-A samples was prrformrd in I ,.jC’,, agarose gel ((a) and (b)) and in 1.2:/, ((c)) containing 0.5 pg ethidium bromidejml. I)EjA samples were loaded on the gels in 1 x RglI buffer (50 mM-Tris.HCl, pH 8.0, 10 m&l-MgCl,. .50 mn-NaCl). Slots a are I)?rjA markers obtained 1)) rrstrict’ing pBR322 with HpaII and slot b contains 1 DNA restricted with HilrdITI. The numbers on the right side of the autoradiograph indicate the lengths of the A/Hind111 restrirtion fragments in kb.

livvr AI nuclei (slot 1 to 3). Sublease digestion of this part of the gene also generated a series of bands of low intensity between 0.8 kb and 2 kbt. The fragment,s of 0.4 kb and 0.35 kb (slots 5 and 6) are also present in the control DNA (slot 4), although the shorter one has a lower intensity. Overexposure of the same filter shows that both the low intensity hands (0.X to 2 kb) and the fragments at 0.1 to 0.35 kb are also present> in the liver DNA restriction pattern (slots 1 t,o 3). The bands may be due to sF)ecif(- cut,ting sites within the genes. Figure 5 shows that micrococcal nuclease attack on the 28 S caoding region (probe IV. Fig. 1) of blood cell

t Abbreviations used: kb. 10” bases or basr-pairs: ITS. internal transcribed sl)acpr: KTS. non-tranx~ri~,rd sy,acPr.

chromatin also generates a shifted restriction pattern. Tn (*ontrast the restriction pat’trrn of 1his region in liver chromatin remains unmodified under the same r~xperimrntal conditions (not shown). ;\s far as we can determine, the only portion of the ribosomal repeat unit of blood chromatin which appears not to shift when digested wit,h mic~roc*occ~al nuc~least~. is t)he largest DNA fragment shown in Figurtx 3( 1)). This fragment contains sequences bet.wwn t Iw 3’ end of the 28 S gem’ and the first Kam Islands (Koseley rt nl.. 1979). It) must be pointed out however. that the esac*t tnapping of the restriction fragments is often difficult because of the heterogeneity of the ribosomal repeat units, both in Iengt~h and sequence. We want to emphasize two points: iirst. these experiments havt~ been repeated on blood and liver

X. laevis

a

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Ribosomal

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2

3

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Figure 3. The same filter as in Fig. 2(a) and (b) was stripped of the first hybridization probe and was rehpbridizrd with probe 1. (a) Liver nuclei micrococcal nuclease digestion time course. (b) Blood nuclei microc>ocacal nucleasr digestion time cpourse. I. IT and III indicate restriction fragments discussed in the text.

chromatin of at least ten individuals, and all show that nuclrase digestion of blood nuclei modifies the restriction pattern of almost the entire length of the ribosomal repeat unit, while in liver only the spacer region between the two Barn Islands appears slightly affected. Second, the shift is not caused only bv extensive nuclease digestion of the chromatin. In fact, under our conditions, the shift’ of the restriction patterns occurs within the first 30 seconds of digestion and remains unmodified afterwards. The shift does not increase with time. ruling out a slow. non-specific degradation process. (h) Micrococcal

nuclease digestion of embryonic rhwmatin qf X. laevis

The different responses to nuclease digestion of blood and liver chromatin prompted us to study the ribosomal chromatin in a third tissue where the ribosomal genes are fully transcribed, i.e. X. Zaevis embryos at stage 40 (Brown & Littna, 1966). The

patt,erns presented in Figure 6(a) and (b) show that’ restriction fragments derived from micrococcal nuclease-treated nuclei and non-treated nuclei have identical electrophoretic mobility. not only in the coding part of the gene (Fig. 6(b)) but also in the non-coding part (Fig. 6(a)). This result differentiates the embryonic chromatin not’ only from the blood: where ribosomal genes are switched off (Thomas & McLean, 1975), but also from liver where there is low activity (Davis et al.. 1983). We cannot exclude that small differences may exist in t’he elect,rophoretic mobility, but in this case they are very small and below the resolution of our electrophoretic syst,em. (c) Micrococcal n&ease digestion of ribosomal chromatin from embryos at different stages of development The finding that the structure of the ribosomal chromatin in blood cells is different from the

