18 Biochimica et Biophysica A cta, 781 (1984) 18-29 Elsevier

BBA 91305

ISOLATION AND PURIFICATION OF TRANSCRIPTIONALLY ACTIVE RIBOSOMAL CHROMATIN FROM THE SLIME MOULD, PHYSA RUM POL YCEPHA L UM

MARTIN CUNNINGHAM *, THOMAS SEEBECK and RICHARD BRAUN

lnstitote of General Microbiology, University of Bern, Baltzer-Strasse 4, CH-3012 Bern (Switzerland)

(Received June 6th, 1983) (Revised manuscript received October 26th, 1983)

Key words: Ribosomal gene; Nucleoprotein particle; Active chromatin; (P. polycephalum)

In the acellular slime mold Physarum polycephalum the ribosomal genes are all located on linear, extra- chromosomal DNA molecules which are clustered in the nucieolus. This report describes the isolation and purification of these ribosomal genes as functionally active chromatin particles. Nucleolar lysates are fractionated by gel filtration to remove ribosomal precursors and other soluble material. The ribosomal chromatin is subsequently separated from contaminating nuclear chromatin by a sucrose gradient centrifuge- tion step. This procedure allows the isolation of the ribosomal genes as intact nucieoprotein particles, which are now amenable to a biochemical analysis of their structural and functional properties.

Introduction

The bulk composition of chromatin and its gross structural features are well known by now. However, important cellular functions such as the programming of transcription and replication of specific genes cannot so far be understood as a consequence of specific changes of chromatin. While recombinant DNA techniques have allowed a very rapid analysis of the primary structure of many specific nucleic acids, their complexes with histones and other proteins are in general impossi- ble to isolate in pure form and in the relatively large amounts required for a detailed composi- tional, structural and functional analysis.

There are, however, some fortuitous exceptions to the above situation, in which specific chromatin

* Present address: 116 Plantation Road, Amersham HP6 6H2, Bucks, U.K.

Abbreviations: EGTA, ethyleneglycol bis(//-aminoethyl ether)- N,N'-tetraacetic acid; PPO, 2,5-diphenyloxazole.

0167-4781/84/$03.00 © 1984 Elsevier Science Publishers B.V.

species exist in multiple copies which are not in- tegrated into the large nuclear chromosomes, and which are sufficiently small or homogeneous to lend themselves to more detailed investigations. An example of these are the minichromosomes formed when SV-40 or polyoma virus lytically infect an animal cell [1]. The ribosomal genes of some organisms also fulfil these criteria. For exam- ple, the amplified extrachromosomal rDNA of amphibian oocytes can constitute up to 70% of the DNA present [3].

More recently it has become clear that many lower eukaryotes have ribosomal genes that, al- though often situated in the nucleolus like those of higher organisms, are present as multiple mini- chromosomes separate from bulk nuclear chro- matin.

These are found in Tetrahymena [4], Paramecium [5], the cellular slime mould Dictyostelium dis- coideum [6] and the acellular slime mould Physarum polycephalum [7,8]. Fractions containing substan- tially pure ribosomal chromatin have been isolated from Tetrahymena [9,11] and purified nucleoli have

19

been used to investigate the histone complement of the amplified ribosomal genes of Xenopus oocytes

[121. As all extrachromosomal rDNA molecules of

Physarum are located in the nucleolus and appear to constitute the only DNA species present, it is feasible to achieve a substantial purification of ribosomal chromatin simply by isolating nucleoli. This organism has several advantages for an inves- tigation using this approach. It is a simple matter to grow relatively large amounts of multinucleate plasmodia [13] either in liquid or in surface culture [14], nueleoli have been previously isolated by several methods [15-18] and the ribosomal genes themselves have been the subject of intensive in- vestigation at the DNA level [19-23]. Further- more, electron microscopic visualisations of nucleoli [24-26] have revealed a matrix granular structure within which are embedded fibriUar structures. These latter have been shown to con- tain chromatin [25], to incorporate uridine [25] and to persist during mitosis as distinct bodies [25,26] which appear to be identical to ribosomal minichromosomes. Indeed, minichromosomes which actively synthesize ribosomal RNA using an endogenous RNA polymerase I activity have been successfully obtained from Physarum by the lysis of isolated nucleoli [27]. They are located on linear palindromic DNA molecules of 60 kb length [7,8], of which there are approx. 200 copies/haploid genome [28] constituting about 2% of the total DNA.

The present paper describes an extension of the above method so as to produce purified ribosomal chromatin in relatively large amounts. The major problems to be overcome were firstly contamina- tion of the initial nucleoli with nuclei, secondly to develop a method to gently lyse nucleoli at high concentration and finally to remove from the crude lysate contaminating free proteins, ribosomal pre- cursors and nuclear chromatin. In addition, it is shown that a partial separation of actively-tran- scribing and inactive minichromosomes can be achieved.

