Visualization of nonribosomal transcriptional complexes after cortisol stimulation of isolated rat liver cells

JOURNAL OF ULTRASTRUCTURE RESEARCH

63, 118-131 (1978)

Visualization of Nonribosomal Transcriptional Complexes after Cortisol Stimulation of Isolated Rat Liver Cells FRANCINE PUVION-DUTILLEUL, ~ EDMOND P U V I O N , AND W I L H E L M BERNHARD

Institut de Recherches Scientifiques sur Ie Cancer, B. P. No. 8, 98400 ViIlejuif, France Received August 10,1977, and in revised form, November 14,1977 The organization of transcriptional complexes in chromatin of isolated rat hepatocytes was investigated. Two distinct types of patterns were found both with and without cortisol stimulation of template activity. (1) Nucleolar transcription was of the "Christmas-tree" type. It occurred rarely in nonstimulated controls. After hormonal treatment, however, the presence of masses of coiled m a t r i x units revealed a strongly increased transcriptional activity of the nucleolus. (2) Nonribosomal transcription figures also were rare in unstimulated nuclei and more frequently were found after hormonal treatment. Analysis of the RNP fibril lengths and of the RNA polymerase molecule densities revealed a high degree of heterogeneity. In these nonribosomal transcription arrays the RNP fibrils were, as a rule, of irregular length; only rarely did they show a regular gradient of growth. The morphology of the DNP fibers as observed under our preparative conditions was either "smooth" or "rought." Extended chromatin could be observed between the RNA polymerase molecules when the density of the RNP fibrils was higher t h a n four transcripts per micrometer of DNA fiber.

Miller and Bakken's technique (1972a) RNA synthesis: lampbrush chromosomes of spreading nuclear contents facilitated of amphibian oocytes, primary nuclei of the analysis of transcriptionally active green algae (Scheer et al., 1976a), and chromatin. The transcriptional organiza- embryos or spermatocytes of various intion of the nucleolar DNP has been well sects (Amabis and Nair, 1976; Foe et al., characterized and the redundancy of the 1976; Glatzer, 1975; Laird et al., 1976; Laird ribosomal genes favored their visualiza- and Chooi, 1976; McKnight and Miller, tion and quantitation (Angelier and Lac- 1976). roix, 1975; Berger and Schweiger, 1975a,b; In mammalian cells, on the other hand, Franke et al., 1976a; Hamkalo and Miller, transcriptional complexes have been very 1973; McKnight and Miller, 1976; Meyer rarely visualized. Only a few figures were and Henning, 1974; Scheer et al., 1973, detected in somatic or germinal cells 1976a; Sommerville and Malcolm, 1976; (Kierszenbaum and Tres, 1975; Miller and Spring et al., 1974, 1975, 1976; Trendelen- Bakken, 1972b; Oda and Omura, 1975). burg, 1974; Trendelenburg et al., 1973, Recently, we were able to characterize two 1974a,b, 1975). Recently, a quantitative different types of transcriptional comautoradiographic study of nucleolar chro- plexes by direct spreading of isolated rat matin was undertaken (Angelier et al., liver cells (Puvion-Dutilleul et al., 1977c) 1976). The characterization of the nonribo- but in this model the degree of transcripsomal RNP, however, has been generally tion is extremely low. In a further study based on biochemical difficult because this type of structural gene is usually nonredundant in somatic identification and spreading of purified cells. Nevertheless, interesting results nuclear fractions, we confirmed that these were obtained from certain biological ma- two types of transcription patterns were terials with a high level of heterodisperse specifically linked to ribosomal RNA and to heterodisperse RNA (Puvion-Dutilleul 1 To whom correspondence should be addressed. et al., 1977a,b). 118

