non-nucleolar transcription complexes of ra...

J. Cell Sci. 40, 181-192 (1979) l 8 lPrinted in Great Britain © Company of Biologists Limited

NON-NUCLEOLAR TRANSCRIPTIONCOMPLEXES OF RAT LIVER AS REVEALEDBY SPREADING ISOLATED NUCLEI

FRANCIS HARPER* AND FRANCINE PUVION-DUTILLEUL

Institut de Recherches Scientifiques sur le Cancer, B.P. n. 8, 94800 Villejuif, France

SUMMARY

Miller's technique was applied to isolated nuclei of rat liver. Both the usual nucleolar andnon-nucleolar transcription complexes were visualized. In addition, an unusual type of putativenon-ribosomal transcription unit was revealed. It was characterized by a high density of thelateral ribonucleoprotein (RNP) fibrils. Although these particular units exhibited a regularincrease of fibril lengths, the length of the transcript-covered deoxyribonucleoprotein (DNP)fibres and the morphological aspect of the RNP fibrils distinguished them from the nucleolar' Christmas-tree '-like figures. The linear and granular configuration of the transcripts and theabsence of terminal knobs made them similar to non-nucleolar nascent RNP fibrils.

INTRODUCTION

In contrast to the large volume of literature on the morphology of ribosomaltranscription units (Angelier & Lacroix, 1975; Berger & Schweiger, 1975; Frankeet al. 1976a, 1977; Franke & Scheer, 1978; Hamkalo & Miller, 1973; McKnight &Miller, 1976; Meyer & Hennig, 1974; Miller & Bakken, 1972; Scheer, Trendelenburg& Franke, 1973, 1976a; Scheer, Trendelenburg, Krohne & Franke, 1977; Sommer-ville & Malcom, 1976; Spring et al. 1976; Trendelenburg, 1974; Trendelenburg,Spring, Scheer & Franke, 1974) much less information exists concerning those ofheterodisperse RNA (HnRNA) synthesis. Transcription complexes from activenon-nucleolar genes have been described, however, in various biological materials witha high level of HnRNA synthesis: these include lampbrush chromosomes of amphibianoocytes (Angelier & Lacroix, 1975; Franke et al. 19766; Hamkalo & Miller, 1973;Keyl, 1975; Miller & Bakken, 1972; Miller & Hamkalo, 1972; Scheer, Franke,Trendelenburg & Spring, 19766), primary nuclei of green Alga (Spring et al. 1974;Spring, Scheer, Franke & Trendelenburg, 1975) and embryos and spermatocytes ofvarious insects (Amabis & Nair, 1976; Foe, Wilkinson & Laird, 1976; Glatzer, 1975;Laird & Chooi, 1976; McKnight, Bustin & Miller, 1977). Detection of non-nucleolartranscription units is more difficult in somatic mammalian cells on account of theabundant condensed chromatin. Until now it has been carried out only in culturedcells, that is, HeLa cells (Hamkalo & Miller, 1973; Miller & Bakken, 1972), isolatedcultured rat liver cells (Puvion-Dutilleul, Bernadac, Puvion & Bernhard, 1977c;

• To whom all correspondence should be sent.

182 F. Harper and F. Puvion-Dutilleul

Puvion-Dutilleul, Puvion & Bernhard, 1978), Chinese hamster ovary (CHO) cells(Puvion-Dutilleul, Bachellerie, Bernadac & Zalta, 1977 a; Puvion-Dutilleul, Bac-hellerie, Zalta & Bernhard, 19776) and isolated cultured mouse kidney cells (Puvion-Dutilleul & May, 1978). High-resolution autoradiographic studies demonstratedthe ribonucleoproteic nature of the growing nascent transcripts (Angelier, Hemon &Bouteille, 1979; Villard & Fakan, 1978). However, visualization of measurabletranscription units in non-cultured mammalian tissues has not been reported heretofore.