Materials and Methods

Isolation of nucleoli and nuclei The strain of Physarum polycephalum used in

these studies was M 3 CVIII. It was grown in N + C medium either in liquid cultures as micro- plasmodia, or as macroplasmodial surface cul- tures, essentially as described by Daniel and Bal- dwin [14]. However, when nucleoli were to be isolated, plasmodia were grown in N + C medium containing 10 mM EGTA for at least 12 h before harvesting. This produced no detectable inhibition of growth [29].

Nucleoli were isolated from macroplasmodia as follows: 2-3 cm diameter cultures approx. 20-h-old and in the G2 phase before M3, were washed for several seconds at room temperature successively in deionised water and 10 mM Tris-HC1, pH 7.5. The outer, growing edges of three or four cultures were scraped into 200 ml homogenisation medium (250 mM sucrose/10 mM Tris-HC1, pH 7.5/0.1% Triton X-100) containing about 1 mM freshly ad- ded CaCI e. The exact concentration of CaC12 was optimised for each preparation by carrying out a few preliminary small scale trials. The plasmodia were blended for 45 s at 140 V in a 1 1 Waxing Blendor cup. For the final 5 s, 2 ml of a 1M CaC12 stock solution (refractive index 1.3559) were ad- ded. Homogenates were left in ice for 5-10 min to allow the foam to settle, and were then filtered through two layers of milk filter and centrifuged at 2000 × g for 30 rain at 2°C to pellet nuclei. Fol- lowing the homogenisation, all steps were carried out on ice.

To succesfully isolate transcriptionally-active nucleoli from liquid cultures at normal blending speed, it was found essential to avoid slime and to use rapidly growing cultures. Thus, wellgrowing cultures were diluted 10-fold with N + C medium containing 10 mM EGTA, and growth was al- lowed to continue overnight. Microplasmodia were then harvested by centrifugation at 50-100 x g for 30 s after they had already been allowed to par- tially settle under gravity. These were then washed once in resuspension medium (250 mM sucrose/10 mM Tris-HCl, pH 7.5). It was found that the yield of packed cells should be no more than 2.5 ml/100 ml cultures. Thicker cultures tended to give a high contamination with nuclei. Aliquots of 2-ml packed microplasmodia were blended for 45 s at 140 V in 200 ml ice-cold homogenisation medium con- taining freshly-added 1 mM CaC12 and nucleoli were recovered as above after addition of 10 mM

20

MgCI 2. More vigorous blending was found to increase the proportion of nucleoli, but these had minimal transcriptional activity and were heavily contaminated with pigment granules.

Nuclei were isolated in exactly the same manner as above except that 10 mM MgCI 2 and 1 mM CaCI 2 were present throughout the blending step and it was possible to use up to eight small surface cultures or 5-ml packed microplasmodia per 200 ml homogenisation medium.

Lysis of nucleoli and nuclei The nucleolar pellets obtained as above were

suspended in one-fortieth of the initial homogeni- sation volume of resuspension medium containing 10 mM MgC12 in a 50 ml conical centrifuge tube, and slime was partially removed using Percoll (Pharmacia Fine Chemicals) as previously de- scribed [16]. The resulting pellet was then washed in half this volume of resuspension medium con- taining 5 mM MgC12 and nucleoli were pelleted at 2000 × g for 10 min at 2°C. The pellet was care- fully suspended, using a siliconised Pasteur pipette, in 1 ml cold resuspension medium containing 2 mM MgC12 and nucleoli were pelleted by centrifu- gation for 3 min in an Eppendorf centrifuge. The pellet was again carefully resuspended this time in 0.5 ml hypotonic medium (10 mM Tris-HC1, pH 7.5 + 1 mM EDTA) and left on ice for 5 min. Phase contrast microscopic examination generally showed most if not all nucleoli to have lysed almost instantaneously. Debris and remaining nudeoli were pelleted by a further 3 min spin and, if necessary, the resulting pellet was extracted again in 0.5 ml hypotonic medium and cleared by a further 3 min spin. The hypotonic supernatants contained ribosomal chromatin capable of tran- scription in vitro as shown by assays for RNA polymerase in the presence of a-amanitin. Before further purification the chromatin solution was given a further clearing spin by centrifugation at 60000 x g for 30 min at 2°C (30000 rpm in a Beckman Ti50 rotor).

The lysis volumes given above were found to be suitable for nucleoli from up to 150 9-cm Petri dishes or 30-ml packed microplasmodia. Nuclei prepared as described above could also be success- fully lysed by this method to prepare total chro- matin.