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Figure 4. Micrococcal nuclease digestions of X. lneris liver (slots 1 to 3) and blood (slots 4 t,o 6) nuclei and hybridization with probe III. D?JA samples were restrict,ed kvith BgII. Micrococcal nuclease digestions were performed as described. Liver nuclei were digested for the following lengths of time: slot I. 0 s: slot, 2. 5 min: slot 3. 20 min. Blood nuclei for: slot 1. 0 s; slot, 5. 2 min; slot 6. IO min. Slot a contains E./Hind111 marker and slot t) contains pRR322/HpaII. The numbers on the left side of the autoradiograph indicate the lengths of the i/HindJJJ restriction fragments in kb.

ribosomal chromatin of embryos at stage 40. prompt,ed us to study the ribosomal chromatin structure in embryos at) earlier stages of development when the activity of these gents is vcr\- low or absent (Davidson, 1976). Figure 7 shows the influence of micrococcal nuclease digestion of nuclei

Figure 5. Micrococ~cal nuclrase digestion of‘ blood nuclei and hybridization with lnobe TV. Slot 1 contains I)KA prepared from undigested nuclei. Slot, P cnontainx 1)X-A prepared from nuclei digest,ed with micrococcal nucleasr for 1 min. Restriction was with Hg/T. The numbers on the left side indicate t)hr positions of the ;I:HindIlI restriction fragments in kh.

on restriction fragment mobility. ‘l’hc 11robc uwrtl (II) is from the non-transcribed spacer region 01’ the ribosomal gene. Suclei were prepared from embrvos at. st,age 9 (slots 1 and 2), 13 (slot,s 3 and 4) an(j -CO (slots 5 and 6). stages t>he rrstric*tiotr f’rapntents In all thrrc obtained from control and micrococcal nuclc~rset,reated DSA have identical t+ctropboret ic mobility even at stage 9 and 13 when \cry littlc or . no ribosomal synthesis is occurrmy.

X. laevis Ribosomal

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Figure 8 shows the results obtained after digesting blood chromatin with micrococcal nuclease (slot 2). DNase I (slot’ 4) and DNase II (slot 7). D?;A samples were restricted with BgZI and the filter was hybridized with probe II (non-transcribed spacer). The three enzymes generate essentially the same digest’ion patterns which are clearly shifted with respect, to their relat’ive controls (slots 1. 3 and 6). Analogous experiments performed on embryonic chromatin confirm t’hat no shift in patterns are generated upon digestion (not shown). (e) Micrococcal

202.3

(a)

I and

DYVase II digestions chromatin

in the electrophoretic mobility oj ~fragments from ribosomcrl DLVA4 blood, cells lit.er and emb,ryonir

(f) Diferences

Figure 6. Micrococcal nuclease digestion of nuclei prepared from X. laevis embryos at stage 40. Hybridizations were performed using probe II (a) and probe III (b). Slot 1 of (a) and (b) contains DNA prepared from nuclei of embryos at stage 40. slot 2 of (a) and (b) contains DP;A from embryonic nuclei digested with micrococcal nuclease for 1 min. All DNA samples were restricted with BgZI. Micrococcal nuclease digestion was performed using 0.1 unit of nuclease/pg of chromatin at 37°C for 1 min. Slots a are L/Hind111 markers and the numbers indicate the lengths in kb of the restriction fragments.

ribosomal

of DAv=l extracted rell<~

In order tjo establish whether the unusual response t80 nuclease degradat,ion is a feature of t’he chromatin structure or can be obtained also with purified DNA, we performed micrococcal nuclease digestions on DNA ext’racted from blood and liver cells of the same animal. Portions of the two D?I;A preparations were digested at 0°C for one minute, the reactions were stopped and the D?\‘A was extract,ed and precipitated with ethanol. DNA samples were then rest’rict’ed with RglI. Figure 9 (hybridization with probe III) shows that rest’ric>tion fragments between 2 and 1 kb of the micrococcal nuclease-treated DNA of blood cells (slots 2 and 3) are clearly shifted in comparison to the fragments of the control DNA (slot 1). Tn contrast a similar shift is not obt’ained after micrococcal nuclease degradation of liver cell DNA under identical digestion conditjions. In fart RglI restriction fragments of control liver DNA (slot 4) and of microcaccal nuclease-treated liver DNA (slots 5 and 6) have an identical mobility. This result demonstrates that ribosomal DNA extracted from blood and liver cells of the same individual have a different response t’o micrococcal nuclease degradation. ( b)

(d) DlVase

nuclease digestion from blood and liver

of blood

of X. laevis

To rule out the possibility that the particular digestion patterns obtained on the different tissues were caused by some specific preference of the micrococcal nuclease for ribosomal DNA sequences we tested the effect of DNase I and DNase II.