In the present paper, we describe non-nucleolar transcription complexes derivedfrom isolated nuclei of rat liver and, in particular, the morphology of a type of complexrarely seen in other cells and more abundant in this material.

MATERIALS AND METHODS

Male Wistar rats weighing 200 g were used in all experiments. The 2 largest lobes of theliver were removed under ether anaesthesia. They were immediately washed and minced inice-cold phosphate-buffered saline (PBS) containing 0-04 M NaCl.

Preparation of nuclei

Isolation of nuclei wa3 carried out according to a modification of the method of Zalta (Zalta,Zalta & Simard, 1971). All procedures were performed at 0-4 °C. The minced liver was rinsedin the chilled homogenizing medium and placed in a Dounce homogenizer containing 15 ml ofbuffer N (0-25 M sucrose, 10 raM Tris-HCl pH 7-4, 2-5 ITLM MgCls, o-i DIM CaCla and 20 /tg/mlpotassium polyvinyl sulphate). After 10—15 strokes with a loose pestle, the resulting homo-genate was filtered through 4 thicknesses of gauze. The non-ionic detergent, cemulsol NPT 6(Rhone-Progil, France), and collagenase (Sigma Chemical Co., USA) were added to obtainfinal concentrations of 0-3 % and 0-3 mg/ml, respectively. Many strokes with a tight pestlewere given in order to disrupt every cell as monitored by phase-contrast microscopic controls.This suspension was centrifuged at 400 g for 5 min (Jouan, E 96 centrifuge). The crude nucleiwere resuspended and washed in 15 ml of buffer N without detergent and collagenase. Purifiednuclei were collected by a 5-min centrifugation at 400 g.

Spread preparations

As previously described (Puvion-Dutilleul et al. 19776) the nuclear contents were spread ina hypotonic medium with or without 5 mM ethylenediamine tetra-acetate (EDTA) accordingto Miller's technique (Miller & Beatty, 1969; Miller & Bakken, 1972) with slight modifications.

Electron-microscopic techniques

All the preparations were stained with ethanolic phosphotungstic acid (PTA) for 1 min andrinsed in 95 % ethanol. Then, they were rotary-shadowed with platinum at an angle of 5-8°.Micrographs were taken with a Philipps EM 200 electron microscope at 60 kV. The magni-fication was checked by comparison with a grating replica (Fullam).

Length measurements

The length measurements were made with a Hewlet-Packard 9864 A digitizer interfacedwith a 9820 calculator.

Rat liver transcription complexes 183

RESULTS

Nuclear contents were always well dispersed and resulted in innumerable DNPfibres. When an EDTA-free spreading medium was used, generally the appearanceof these fibres was 'rough'; granules were contiguous or sometimes irregularlyspaced on the same DNA axis. The beaded structures were less frequent when thespreading technique was performed with an EDTA-containing solution. As usual,DNP fibres with attached RNP fibrils appeared either as ' Christmas-tree'-like figuresof nucleolar transcription or as units covered with widely and irregularly spacedtranscripts which are typical of non-nucleolar transcription in somatic mammaliancells. In addition, special units with closely spaced polymerases were observed whichseem to be related to HnRNA transcription. These atypical non-ribosomal transcrip-tion units were observed only one-third as often as the typical ones (Table 1).

Table 1. Ultrastructural data for non-ribosomal transcription units

No. ofindividualmeasure-

ments Range Mean s.D.