Purification of ribosomal chromatin Ribosomal chromatin was purified from the

crude lysate by the following two techniques. Agarose gel filtration. The cleared nucleolar

lysate was loaded onto a 40cm-7 mm ~ column containing 1% agarose gel filtration beads (Bio-Rad A-150 m; exclusion l imit= 150-106 Da for a spherical protein) and run in 10 mM Tris-HC1, pH 7.5/1 mM EDTA overnight at a flow rate of 1 ml /h . Fractions of 0.5 ml were collected. To de- termine the included volume, a radioactive marker such as [3,-32p]ATP was run with the lysate.

Sedimentation through sucrose. 3.8 ml 20% (W/V) sucrose in 10 mM Tris-HC1, pH 7.5/1 mM EDTA was layered over 0.75 ml 70% (w/v) sucrose in the same buffer in a 5 ml polypropylene tube. This was overlaid with up to 0.5 ml sample and centri- fuged at 45000 rpm (180000 × g) in a Sorvall TV 865 vertical rotor at 2°C for 90 min. Similar results were obtained by sedimentation in a Beck- man SW 50.1 swing out rotor at 45000 rpm (190000 × g) for 4 h.

Assays for ribosomal chromatin Ribosomal chromatin was assayed in three dif-

ferent ways. RNA polymerase I assay. 10 /xl aliquots were

assayed for RNA polymerase activity in the pres- ence of 20/ag. ml-1 a-amanitin as previously de- scribed [27], both to check nucleolar lysis and to detect fractions, containing transcriptionally-active ribosomal chromatin during the subsequent purifi- cation.

Restriction enzyme analysis. Suitable aliquots were generally digested with the restriction endo- nuclease EcoRI (Boehringer, Mannheim) without prior deproteinisation, although in some cases it was necessary to concentrate the DNA by ethanol precipitation. Digestion products were analysed by electrophoresis through 0.8% agarose in 40 mM Tris-HC1, pH 8.3/5 mM sodium acetate/0.1 mM EDTA using 3 mm slab gels. This method was sometimes used to check lysis and to identify fractions containing ribosomal chromatin during purification. It also gives an indication of the degree of contamination of ribosomal chromatin with other chromatin.

Hybridisation. This was used as a more sensitive method for detecting both ribosomal gene se-

21

quences and total chromatin. Two procedures were employed. In a quantitative method. DNA was denatured with alkali and loaded onto nitrocellu- lose filters (Millipore, 13 mM diameter) as previ- ously described [27,28]. After baking at 80°C for 2 h, all filters were placed into a sterile, siliconised scintillation vial and prehybridised at 37°C for 4 h with a solution containing 50% deionised for- mamide, 6 × SET (0.9 M NaCI/0.18 M Tris-HCl, pH 8.0?6 mM EDTA), 1 × Denhardts, 25 #g- m1-1 sonicated, denatured calf thymus DNA. 0.2% SDS, 0 .1% sodium pyrophosphate, using 2 ml of this solution per 20 filters. This was then replaced with a half volume of the same mix containing 106 cmp (Cerenkov) of heat-denatured, nick-translated probe per 20 filters and overlaid with paraffin. Hybridisation was for 36-72 h at 37°C. Filters were washed successively in chloroform, 2 × SSC (0.3 M NaCI/0.03 M sodium citrate, pH 7.2)+ 0.1% SDS and in 0.1% SSC + 0.1% SDS at room temperature (20 ml /20 filters). They were dried and counted by scintillation spectrometry.

In a qualitative method [31] 1-5-#1 aliquots of each fraction were spotted onto a 1% agarose plate containing 2 #g- ml-1 ethidium bromide and dried. Examination of these plates under ultraviolet light revealed the overall distribution of DNA and RNA. The DNA from these samples was then denatured and blotted onto nitrocellulose filters [31,32]. After baking, these were sealed into plastic bags and hybridised as above using 50 #1 prehybridisation mix and 20 #1 hybridisation mix /cm 2. Filters were washed as above, again sealed into bags, placed in contact with X-ray film (Curix) backed by an intensifier screen (Du Pont) and exposed for 1-2 days at - 70°C.

The hybridisation probes used were either total nuclear DNA from Physarum or a plasmid, pPHR 106, containing only sequences from the tran- scribed 19 S gene region of rDNA. Probes were labelled with 32p by the nick-translation reaction [33] using [~x-32p]dCTP (Amersham, U.K.).

Analysis of proteins Protein was extracted from chromatin-contain-

ing fractions as follows. The fraction was made up to 1 ml with H20 and transferred to an Eppendorf tube. 0.1 ml fresh 0.15% sodium deoxycholate was added, the solutions mixed and left at room tem-

perature for 10 min. 0.1 ml 72% trichloroacetic acid was then added, the solutions mixed and left on ice for approx. 30 min. The tube was then centrifuged in an Eppendorf centrifuge for 20 min, the supernatant gently poured off and the pellet carefully dried with a tissue.