0022-5320/78/0621-0118502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

TRANSCRIPTIONAL COMPLEXES IN RAT LIVER CELLS

We now have extended our work to develop experimental conditions which permit stimulation of the template activity of the liver cell DNA. Cytochemical and autoradiographic methods have demonstrated that cortisol strongly stimulates this activity (Moyne et al., 1977; Nash et al., 1975) in increasing the number of the perichromatin fibrils and the radioactive labeling over these fibrils. We describe here new data on the organizational pattern of DNA fibers and on the structure of transcriptional complexes in mammalian cells in which RNA synthesis is stimulated by hormonal treatment. MATERIALS AND METHODS

Cell preparation. WAG r a t liver cells were isolated by perfusion of the liver with a mixture of collagenase and hyaluronidase in Hanks' calciumfree solution as described elsewhere (Puvion et al., 1974). The dissociated cells (1.106) were suspended in 3 ml of MEM medium containing 10% calf serum and placed in 30-ml Falcon flasks. Hormone treatment. After 24 h r of incubation at 37°C in the presence of 5% CO2, the culture medium was discarded and replaced by the same nutritive medium containing 20 t~g/ml of hydrocortisone-21phosphate (Serlabo). Cells were examined after I to 5 h r of t r e a t m e n t . Controls consisted of cells incubated for the same periods in a cortisol-free medium. Spread preparations of liver cells. Our procedure for spreading r a t liver cells was previously described (Puvion-Dutilleul et al., 1977a). It is a n adaptation of the original Miller technique (Miller and Beatty, 1969; Miller and Bakken, 1972a) to our biological material. In this study two different spreading media were used. Both were hypotonic but one contained the commercial detergent Joy, while the other containing the same detergent without perfume was known as surfactant (Procter and Gamble, Cincinnati, Ohio). The concentration of both detergents was considered to be 100% before dilution and was decreased to 0.50% (v/v) with bidistilled water. The culture medium was removed and the cells were rapidly scraped off t h e i r substrate in the presence of 0.2 ml of 0.50% detergent. They were allowed to disperse in the spreading solutions (0.43% detergent) for 5 m i n and t h e n for 5 more m i n in 0.17% detergent. Copper grids coated with a carbon film and freshly glow discharged were deposited in microcentrifuge chambers which were filled with 1% formaldehyde containing 0.1 M sucrose (pH 8.5). The dispersed m a t e r i a l was lightly fixed (two drops

119

of 1% formaldehyde adjusted to pH 8.5 with borate buffer per milliliter of biological material) before layering onto the fixative cushion in the microcentrifuge chamber. After centrifugation (8 m i n at 2300 g and 10°C), the grids were washed for 30 sec in a Photo-Flo (Kodak) solution (0.40%, pH 7-8). Electron microscopic techniques. The dried grids were stained with 1% phosphotungstic acid (PTA) in 70% alcohol for 1 rain, t h e n rinsed in 95% alcohol. In addition, most of t h e m were rotary shadowed with platinum at a n angle of 6-8 ° (Edwards coating unit). Micrographs were t a k e n with a Siemens Elmiskop 1 A electron microscope at 40 kV. The magnification was checked with a g r a t i n g replica (Fullam). Length m e a s u r e m e n t s were performed with a Hewl e t t - P a c k a r d 9864 A digitizer interfaced with a 9820 calculator. Calculations and distribution analyses were made with a computer program kindly provided by G. Moyne (Villejuif). RESULTS

Commercial Joy and surfactant were used to detect a possible action of the perfume on the ultrastructure of the nucleic acid fibers. Our results were identical after use of either Joy or surfactant; therefore, these results shall be described together. All micrographs represent structures of stimulated cells, except Fig. 3 which is a control. After spreading whole liver cells, unstimulated or stimulated with cortisol, two major configurations were always visualized in a network of deoxyribonucleoprotein (DNP)fibers: (1) "Christmas-tree" figures were interpreted as ribosomal transcriptional matrix units. We already have presented our observations on ribosomal RNA complexes in rat liver cells elsewhere (Puvion-Dutilleul et al., 1977c), so we shall mention them only briefly in this paper in order to demonstrate the very different aspects of nonribosomal RNA transcripts which we are presenting here in detail; (2) arrays of twisted fibrils were organized differently from the "Christmas-trees" and were not serially repeated along the DNP fibers: They were single. In addition, isolated RNP fibrils were frequently observed. These structures were thought to represent nonribosomal RNP synthesis.