Typical non-ribosomal Lengths of fibril- 119 10-15-5 29 2-3transcription units covered DNP fibres,

/imRNA polymerase, 118 0-9-11-3 4-3 23

mol//tm of DNPfibre

Atypical non-ribosomal Lengths of fibril- 39 1-7—6-4 3-1 1 0transcription units covered D N P fibres,

fimRNA polymerase, 24 10-4-29 193 4-2mol//*m of DNP fibre

Ribosomal transcription complexes

Christmas-tree-like figures (Fig. 1) were similar to those previously described inisolated rat liver cells and CHO cells (Puvion-Dutilleul et al. 1977 b, c). The DNA axiswithin each matrix unit was 'smooth' between the RNA polymerase molecules.Measurements of 18 of these complexes revealed that the transcriptional units wererelatively constant in length: 3-76 ± 0-50 /tm (mean + s.D.) and their RNA polymerasemolecule density was 27 ± 4 enzyme mol//tm of DNP fibres. At a short distance fromthe initiation site the characteristic knob was observed at the free end of each trans-script (Fig. 4). The lateral fibrils reached a maximum length of about 0-25 /tm. Owingto the large number of matrix units in the vicinity of partially spread nuclei, we havenot been able to make a quantitative study of the length of the tandemly repeatingunits (matrix unit + spacer intercept).


X

• - • - * -


Typical non-ribosomal transcription complexes

Non-ribosomal transcripts were either solitary or clustered on DNP fibres. In thispaper, a transcriptional unit is defined as 2 or more nascent RNP fibrils carried by aDNP fibre larger than 1 /tm. As already described in cultured mammalian cells(see references in Introduction), these fibrils were heterogeneous in length and fre-quency and were either linear or twisted (Fig. 2). Occasionally, a regular increase ofthe nascent fibril lengths was observed (Fig. 7). The length of the transcripts mightreach up to 2-6 /im but 70% of them were shorter than 1 /im (mean value of 600measurements, 0-58 ± 0-43 /im). Inside the units, the DNP axis was often punctuatedby nucleosome-like particles and sometimes exhibited a 'bead on a string' appearance(Fig. 2). The high variability of the lengths of fibril-covered DNP fibres and of RNPdensities is confirmed by the quantitative analysis (Table 1).

Putative non-ribosomal transcription units with unusually high RNP fibril density

This peculiar pattern of transcription activity (Fig. 3) was more frequently observedin this biological material than in similar previous studies. It did not occur in clusters.It was characterized by closely spaced transcripts showing a regular gradient ofgrowth from an initiation site to a terminal site. Nevertheless, abnormally short RNPfibrils were sometimes observed inside the unit. The lengths of DNP fibres carryingtranscripts were distributed between 1-7 and 6-4 fiva but about 40% of them measuredbetween 2-5 and 3-0 /tm. This relative homogeneity in the lengths of the matrix unitswas confirmed by the low standard deviation of the mean value (Table 1).

The base of each RNP fibril was marked by a molecule of RNA polymerase (Fig. 6).In these atypical units, the density of transcripts was higher than in the typicalnon-ribosomal transcription units (Table 1). However, the RNA polymerase moleculeswere not so closely packed as in ribosomal transcription units. Between the RNApolymerase molecules, the DNP axis exhibited a smooth configuration (Figs. 3, 8).Nascent RNP fibrils showed a knobby structure of constant diameter. They wereoften superposed on adjacent transcripts and measurements were difficult. The longestmight reach up to i-2/im.

Figs. 1-3. Three types of transcription matrix units in isolated nuclei of rat liverspread with an EDTA-containing solution, x 30000. Bars represent 1 /im.

Fig. 1. Nucleolar matrix unit. Length of the DNP axis carrying transcripts, 4-0 /im.Density of the RNA polymerase molecules, 25/fim of DNP fibre. Note the 'smooth'configuration of the DNP axis in gaps (arrows).

Fig. 2. Typical non-ribosomal transcription unit. Low density of RNA poly-merase molecules, 43//im of DNP fibre. Between the transcripts the DNP axisexhibits a 'bead on a string' aspect (arrow). RNP fibrils are heterogeneous in lengthand spacing. Some of them are linear while others are twisted.