Proteins were then labelled with 3H as follows. The pellet was suspended in 38 #1 0.1 M b o ra t e , pH 8.5/1 mM E D T A / 2 #1 20% SDS (BioRad). Samples were boiled for 10 min. To test that trichloroacetic acid had been effectively removed, after cooling to room temperature a small drop of solution was applied to pH paper. If the solution remained acid, it was necessary to repeat the pre- cipitation step, but with careful removal of acid this was very rarely required. 30 #1 of a 2 mCi. m1-1 solution of N-succinirnidyl [2,3-3H]pro - pionate (NSP) in toluene (Amersham, U.K.) were evaporated to about 2-3 #1 in a gentle stream of air in another Eppendorf tube to which the protein solution was then added. The mix was left at room temperature for about 1 h. Then an appropriate aliquot was removed and added to 3 × SDS sam- ple buffer (0.188 M Tris-HCl, pH 6.8/15% 2- mercaptoethanol/30% glycerol/6.9% SDS) con- taining Bromophenol blue. The remainder was stored at - 70°C.

Samples prepared as above were run directly on 0.7 mm SDS-polyacrylamide slab gels [34,35] with either a 6-12% or 8-15% gradient of polyacryla- mide. Running buffer was 0.2 M glycine/25 mM Tris base /2 mM EDTA/0.1% SDS. Gels were stained with Coomassie blue and subsequently treated for fluorography either by impregnation with PPO [36] or by soaking for 30 min in 1 M sodium salicylate [37]. The dried gels were fluoro- graphed at - 70°c.

Results

Isolation of nuclei and nucleoli Nucleoli were routinely isolated from either

young shake cultures or from macroplasmodia. To avoid excessive contamination with nuclei, it was found to be essential to maintain a low concentra- tion of divalent cations during homogenisation. For this reason, 10 mM EGTA was included in the growth medium, thus chelating Ca 2÷ in a form that still appears to be accessible to the slime mold

22

[29,30]. Furthermore, it was necessary to use a substantially lower ration of packed cell volume to homogenisation medium than is normally used for nuclear isolation (approx. 1:100 compared to 1 : 40), presumably to reduce the contribution from lysed cells to the overall concentration of Ca 2+ or Mg 2+. Significant amounts of slime also appeared to increase the proportion of nuclei, a problem particularly acute when using shake cultures as source material. Even under optimal conditions, nuclear contamination from liquid cultures tended to be higher (approx. 5%) than from surface cul- tures (less than 1%). In vitro transcriptional activ- ity, as determined by an RNA polymerase I assay, also tended to be substantially lower in nucleoli from liquid cultures, and was again very depen- dent on the age and slime content of the cultures. Nucleoli from both sources are compared in Table I.

Because the nuclear contamination in the pre- paration from packed cells is in the higher range of values obtained and the RNA polymerase I activity is the lowest, it was clear that this source was consistently inferior to macroplasmodia which were therefore used for small-scale preparations. However, for the preparation of purified ribo- somal chromatin in significant amounts, it was necessary to use liquid cultures as these were far more convenient to prepare in large batches.

Lysis of nuclei and nucleoli Two methods had been previously used to lyse

nucleoli from Physarum under conditions in which chromatin remains intact. In the first, lysis was obtained by treatment with 10 mM EDTA [27] but results were very variable between experiments. In the second, chromatin was solubilised in 2.5 mg. m1-1 lysolecithin/2 mM CDTA/2 mM EDTA (Seebeck and Bindler, unpublished data). Al- though more consistent, only a fraction of nucleoli were lysed at high concentrations by this method.

In the present method, nucleoli are progres- sively destabilised by gradually reducing the con- centration of divalent cations, in this case Mg 2+, in a stepwise manner which, it is presumed, allows the internal concentration to equilibrate with that in the external medium. This reduction is accom- panied by a visible swelling and darkening of the nucleoli when observed under phase contrast mi- croscopy. In 2 mM Mg 2+ they have become suffi- ciently destabilised to lyse when transferred to hypotonic medium. A more rapid reduction of the Mg 2+ concentration does not produce efficient lysis presumably due to retention of ions within the nucleoli.

Like previous methods of lysis, this method is dependent upon concentration of nucleoli, but is capable of lysing much higher concentrations. It is also possible to extract concentrated nucleolar pel- lets two or three times to achieve complete lysis, while still maintaining volumes sufficiently low to use directly for subsequent purification. The effect of concentration is illustrated by the data in Table II, in which the lysis of nucleoli prepared from

TABLE I

COMPARISON OF N U C L E O L I P R E P AR E D F R O M SHAKE A N D S U R F A C E C U L T U R E S

Equivalent volumes of packed cells from either shake cultures or surface cultures were used as starting material. Nuclei and nucleoli were counted in a haemocytometer, using phase contrast microscopy at a magnification of 400 × . Nucleolar D N A contents were calculated using values given in Ref. 22. R N A polymerase I activity, expressed in clam, is that obtained in the final volume of solubilised chromatin; values are derived from assays on 10 ttl aliquots. The results are typical values from two separate experiments.