redriction

c?f

In order to test whether micrococcal tiuclease treatment of chromatin or DNA is a prerequisite to visualize the difference between blood. liver and embryonic DKA, we rest’ricted D1\A samples of the three different types of cells (blood and liver cell DNA were from the same animal) wit,h several restriction nucleases without prior miwococcal nuclease digestion. The samples were fractionated on a 1.2Oj agarose gel and, aft’er blotting, filters were hybr:!dized with probe ITT (Fig. IO). Regartlless of the restriction enzymes used. the ribosoma] DSA fragments of blood cells (slots 1 of all panels) always have a faster electrophoretic migration than the corresponding fragments from liver (slots 2 of all panels) and from embryonic cells (slots 3 of all panels). An exception is t,he fastest migratitig DSA band of the BamHI digests (slots I, 1 and 3, panel (b)) which has very similar mobility in the three

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Figure 7. Micrococcal nuclease digestion of ribosomal chromatin in nuclei of S. Inrc+s embryos at different stages of development. Nuclei were prepared from embryos at 3 different stages of development: 9. 13 and 40. Nuclei of stage 9 anti 13 were digested with 100 units micrococcal nuclease/ml for 1 min at. 37°C. Nuclei from stage 40 were digested for I tniti with 0.2 unit/pg of c-hromatin at 37°C. After extraction DNA samples were restricted wit,h BglI. Slot 1. Dh’;\ prepared from nuclei at, stage 9; slot 2. DSA from micrococcal nuclease-digested nuclei, stage 9; slot 3. 1)X.A from nuvlri. stage 13: slot 4. DNA from nurlease-digested nuclei. stage 13: slot .i. DSA frotn nuclei, stage 40: alot 6. I )S;\ from nuclease-digested nuclei. &age 40. Hybridizat,ion was with probe IT. Slot a contains ijHindlT1 ma.rkt*rs.

different types of DNA. When the same samples were fractionated under denaturing conditions, the restriction patterns of the t,hree I>NAs are indistinguishable (not, shown). This finding confirms t,hat ribosomal DXA in blood cells has a different conformation than in liver a.nd embryonic cells a.nd rules out, the possibilitthat the strong effect on electrophoretic mobility induced by nuviease treafrnent of chromatin or IlKA may be caused by length or sequence heterogeneity or hy nuclease shortening of the ribosomal repeat units.

Our first intjerpretatjion of the results reported above was that nuclease cleaves preferentially at a

number of precise positions a101lg tltcl riboson~al repeat unit. of blood chromatin and only in the non-transcribed spacer of liver c-hrotnatm. Sucsh interrupt~ions must be located at it cvrtain dist~attc*c~ from RqlT restriction sites, since the restriction fragmenk of tiucltase-treated chrornat.in appeared t,o be shortened in c~omparison to the, cont,rol DN:S. fra,gments. \l:e tried to map the sitths of trtic~roc~oc~c~al digestion on the repeat unil using thr indirrcat etrtllabelling tec~hniclue (Wu, 1980). 1)s X pt~pared from nurleasr-treated and non-treat,ed ttttclei was tvst.ric%ed with EcoRT. The hybridization probes used (probe T (2X S 3’) and probe TTT (I8 SITTS)) \vt’t’(’ flush wit.h EroRT ends (see Fig. 1 ). The t~xpcriments demonst,rat,ed that the rest.ricat iort fragmettt.~ derived from rtucleast~-trratct1 vhromatitt hatl the same length as the cont,rol fragments (no! S~OWYI).

X. laevis Ribosomal Chromatin

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Figure 8. DBase I and DBase II digestions of ribosomal chromatin of blood cells. Slot 1, DNA prepared from blood nuclei; slot 2, DPU’A prepared from blood nuclei digested for 1 min with micrococcal nuclease; slot, 3, DXA from blood nuclei: slot 4, DPU’A from blood nuclei digested for 5 min with 10 units DBase I/ml; slot 5. empty; slot 6. DKA from blood nuclei; slot 7, DNA from blood nuclei digested for 1 min with 0.5 unit DBase II/ml. All DIVA samples were restricted with BgZI. Slot a contains L/Hind111 markers. Hybridization was with probe II.

and no new bands due to site-specific cutting by micrococcal nuclease were observed. Micrococcal nuclease does, of course, introduce double-stranded cuts, causing the gradual fading in restriction fragment int,ensit’y with increasing digestion time seen in Figures 2, 3 and 4. However, the indirect end-labelling experiment’ rules out’ thP possibility that micrococcal nuclease introduces