Fig. 3. Putative non-ribosomal transcription unit. Note the unusual high densityof the transcripts (21 RNP fibrils//im of DNP fibre). In regions free of fibrils (arrow)the DNP axis is also granule-free. Note the regular increase of the fibril lengths. Never-theless, short transcripts (long arrow) are inside the unit. Solitary RNP fibrils(arrowheads) are located on DNP fibres punctuated by some beads.

i86 F. Harper and F. Puvion-Dutilleul


In contrast to typical non-nucleolar arrays, DNP fibres of these putative non-ribosomal units always appeared ' smooth' and lateral RNP fibrils were never twisted.The high density of the latter and the triangular shape of the unit resemble thenucleolar matrix units. Nevertheless, the ultrastructural aspect of the transcripts,that is, the constant knobby structure without a distinct terminal knob, was not inagreement with such an origin. Fig. 8 corroborates this assumption. Indeed, itsterminal region exhibits a typical non-nucleolar array although the initiation parthas a 'Christmas-tree'-like appearance.

DISCUSSION

In this work we demonstrate that transcription complexes from a fresh mamma-lian organ can be examined in detail, and that transcription complexes of hepatocytesin vivo may differ from those of hepatocytes which are isolated and maintained invitro. Such somatic mammalian tissue has already been used (Laval, Bouvier & Bouteille,1978) but, in this preliminary work, the coiled non-nucleolar RNP fibrils did not allow adetailed study. The abundant condensed chromatin in liver cells is not convenientfor an easy spreading of matrix units. Therefore, we introduced slight modificationsto the original technique of Zalta et al. (1971) for isolation of nuclei in order tofacilitate the spreading of their contents by our modification of Miller's technique(Puvion-Dutilleul et al. 19776). Under our preparative conditions, the results werereproducible. Nucleolar matrix units were frequently coiled and clustered. WhenEDTA was added to the spreading medium, some of them were well extended andmeasurable. On the other hand, the spreading of the non-nucleolar transcriptionunits was not modified by the presence of EDTA. However, beaded particles becamerare on the DNP axis when the spreading was performed with a medium containingEDTA. Nevertheless, they were sometimes contiguous on the active DNP fibres intypical non-nucleolar units as shown in Fig. 2. In contrast, the atypical non-nucleolarunits never exhibited such granules whether EDTA was used or not. This may berelated to a high nucleic acid-synthesizing activity. It is in good agreement with theresults of others (Scheer, 1978; Scheer et al. 1976a, b; Foe, 1977) and with our ownprevious studies which established a correlation between the configuration of the

Figs. 4-6. Comparison between the RNP fibrils of 3 types of units. Nuclei spreadwith an EDTA medium, x 96500. Bars represent 025 /im. Simple arrows indicatethe RNA-polymerase molecules.

Fig. 4. Terminal region of a ribosomal matrix unit. The terminal knobs (arrow-head) are especially well shown.

Fig. 5. Solitary non-nucleolar RNP fibril. Its branched appearance arises fromfolds (barred arrow) of the granular fibrils. Note the beaded aspect of the right-handpart of the DNP fibre (arrowheads).

Fig. 6. Part of a putative non-nucleolar unit. Note the closely spaced lateral fibrilsand their regular diameter. In contrast to the transcripts of Fig. 4, they do not bear aa big knob at their free end.

i88 F. Harper and F. Puvion-Dutilleul

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f V ; ; ; : ; • ; . . • ' •••'..:. ' • • : : - \ ' . * "


active DNP axis and the level of transcription activity. Our morphological work doesnot permit us to equate the beaded particles with nucleosomes. Nevertheless, H2B andH3 histone proteins were demonstrated by immunoelectron microscopy (McKnightet al. 1977) to be in similar granules.