Source Number of Nuclear Final Number of Nuclcolar R N A polymerase I blendings contam, vol. (ml) nucleoli D N A (/zg) activity

32 rid cells f rom shake culture

140 small surface cultures

16 6.8% 2.0 1" 109 10-20 5.2' l0 s

35 0.25% 1.0 4.108 5-10 7.2.106

23

TABLE II

DISTRIBUTION OF RNA POLYMERASE I ACTIVITY DURING NUCLEOLAR LYSIS

In both experiments shown in this table nucleoli were washed in 2 ml resuspension medium containing 2 mM Mg 2÷ and pelleted by centrifugation (see Materials and Methods). 10/~1 aliquots of the supernatant were assayed for RNA polymerase I activity [2]. The nueleolar pellet was then lysed in 0.5 ml TE, 10 ~1 aliquots again assayed [31, then solubilised chromatin was obtained by a brief centrifugation. 10/d aliquots of the supernatant were assayed [4], while the pellet was reextracted in a further 0.5 ml TE, centrifuged and the supernatant again assayed. [5]. In some cases the initial nuclcoli in resuspension medium containing 2 mM Mg 2÷ [1] and the final pellet in TE [6] were also assayed.

Source (1) Intact (2) Total (4) (5) (6) Final nucleoli Mg 2+ sup. lysate -. Extract 1 Extract 2 pellet

25 ml cells from shake cultures

60 small surface cultures

2.8.106 1.2.105 1.1.10 6 7.4- l0 s 9.3. l0 s 1.5- l0 s

5.5. l0 s 2.9.106 3.1.10 6 2.6. l0 s

small surface cultures is compared with that of a much more concentrated preparation from liquid cultures. The near quantitative lysis seen during the first extraction of the less concentrated nucleoli in hypotonic medium was paralleled by an almost complete disappearance of nudeolar structures as visualised by phase contrast microscopy.

Chromatin can be solubilised from whole nuclei prepared in homogenisation medium containing 1 m M Ca2+/10 m M Mg 2+ using an identical proce- dure. However, in this case some lysis occurs dur- ing the 2 mM Mg 2+ wash stage and a significant proportion of nuclei do not lyse at a l l After lysis of nucleoli, only contaminating nuclei can be seen to remain intact. Thus, this method of lysis pro- vides a partial purification of ribosomal chromatin f rom nuclear chromatin.

Sedimentation of lysate through 20 % sucrose To achieve a separation of ribosomal chromat-

ing from contaminating nuclear chromatin and ribonucleoprotein, several methods were tested. One of the most potentially useful was to purify minichromosomes on a sucrose gradient but the peak of RNA polymerase I activity obtained was inconveniently broad, possible due to heterogene- ity within the population of minichromosomes. To avoid this, the present method was adopted in which ribosomal chromatin was sedimented through 20% sucrose and concentrated at the inter-

face with a 70% sucrose cushion. The method also allows the use of relatively large sample volumes.

Effect of divalent cation concentration. The be- haviour of nucleolar lysates sedimented through 20% sucrose was found to depend on the con- centration of divalent cations present. Nucleori were lysed in 10 mM Tris-HC1, pH 7.5, the lysate sprit into three and each aliquot sedimented through 20% sucrose containing a different effec- tive concentration of free Mg 2+ ions (Fig. 1). As the divalent cation concentration decreases, ribonucleoprotein complexes (as assayed by ab- sorbance at 258 nm) sediment less far. At zero added Mg 2+ ribonucleoprotein forms a reproduci- ble biphasic profile, suggesting the presence of two populations of ribonucleoprotein complex. The ef- fect of cation concentration on the sedimentation of ribosomal chromatin is less pronounced, but the progressive removal of Mg 2+ appears to allow a greater incorporation of [3H]UTP in a subsequent R N A polymerase I assay. For further experiments, conditions were chosen so as to obtain ribosomal chromatin at the 20/70% sucrose interface with maximal separation from ribonucleoprotein. These criteria were met at a concentration of 1 mM EDTA.

Separation of ribosomal chromatin from total nuclear chromatin. Besides producing a substantial s epa ra t ion of r i b o s o m a l c h r o m a t i n f rom ribonucleoprotein, sedimentation through 20%

24

A B C , ~ k l m M Mg2+ ~o Mg added k 2 mM EDTA

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Fig. 1. Sedimentation of nucleolar chromatin: effect of divalent cation concentration. Nucleoli were lysed in 10 mM Tris, pH 7.5 and equal aliquots sedimented through 20% sucrose con- taining different effective free Mg 2+ concentrations. A, 1 m M Mg2+: B, no added Mg 2+ or EDTA: C, 2 mM EDTA. • • , relative R N A polymerase I activity (incorporation of [3H]UTP into cold trichloroacetic acid-insoluble material); O- - -O , absorbance at 258 nm. Upper continuous line repre- sents the percentage of sucrose (w/w) in each fraction. ~, peak position of r D N A sequences as determined by a spot blot assay using pPHR 106 as probe, v, peak position of nuclear D N A as determined by a spot blot assay using total nuclear D N A as probe.

sucrose also produces a partial separation from total nuclear chromatin. This was demonstrated in three ways.