Figure 9. Micrococcal nuclease digest’ion of DNA extracted from blood and liver cells. All micrococcal nuclease digestions were carried out, on purified DNA. Slot 1. DNA prepared from blood nuclei; slot 2, DKA prepared from blood nuclei and then digested with 1 unit of micrococcal nuclease: slot 3, DKA from blood nuclei digested with IO units of micrococcal nuclease; slot 4, DKA prepared from liver nuclei; slot 5. DNA from liver digested with 1 unit of micrococcal nuclease: slot 6, DKA from liver digested with 10 units of micrococcal nuclease. Yricrococcal nuelease digestions were performed at 4°C for I min in 100 ~1 digestion mixtures containing 1 mM-Call, wit,h the indicated amounts of nuclease. Reactions were stopped bv adding EDTA to a final concentjration of 10 mM. DkA samples were extracted with phenol and chloroform and precipitated with ethanol. and t’hen restricted with Bgn. Hybridization was with probe III. Xumbers in the left side indicate the positions of the i/Hind111 restriction fragments in kh.

752

L’. Spadafora

(a)

(b)

and P. tliccnrdi

(cl

(di

Figure 10. (‘omparat,ive analysis of DKA extracted from blood. livet, and erniq-onic t~ells rrstrit~trcl wrth tfiflercal1t restrit+ion nucleases. Slots 1. 2 and 3 contain blood, liver and rmbryo DISi\. resprt~tively. (a) ,IlrcI restric~tiou: (h) BrrnrHT r&ritstion: (r) SbnT restriction: (d) PstT restriction. Fractionation \\-a~ ljerformed under nati\-tx t,onditiolts on a 1.Y?t, agarosr gel. containing 0.5 pg ethidium bromide/ml. The 1>2u’.L~ samples were loaded with a huti& t*ontainitq 100 m,n-?Sa(‘l. Xumbers on the left side indicat,e positions of 2jHirLdTII restriction fragments in kb. Thr h>-hridizatiotl I)robr wxs III 18 SjTTS

prfyise double-strand deavages in ttirl repeat units. and thereby exc~tudes t.hr> that the observed differenws in t4ect migration w-as due to a shortening of fragments.

ribosornal

possibilil! rophoret it, the I)XA

conditioris

An alternative hypothesis which t~)ntd explain the difference in t.hr elert rophorct iv migration is that mic*rococcal nurteasr introduces single-strand nicks into the DSA. To test this hypothesis we compared the micrococcal nuclease-treated and patt,erns on IlKA restriction non-treated denaturing gels. For this purpose we selected the DNA of an animal showing a considerable difference in the rte&rophoretic mobility between micrococcal nuclease-treated and control DNA. Figure 1 l(a) and (b) shows t,he pat,terns of tjhe two DNA populations fractionated (a) under native conditions and (b) under denaturing conditions and hybridized wit,h the same probe (II). DNA of bot,h treated and non-t’reat,ed fragments identical elrctrophoretical chromatin have an

mobility under tienatjuriny conditions and onI>tninor differences are visible, for inst)ance, slightI! more degraded IIPI’A running at the front, of tht> denaturing gel (taf. slots 1. 2. Fig. 1 I(a) and (b)). Our intrrpreta,tion of this result is that mit*rot~oc4 nuclrasr has introduced sin&-stranded nicks. The sa,me results are obtained by hybridizing with probe TIT ( 18 S/TTS): t)he patterns are shift,ed under nativts conditions (Fig. 11 (c)). but are identical after drnaturation (Fig. 11 (d)). Identical results were obtained hvbridizinp under native and by denaturing co&ions with the 2X $ probta (not shown). The cxistener of single-stra,nd nicks is supported addit,ionat evidence. Following hy incubation of the nuctcase-treat’rd I)KA with tigase the t,trt.troE)llc)rc,tit. mobility of’ thca restriction fragments is restjortatl to that of’ t~ontrol D?;A. Figurtl 12(a) and (b) shows the results of’ such experiments. DNA prepared from nuclease-treated chromatin was incubated with Klenow enzyme. with Klenow enzyme and ligase (Fig. I%(a). slots 3 and 1). and with ligasc alone (Fig. 12(b). slot 3). t,hen restricted with I&$1 a,nd fractionnt8rcl on agarose gels. The filters were then hybridized with probe II (NTS). It is clear t,hat the restriction derived from thr. nut~leas~-1 reated fragn1ent.s

X. laevis

1

(5)

2

al

Ribosomal Chromatin

2

(b‘l

12

753

12

(c)

id)