Typical ribosomal and non-ribosomal matrix units were similar in their patternand in the ultrastructure of their nascent RNP fibrils to those described in othermaterials (see Introduction). As a rule, the spacing and the length of the transcriptswere heterogeneous in non-ribosomal units whatever the contents of the spreadingsolution. Indeed, similar appearances were obtained whether detergent was added(Miller & Bakken, 1972; Villard & Fakan, 1978) or not (Foe et al. 1976; Laird & Chooi,1976; Laird, Wilkinson & Foe, 1976). The length heterogeneity of DNP intervalslocated between RNA polymerase molecules can be related to a variable frequencyof initiation.

The unusual pattern of non-nucleolar transcription activity which we obtainedafter spreading the isolated nuclei of rat liver was characterized by a long triangularshape and by a high density of the lateral RNP fibrils. We had not observed it inpreparations of isolated whole rat liver cells examined after culture (Puvion-Dutilleulet al. 1977c), but it was occasionally detected after cortisol stimulation of the culturedhepatocytes (Puvion-Dutilleul et al. 1978). Similar units also were obtained in cellsabortively infected with SV40 virus (Puvion-Dutilleul & May, 1978) and, recently,in sea-urchin embryos (Busby & Bakken, 1979, fig. 3 a). In the latter material, however,the transcripts were more widely spaced. A decrease of RNP synthesis by cell culturemight be inferred from these findings; the initial frequency of transcripts would beachieved by hormonal or viral stimulation. This phenomenon remains to be demon-strated conclusively. The special transcription units were interpreted as non-ribosomalarrays on account of the ultrastructural appearance of their RNP fibrils whichexhibited a constant beaded structure, whatever the spreading medium used. Incontrast with ribosomal transcripts, RNP fibrils in putative and typical non-nucleolarunits always exhibited a granular configuration. This knobby configuration seems tobe a general feature (Angelier & Lacroix, 1975; Keyl, 1975; Kierszenbaum & Tres,1975; Laird & Chooi, 1976; Laird et al. 1976; Oda, Nakamura & Watanabe, 1977;

Fig. 7. Typical non-nucleolar array spread from nuclear fraction with an EDTA-freesolution, x 38000. The transcripts exhibit a visible gradient of length. As usual, RNPfibrils are irregularly spaced and short transcripts are inside the unit (barred arrows).Density of transcripts, 8-s//im of DNP axis. The longest lateral fibril (arrowhead)measures 06 fim. Note the non-beaded structure of the DNP fibre between thetranscripts. Scale bar, 1 /(m.Fig. 8. Putative non-ribosomal transcription unit. Spreading with an EDTA-freesolution, x 50000. The DNP fibre covered by transcripts is 5-4 /ira long. In theinitiation region, closely spaced RNP fibrils (density io.//tm of DNP axis) cover2-7 /tm of DNP fibre. Then, the spacing of the transcripts is very heterogeneous as inthe typical non-nucleolar units. If we consider the entire unit, the density of the RNPfibrils is only 10-5 hbrih/fim. The longest transcript (arrowhead) measures 06 fim.Scale bar, 1 /im.

13 CIL 40


Puvion-Dutilleul et al. 1978; Puvion-Dutilleul & May, 1978; Villard & Fakan, 1978).

It agrees with biochemical data (Karn, Vidali, Boffa & Allfrey, 1977; Martin et al.

1977) which demonstrated that growing RNP fibrils were constituted by repeated

globular structures connected by ribonuclease-sensitive strands.

In conclusion, our results obtained with rat liver demonstrate that it is now possible

to study transcription complexes from whole somatic tissues. This procedure, which

eliminates the necessity of primary culture of previously isolated cells, makes it

possible to observe genetic activity in tissues submitted to different physiological

or pathological conditions in vivo.

This work is dedicated to the memory of Dr W. Bernhard. The authors acknowledge thetechnical assistance of Mr. R. Cousin who performed the shadow-casting. They thank Prof.E. Leduc for critical reading of the manuscript. Mrs Marchant provided efficient secretarialassistance. F. Puvion-Dutilleul is a member of INSERM.

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(Received 25 May 1979)

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