(i) Aliquots of each fraction were digested by the restriction endonuclease EcoRI and analysed by agarose gel electrophoresis. Fractions 2-3, from the 20/70% sucrose interphase, showed predomi- nantly specific rDNA fragments, but fractions from the middle of the 20% sucrose show a smear of material.

(ii) Small surface cultures were grown overnight in the presence of [3H]thymidine, and nuclei and

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Fig. 2. Comparison of the sedimentation properties of ribo- somal chromatin and total nuclear chromatin. Cultures were prelabeled with [methyl-3H]thymidine, either nuclei or nucleoli were isolated, lysed and sedimented through 20% sucrose con- taining 10 m M Tris, pH 7.5/1 mM EDTA. A: nucleolar lysate; B: nuclear lysat¢. • i , relative R N A polymerase I activ- ity; © - - - © , absorbance at 258 nm. Upper continuous line: sucrose profile.

nucleoli were prepared. Lysates of these were sedi- mented on parallel gradients (Fig. 2). The nucleo- lar lysate (Fig. 2A) gave a peak of RNA poly- merase I at the interphase and of absorbance towards the top. This slowly-sedimenting material consists predominantly of ribonucleoprotein, which is present in high concentrations in the nucleolus. Thus, the sucrose gradient sedimentation allows a separation of ribosomal chromatin from the nucleolar ribonucleoprotein. In contrast, the majority of the ultraviolet-absorbing material from the nuclear lysate, which represents chromatin, is found in the middle of the upper sucrose layer

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Fig. 3. Sedimentation profile of ribosomal and nuclear DNA sequences. A crude nucleolar lysate was sedimented through 20% sucrose in 10 mM Tris, pH 7.5/1 mM EDTA. The distribution of ribosomal and total nuclear DNA sequences was determined using a quantitative filter hybridisation a s s a y with, respectively, pPHR 106 and total nuclear DNA as nick- translated radioactive probes. • • , relative RNA poly- merase I activity; O- - -O, absorbance at 258 nm; A A, sequences hybridisable to pPHR 106 (i.e., ribosomal DNA); A- - -A, sequences hybridisable to total nuclear DNA.

(Fig. 2B). This co r responds to the pos i t ion of the 3H-label led D N A . It is not yet clear why the bulk o f R N A po lymerase I act ivi ty cosediments with the bulk ch romat in in the nuclear lysate; non- specific t r app ing of r ibosomal chromat in within bu lk ch romat in might be a poss ib le exp lana t ion for this appa ren t d iscrepancy.

(iii) In several cases, the D N A from the frac- t ions of such sucrose gradients were analysed by hybr id i sa t ion ei ther to r ibosomal gene-specif ic or

25

to total nuclear D N A probes . The result of such a hybr id i sa t ion analysis of a g rad ien t prof i le of a nucleolar lysate is given in Fig. 3. The bulk of the r ibosomal chromat in sediments faster than the con tamina t ing nuclear chromat in . Interest ingly, the peak of R N A po lymerase I act iv i ty sediments sl ightly ahead of the bu lk of the r ibosomal chro- m a t i n . T h e s l o w l y s e d i r ~ e n t i n g p e a k o f u l t rav io le t -absorb ing mater ia l again consists pre- d o m i n a n t l y of nucleolar r ibonucleoprote ins .

Removal of ribonucleoprotein Figs. 1C and 2A show that the separa t ion of

r ibosomal ch romat in f rom r ibosome precursors is

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Fig. 4. Ef fect o f r i b o n u c l e a s e t rea tment on the s e d i m e n t a t i o n o f crude nucleolar lysates . A crude nucleolar lysate was treated with 50 /~g.ml-1 ribonuclease A for 1 h on ice, then sedi- merited through 20% sucrose containing 10 mM Tris, pH 7.5/1 mM EDTA. • • , relative RNA polymerase I activity; O- - -O, absorbance at 258, nm. Upper continuous line: sucrose profile. Inset: ethidium bromide spot plate, fractions 1-5 (top row) and 6-10 (bottom row), both left to right.

26

by no means complete, a situation that worsens at higher concentrations of material. It was found that a longer distance of sedimentation served only to increase the spreading of ribosomal chromatin, while replacement of the 20% sucrose layer with a 15-30% sucrose gradient produced no significant improvement of the profile. Two alternative meth- ods were explored for a more efficient removal of RNP contaminations.