Figure 11. Comparison of the DNA restriction fragments from X. Zaecis blood nuclei and micrococcal nuclease-treated blood nuclei. under native and denaturing conditions. (a) BgZI restriction fragments of DNA prepared from blood nuclei (slot 1) and micrococcal nuclease-treated blood nuclei (slot 2). Electrophoresis was performed under native conditions in a 1.2q’, agarose gel. The filter was hybridized with probe II. (b) The same samples as in (a) were fractionat,ed in a 1.2:/b agarose gel under denaturing conditions. Hybridizations were performed with probe II. Slot a contains J/Hind111 markers. (c) The same DNA samples as in (a) and (b). but the hybridization was with probe III. Electrophoresis was under native conditions. Arrows indicate the positions of the 2 BgZI restrictions fragments of the micrococcsal nucleasetreated DPr’A samples. ((1) The same DNA samples as in (c) were fractionated in a 1.23, agarose gel under denaturing caonditions. hybridization was with probe III.

ohromatin after ligase treatment (Fig. 12(b), slot 3), and Klenow enzyme plus ligase treatment (Fig. 12(a), slot 4), migrate the same as restriction fragments of control chromatin (slots 1 in (a) and (b)). Klenow enzyme treatment alone does not have an.y visible effect (Fig. 12(a), slot 3). Two questions arose: are these nicks concentrated on specific .‘hot” DNA sequences and are they strand-specific? In order to answer these questions we analysed the restriction patterns of micrococcal nuclease-digested and non-treated blood chromatin under denaturing conditions and hybridized them w&h single-stranded probes corresponding to the non-transcribed spacer regions and the 18 S/ITS (not shown). We have found that nicks were

distributed on almost the entire length of the repeat unit and that both strands were equally nicked. (i) Effects of ionic strength and ethidium bromide on

the electrophoretic migration of ribosomal DNA restriction fragments All native gels reported in this paper contain 0.5 pg of ethidium bromide/ml and DNA samples are loaded in a buffer containing 100 mi%-Nacl (Materials and Methods) if not otherwise indicated. Ionic strength has a considerable effect on the differential electrophoretic mobility of the DNA restriction fragments from micrococcal nucleasedigested and non-treated chromatin. Figure 13

754

C’. Spadafora

and I’. Kiccardi

2.32.02.32Q-

o-5-

(b)

(a)

Figure 12. Effect of ligase treatment on the mobility of DKB restriction fragment,s from microc~occal nrcc*leasedigested chromatin. (a) Hybridization patterns obtained with probe TT of DK’A restriction fragments of blood nuc4ri (slot 1) and of nuclei digested for 1 min with micrococcal nuclease (slot 2). Slot 3 contains the same DS=\ as in slot 2. but incubated with Klenow enzyme (see Materials and Methods). Slot 1 contains the same DPU’Asamples as in slot 2. but treated with Klenow and wit,h ligase (see Materials and Methods). Restriction was with BgII. Slot a contains E./Hind111 markers. (b) Hybridizations patt,erns using probe II of DX’A rest,riction fragments of blood nuclei (slot 1) and of nuclei digested for 1 min with micrococcal nuclease (slot. 2). Slot 3 contains the same DKA4 samples as in slot 2. but incubatrtl with ligase. Restriction was with BglI. Slot a contains A/Hind111 markers.

shows the relative migrations of XTS restrict~ion fragments (probe II) of treated and non-treated DSA in: BgZT buffer (slots I and 2). water (slots 3 and 4). 50 m&t-NaCl (slots 5 and 6). 100 m&r-NaCl (slots 7 and 8) and 200 mM-NaCl (slots 9 and 10). In the absence of salt (slots 3 and 4) the difference in the electrophoretic mobility is reduced, although still present, whereas it is well-pronounced at the three NaCl concentrations tested. This gel con tained 0.5 lug of ethidium bromide/ml. Similarly. ethidium bromide modifies the electrophoretrc migrat,ion of the two DNA fragment populations. In order to test. the effects of et,hidium bromide. DIVA samples were mixed with increasing concent.rat.ions of the intercalating agent and fractionat,ed on a native agarose gel prepared without et,hidium

bromide.