Treatment with ribonuclease. Before sedimenta- tion through sucrose, a crude nucleolar lysate was treated with 50 #g . m1-1 ribonuclease A for 60 rain on ice. During sedimentation the nuclease remains at the top of the sucrose, and it was found to be possible to subsequently assay fractions for in vitro RNA polymerase I activity, suggesting that, after this treatment, some nascent RNA chains still remain. However, the incorporation obtained was generally reduced to about 50% of controls although the position of the peak of RNA polymerase I activity was not altered (Fig. 4). It may be seen that the peak of absorbance was shifted, compared to Fig. 3, to the top of the gradient, and there was no significant crossing of the two peaks. Spotting of aliquots of each frac- tion on ethidium bromide plates showed that the ultraviolet-absorbing material, unlike intact RNA, does not intercalate ethidium bromide, demon- strating that the RNA has been substantially de- graded. A spot blot hybridisation to such a plate showed the position of the ribosomal genes to correspond to that of the actively transcribed ribosomal chromatin.

This treatment, although quick and simple, has two major disadvantages. Firstly, it is a rather severe treatment that might disturb delicate tran- scriptional structures and secondly, due to the great excess of ribonucleoprotein over ribosomal chromatin in the nucleolar lysates, it is still possi- ble to overload a short sucrose layer at relatively low chromatin concentrations. Thus, the following technique was more frequently used to remove contaminating ribonucleoprotein.

A garose gel filtration chromatography. The crude nucleolar lysate was subjected to a high speed clearing spin and then loaded directly onto a gel filtration column of 1% agarose beads which have an exclusion limit for globular proteins of 150.10 6 Da. The eluate was assayed for RNA polymerase I

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" ~ _ , ? -o .o

- 2 0

- - 1 5

i

u

- 1 0

< z o

L - 5

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Fraction number

Fig. 5. Elution profile of nucleolar lysate on BioGel A-150 column. Column dimensions were 0.5 x40 cm, flow rate 1 ml/h. 0.5 ml fractions were collected at 4°C, • •, relative RNA polymerase I activity; O---O, absorbance at 258 nm; • •, [32P]dCTP marker, Inset: ethidium bromide spot plate; fractions are from left to fight across each r o w .

activity and scanned for absorbance at 258 nm. A typical elution profile (Fig. 5) showed a virtually complete separation between the large absorbance peak due to ribonucleoprotein and the much smaller peak of chromatin which was found to elute with the excluded volume, together with the RNA polymerase I activity. These results were confirmed by a corresponding ethidium bromide plate (Fig. 5, inset) which shows a complete sep- aration between the clearly-defined spots due to DNA and the more diffuse RNA spots. These results were found to be completely reproducible between experiments.

The major potential advantage of this step is its potential for purifying larger quantities of ribo- somal chromatin. If a larger column is used, the chromatin-containing fractions can be con- centrated before sedimentation through sucrose (see below) by vacuum dialysis which can produce,

27

TABLE II1

C O N C E N T R A T I O N OF RIBOSOMAL C H R O M A T I N BY V A C U U M DIALYSIS

200 lal of crude nucleolar lysate were diluted with 4 ml of 30% sucrose in TE. This solution was dialysed under vacuum against TE for 150 rain. Sucrose concentration was determined using a refractometer. Total R N A polymerase I activity, expressed in cpm, was derived from assays on 10 #1 aliquots.

Time (min) Volume (ml) % sucrose Total R NA Yield (w/w) polymerase I act.

0 3.9 29.1 7.3-10 s -

150 0.3 13.9 7.0. l0 S 96%

within 2 h, a 10-fold concentration with essentially no loss in activity (Table III).

Two-step purification of ribosomal chromatin. A major disadvantage of the gel filtration procedure is that it does by itself not allow a separation between nuclear and ribosomal chromatin, which both elute in the excluded volume. Therefore,the optimal strategy consists in a two-step purification scheme. In a first step, ribonucleoprotein and other soluble components of the nucleolar lysate are removed by the gel filtration step. The chromatin fraction, which elutes in the excluded volume, is then fractionated into ribosomal and nuclear chro- matin by sucrose gradient centrifugation. The pro- file of such a sucrose gradient is given in Fig. 6. Absorbance now coincides almost completely with the RNA polymerase I activity. A comparison of this profile with the sedimentation pattern ob- tained from nucleolar lysates without a preceeding gel filtration step (see Fig. 3) clearly illustrates the power of this method. Spot blot hybridisation of the DNA from gradient fractions with a ribosomal gene-specific DNA probe (Fig. 6, inset) further demonstrates the comigration of RNA polymerase I activity, absorbance at 258 nm and ribosomal genes. In agreement with these observations, an EcoRI restriction nuclease profile showed only fragments derived from rDNA with a peak of material in fraction 3 (data not shown).

Discussion

The procedure outlined above describes one route by which a unique species of chromatin,

40

30

20

g 40 "~

o.o

2O

~ ~o-

I I I I I I I I 2 3 4 5 6 7 8 9 10

Fr(:lCtiOn number

Fig. 6. Sedimentation profile of partially purified ribosomal chromatin. Ribosomal chromatin eluted from a BioGel A-150 column (see Fig. 5) was layered directly onto 20% sucrose and sedimented. • • , relative RNA polymerase I activity; O- - -O , absorbance at 258 nm. Upper continuous line: sucrose density profile, Inset: spot blot hybridisation using the rDNA- specific plasmid pPHR 106 as radioactive probe. Fractions run from left to fight across each row (fraction 1 is the upper leftband corner).

Physarum rDNP, can be purified. It has been found to depend critically on the concentration of divalent cation at three points: (i) during the ini- tial isolation of nucleoli, (ii) during the lysis of nucleoli and (iii) during sedimentation of chro- matin through sucrose. In the last stage, the pres- ence of Mg 2+ apparently causes some aggregation of nucleic acid-protein complexes, probably due to the neutralisation and bridging of negatively- charged phosphate groups of DNA and RNA.

It is difficult to determine to what extent this aggregation is specific. For example, it is possible that this process is to some extent involved in the maintenance of nucleolar structure in vivo in the absence of a bounding membrane. The removal of

28

divalent cations from isolated nucleoli certainly does render them more labile to osmotic shock and the effect of Mg 2+ on ribonucleoprotein, the major component of the nucleolus, is particularly dramatic as judged by sedimentation through sucrose. Thus, taking into account the picture sug- gested by electron microscopy [24-26], it might be possible to visualise the nucleolus as a matrix-like structure formed by the ion-mediated, cross-link- ing of molecules of ribonucleoprotein. At the op- posite extreme, it might also be possible that the apparently cation-dependent nucleoli isolated un- der the present conditions are actually produced by nonspecific aggregation induced by the rela- tively high concentrations of Ca 2+ and Mg 2+ used. The gradual removal of Mg 2+ might then at least partially restore them to their native physiological structure which, outside the structural support of the nucleus, is rather unstable.

In contrast to the nucleolus, the nucleus is encased by a membrane but the overall structure nevertheless appears to be markedly affected by divalent cations. In the present study the effects seen are an increased resistance to vigorous blend- ing and a contraction in volume visible under phase contrast microscopy, occurring as ion con- centration are increased. However, as with nucleoli, the effects of moderate concentrations of ions (10 mM Mg 2÷) are to some extent reversible. Thus, it is possible to increase the lability of nuclei by a gradual reduction of Mg 2+ concentration. In con- trast, the effect of 10 mM Ca 2÷ is much less readily reversible. Preliminary attempts to induce the lysis of nuclei exposed to this condition have not been successful.

Although an effect on chromatin cannot be ruled out, it is possible that at least the contractile effect of cations is mediated through the nuclear matrix [39-40] i.e., through interactions with pro- teins alone. Both macronuclei and their matrices isolated from Tetrahymena [41] have been shown to contract reversible at 5 mM Ca 2÷ or Mg 2÷.

Not all nucleoli were found to be equally active. The low transcriptional activity of nucleoli from liquid cultures could be due to a variety of factors. An inhibitor of transcription may be present [42] which could be associated with the higher slime content of nucleoli from this source. Some reduc- tion would also be expected because, in contrast to

synchronous surface cultures, only a fraction of nuclei are in late G2 phase at which point ribo- somal transcription appears to be maximal [43]. Using a radioactive isotope dilution method [43], it was also found that the doubling time of liquid cultures as monitored by DNA synthesis is about 50% longer than that of surface cultures (Cun- ningham and Turnock, unpublished data) and this might be reflected in a lower rate of transcription. As might be expected, both growth and in vitro ribosomal transcription are particularly low in old thick cultures.

As shown in Figs. 3 and 6, sucrose gradients partially separate ribosomal chromatin into an active and an inactive fraction. The basis of this separation by sedimentation is unknown. It is likely that an actively-transcribing chromatin- ribonucleoprotein complex would sediment more rapidly than inactive chromatin [2,44] due to the presence of extending RNA chains. This effect might be enhanced if active chromatin is present as a multimeric complex or if it is more subject to aggregation by Mg 2÷ due to its ribonucleoprotein component. It is also not clear whether the ob- served non-transcribing chromatin has been in- activated during isolation, or whether its status reflects the situation in vivo.

Nevertheless, the possibility to isolate a defined eukaryotic set of genes as an intact structural and functional entity will hopefully allow a detailed biochemical analysis of the events involved in the regulation and replication of these genes.

Acknowledgements

We wish to thank H.P. Amstutz for construct- ing and characterizing plasmid pPHR 106. Sup- ported by grant 3.075.81 of the Swiss National Science Foundation.

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