Figure

14 shows tha,t the rntercxlatirrg

agent has very litt,le effect on control DNA, while it

slows considerably the electrophoretic mobilit\- of’ the nuclease-treated DNA fragments. ichcrt et,hidium bromide is omitted (slots 1 and 2) no difference in electrophoretic mobility is seen. Curiously. if et#hidium bromide is rnised with th(a DNA samples. then bhe electrophoretic tnobilit’v is decreased (Fig. 14) but when is added to the gels (standard procedure) t,he electrophoretic mobilitv is increased (Figs 2 to 5 and 8 to 13) in comparisotr to control patterns. The reason of this difference is unknown. The different,ial sensit,irit.x to iotric strength and t!o ethidium bromide of the 1)X-A restriction fragments derived from micrococ~c’al and non-t,rea,ted chromatin. nuclease-digested

X. laevis Ribosomal

1

2

3

4

5

755

Chromatin

6

7

8

9

10

Figure 13. Effects of different ionic strengths on the electrophoretic mobiky of DKA restriction fragment.s prepared from control and micrococcab nuclease-breated blood nuclei. DNA prepared from blood cell nuclei and from blood nuclei digested with micrococcal nuclease for 1 min was restricted with BglI. DIVA samples were loaded on a native agarose gel. containing 0.5 fig ethidium bromide/ml. at different ionic strengths. Slot 1 contains DiYA from blood cell nuclei and slot, 2 from micrococcal nuclease-treated blood nuclei, both in BgZI buffer. Slots 3 and 4 contain the same DXr’A samples as above but were loaded on the gel in water. DNA samples of slots 5 and 6 were loadecl in 50 mM-KaC. Samples of s1ot.s 7 and 8 were in 100 mM-KaCl and 9 and 10 in 200 mM-NaCl. Hybridization was performed with probe II,

suggests t’he presence of some secondary structure in the ribosomal DKA repeat .unit of blood chromatin. The secondary structure may either be released or modified by nuclease activity. analysis of puri$ed (j) Electron microscopic ribosomal DNA prepared from blood and from embryos

The results above predict the secondary st,ructures on the ribosomal

existence of Dn’A repeat

unit of blood and liver cells (this lat,ter only in the Barn Islands region). These structures are not, only present when DSA is packaged in chromatin. but must also persist on the extracted ribosomal DNA as shown above. Similar structures should not be present in embryonic DNA. Vsing the electron microscope. we have analysed ribosomal DKA from blood and embryonic DNA purified through CsCl/actinomycin I> gradients. Figure 15 shows that ribosomal DNA prepared from blood ((a) and (b)) is highly enriched in looped

C’. Spadafora.

12

3

4

5

and P. Riccardi

4. Discussion

6

chromatin

Figure

14. Effect of ethidium

electrophoretic

mobility

bromide on the

of L)Ku‘;z restriction

fragmenk

prepared from control and micrococcal nuclease-treatrd blood nuclei. DK‘A prepared from blood cell nuclei and from blood nuclei digested with micrococcal nuclease fog I min. was restricted with BgZT. DNA samples were loaded on an agarose gel in BgZI buffer without

ethidium

bromide (slots I and 2) or mixed with ethidium bromide to fina\ concent.rations of WF,pg/ml (slot,s 3 and -&) anti 5 pg/ml (slots 5 and 6). Slots 1, 3 and 5 are 1)R’A rontrols: slots 2. 1 and 6 are DPU’Afrom nurlease-treated nuclei. The 8~1did not contain ethidium bromide. H.ybridizatiorr was wit,h probe IT.

structures of various sizes. Some of t,hese slructurea appear completely folded. while others have a stem and loop appearance. Similar st,ructures are not observed in bulk DNA (Fig. 15(c)) nor in purified embryonic ribosomal DNA (Fig. 15(d)).

Our results show that a mild micrococml nucleasc~ digestion of ribosomal chromatin from blood caells causes a considerable modificat’ion in the elrctrophoretic migration of most of the rest~rict.ion fragments of t’he repeat unit tested (Figs 2(b). 3(b). 4 and 5), as compared to control DSA derived from non-treated nuclei. On the other hand. digestion of embrvonic ribosomal c,hromatin does not cause an> signil&ant modification in the migration of the rest.riction fragment,s test.ed (Fig. G(a) and (1~)). The effect of micrococcal nuclease digestion on IivcAr ribosomal chromatin is intermediate to its effects on blood embryonic c>hromatin. and showing differences in electrophorrtic mobi1it.y only i; fragments from the spacer region containing t trr~ Ram Tslands. We ruled out t’he possibility that the differences between blood and embryonic cbhromstin are due to srquencde specificity of’ micrococcal nuclease, since identical results were obtained with DBase J and DBase II (Fig. 8). \Ytl have also found by indirckct, end-lahclling experiments that precisely placed double-stranded cleavages within the ribosomal units art-’ not responsible for t’he faster migration of thfb restri(*tion fragments. Denaturing gels abolish thr shift (Fig. 11) and so does ligase trratrnent (Fig. I%), Our results would t,herefore favour the> interpretation that single-&and nicks are generaW h>- nuclrases along the ribosomal repeat unibs in blood cell chromatin. which modify thr rnohility of J)NX fragments with unusual properties. Jn liver and c~mbryonic chromat.in micrococc+al nucleasc digestion has little or no such eff’ect. The tAntire length of the JINA in the repeat unit in blood st’tlms not to have this pr0pert.y since, for instance. the spa~r region. upstream from the first Ham Islands. does not show a shift, (see Fig. S(b), fragment- I). Our result’s do not rule out the possibility that some specific double-stranded cleavages also may br grnerat,ed by micrococacal nuclease. is reported b? a La Volpe of cd. (1983). They have found hypersensitive sit)e located in the proximity of the first Sam Islands in blood and embryonic cahromatin of S. laths. ITnder out’ nuclease dipest)ion csonditions we generate pat’terns of DNA fragmentation of variable int)cnsity and it ma>’ be that some of these bands are products of’ specitic hypersensit)ive sites (i.1’. Figs 1 and 5). The eflects of ionic st’rength and ethidium bromide

fragments

on the 4ectrophoretic

mobility

of the DI\‘A

cannot he explained simply by the of single-strand nicks. but, imply that presenctt~ st~c~ondar~structure plays a role ill the differential rnobilitv of the I).VA molec~ules. LVe tenta,tively caonclude then. that the diRerent, talec:trophorrt’ic> patterns of J)NA restrict’ion fragmrnts derived from cont,rol and nuclease-digested cahromatin, are caausrd by single-strand nicks which modify an unusual fea,ture of blood cell ribosomal

X. laevis Ribosomal Chromatin

757

Figure 15. F:lectron micrographs of: (a) and (b) ribosomal DPjA purified from blood cells: (c) bulk DIVA of blood. This DKA sample was derived from the same CsCl/actinomycin D gradient, used for the purification of ribosomal DNA shown in (a) and (b); (d) ribosomal DNA purified from embryos at stage 40. Arrows indicate DKA secondary structures. The bar represents I pm

DXA present in specific regions of the repeat unit. In good agreement with our biochemical data is our electron microscopic observation of looped structures on the ribosomal DNA of blood cells but not of embryonic cells. The findings that micrococcal degradation of extracted blood cell DNA also causes the shift of the same restriction fragment’ (Fig. 9, slots 1 t’o 3). and that ribosomal DNA prepared from blood cells generates restriction patterns with a faster electrophoretic migration than the patSterns of the DNA prepared from liver and embryonic cells (Fig. lo), lead us to the conclusion that the shift reflects a feature of the extracted DNA and not only of the chromatin. Micrococcal nuclease treatment and restriction of liver DKA (Fig. 9, slots 4 to 6) and of embryonic DIL’A and cloned ribosomal DNA (not shown) also does not affect, the mobility of the restriction fragments, indicating that t’he shift, is not’ merely a feature of the DNA sequence. One explanation of the results is that chromosomal protein(s) are involved in keeping the ribosomal DNA of blood cells in a particular conformation, and t#hat such protein(s) are stably bound to ribosomal DNA and are not removed during DNA extraction (Xeuer & Werner. 1985). It is interesting to note that the presence of the altered DNA behaviour is inversely correlat’ed with

the pot)ential for ribosomal gene expression. It is absent in embryos, which are active or pre-active, and present in blood cells, which are terminally differentiated and fully active. Strikingly, it’ is present only in the Barn Islands region in liver (as indicated by the shifted restriction pattern) and there only to a small extent. This last tissue is more active in ribosomal transcription than blood cells, but less active than embryos (Davis et nl., 1983: Thomas & McLean, 1975). It is tempting t)o suggest that the altered DNA structures may play a role in a negative control of the ribosomal gene expression. For example, the presence of these struct’ures may permanently inactivate the ribosomal genes in blood cells. Consistent with this hypothesis is the presence of such structures on the Barn Islands region in liver. The spacer region containing the Barn Islands has been proposed to have regulatory sequences for the ribosomal genes and its DSA repeats may function as enhancers (Moss, 1983: Busby & Reeder. 1983; Reeder at al., 1983: Reeder. 1984). Looped structures in this region of the spacer might inactivate the enhancer function. causing a decrease in t’he rate of ribosomal RSA transcription. Ribosomal RKA synthesis could continue, however, at a lower rate as required by liver tissue, under the control of the promoter located at t’he 5’ end of each repeat, unit.

(I’. Spadafora

(b) Ribosomal

chromatin embryonic

structure dwing development

X. laevis

We have detected unusual DNA properties in the ribosomal repeat units of blood cells which are not, transcribed. The question arose whether this phenomenon is a general feature of non-transcribed ribosomal DNA or whether it is a special characteristic of non-transcribed ribosomal DNA found only in terminally different.iated cells. We answered this question by analysing the structure of the ribosomal chromatin in embryos at very early stages of development, when ribosomal RNA transcription has not yet started (Davidson. 1976). Our results (Fig. 7) show t,hat the restriction pattern of ribosomal chromatin from embryos at stages 9, 13 and 40, is identical. This could be interpreted to show that ribosomal chromatin in early embryos, although inactive; is “programmed” for the future requirement, of the cells, and its structure is a,lready in an “active” conformation. Tn ribosomal chromatin in blood cells. contrast, although also inactive, is in a different structure than in the early embryos, perhaps because red blood cells are already terminally differentiated and ribosomal RNA transcription ~111 not’ be required again within the life of t’he cell.

and Y. Riccardi

Davis. A. H.. Keudelhuber. ‘I’. L. & (iarrard. \I- ‘I‘. (1983). J. No/. Hid. 167. 133. 135. Davis. R. \V.. Simon. M. dz Davidson. ?i. (1971). A21uthorl.s Enzyvrwl. 21. 413-1-L% Fritton. H. I’.. Sippel, A. E. & Igo-Kemrnrs. T. (1983). Xucl. =Icidx Rrs. 11. 3467--34&j. Igo-Kemenes. T.. Hijrz. W. 8 Zachau. H. (:. (1982). A nvw Ker. Hiochwvv~.51, 89- 121 Kafatos. F. (‘.. WTeldon Jones. (‘. & Efstratiatiis. .\ (1979). Sucl. Acids Kes. 7. 1.541 155P. Keene. 51.. Forces. V.. Lowenhaupt. Ii. & RIgin. S (1981). Proc. LVat.dcnd. Sci.. l’.S.A. 78. 1X-116. La \‘olpe. ,I.. Taggart. M.. McStay. 13. dt Bird. A. (1983). Sucl. .-ltids Rex 11. 5361- .53x0. MacLeod. I). & Bird. A. (1983). .\‘ntuw (/,~mdo~~), 306. zoo--203. Jianiatis. T.. Fritsch. E. F. B. Sambrook. .I. (19X”). Molecular Cloning. A Laboratory A!lanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. hlathis. D.. Oudet. P. 8z (‘hambon. I’. (1980). Z’roq. ,\-crcl ,-kids RPS. Mol. Kiol. 24. l-.55. Moss. T. (1983). LV~furp (London). 302, L’ZC%L’)3. Xess, I’. ,J.. Labhart. P.. Banz. E.. Keller. T. & I’aristr. R. iv. (1983). -1. Nol. Hiol. 166. 361 38 1. Seuer. H. & Werner. D. (19%). ,/. Xol. Rio/. 181. I5 2.5. Kieuwkoop. I’. D. & Faber. .J. (1956). A’orn~/ Trrblr
We thank Drs Th. Keller, P. J. Ness. S. Gasser and F. Ballivet for critically reading the manuscript. We also thank E. Boy De La Tour for electron microscopy and S. Chrai’ti and H. Felder for help in preparing the manuscript,. This work was supported by the Swiss Bational Funds (grant no. 3.466-0.83).

References &&ey.

1’. (i.. Moss. T.. Mlchler. H.. Portmann. K.. & Birnstiel. $1. L. (1979). (‘(>/I, 17. 1%31. Botchan. P.. Rreder. R. H. C;rDswid. T. K. (1!177). (‘p/I. 11. .599-607. Brown. I). I). & Lit,tna. E. (1964). ./. No/. Hio/. 8. liti!) 687. Brown. I). D. & Littna. E. (1966). ./. .110/. Hiol. 20. X1-m94. Burgoyne, I,. A.. Hewish. I). K. R YIarly. ?I. (1974). Riochem. J. 143 6i- 7”.

[‘tlvardy.

,\.. I,ouih. (I.. Han. S. Cy-Sc,tlf~dl. 1’ (I9X-C) .i 175. 1 13 130. \\:rintrarrb. H. & (:routlint~. J1. (I9ifi). 5c,ir-0c,c. 193. ?(M 856. .Ilol.

Hid

E:ditrd ty I’. C’h.trir/bo,/