120

PUVION-DUTTILLEUL, PUVION, AND BERNHARD

Ribosomal Transcriptional Complexes In both stimulated and nonstimulated cultures the now classical ~Christmastree"-like figures were often grouped near dense unspread chromatin clumps. However, the abundant DNP fibers of the nucleolus-associated chromatin masked them and made their study very difficult. The typical tandem repetition of active matrices was hidden by innumerable DNP fibers intimately linked with the nucleolar body. In treated cells, there was a considerable increase of coiled matrix units. This is the only modification detected after cortisol stimulation. Indeed, although the ribosomal transcripts seemed more frequent, which is in good agreement with autoradiographic studies (Nash et al., 1975; Moyne et al., 1977), the numbers of RNA polymerase molecules per micrometer of DNA were not modified and varied with a mean around 37 (Fig. 1).

Nonribosomal Transcripts In both stimulated and nonstimulated hepatocytes RNP transcripts were roughly perpendicular to linear chromatin fibers. They were either single on the DNP fiber and did not constitute arrays (Fig. 5) or were grouped in solitary arrays (Fig. 2). Isolated RNP fibrils were either linear and easily traceable (Fig. 3) or considerably twisted (Fig. 4). The thickness of the RNP transcripts was variable. High magnification revealed their granular configuration. They were constituted by particles interconnected by a filament in lateral position (Fig. 5). Comparable to ~'beaded" chromatin, their diameter was irregular and larger than that of nucleosomes, measuring between 250 and 300 A. Solitary RNP fibrils were frequently observed, especially after cortisol treatment. In our preparative conditions the mean length of the traceable transcripts was 0.60 _+ 0.4 t~m. However, the transcripts observed in the treated cells were generally longer than those of the controls. The highest value reached was 2.4/zm in stim-

ulated cells and only 1 tLm in controls. In controls, 22% of the solitary transcripts measured between 0.2 and 0.3 t~m while the corresponding value for treated cells was 41% (Chart 1; Table I). Nonnucleolar RNP fibril arrays were never observed as tandem repeats. The number of clustered RNP fibrils was variable from one to another, as well as were the length and spacing. The mean length of the chromatin axes underlying the transcripts was 2.4 _+ 1.3 t~m in controls and 3.5 +__2.1 t~m in stimulated cells (Table II). RNP fibril frequency varied from 1.7 to 25 RNP fibrils/~m of chromatin strand with a mean value of 6.0 _+ 2.5 fibrils in controls and 7.3 _ 5.2 fibrils in treated cells (Table III). However, after hormonal treatment, about 28% of the R N P arrays had a RNP frequency equal or superior to 10; very high fibril frequencies rarely occurred but in this case the mean transcript density was nearly the same as for ribosomal R N P matrices (Puvion-Dutilleul et al., 1977b,c). The morphology of these unusually dense nonribosomal transcription arrays was difficult to determine because RNP fibrils were often superposed on adjacent transcripts (Fig. 2). However, the small number of RNP fibrils present in most transcribing units facilitated the measurements. In our case the mean length of fibrils in arrays was 0.3 _+ 0.2 /zm (Chart 2) while it was 0.6 ___ 0.4 t*m for solitary transcripts (Table I). This variation results from the difficulty in detecting short fibrils isolated in a dense network of DNP axes. Arrays of four or more fibrils were analyzed by the least-square method. As visible from the accompanying micrographs, the path of the twisted RNP fibrils was not always easy to determine. We followed conventions similar to those of Foe et al. (1976) and Laird et al. (1976). Accordingly, granules or a short protuberances were considered as hairpin RNP fibrils. The lengths of the individual fibers were plotted against the chromatin strand lengths

FIGS. 1,2. Comparison between a ribosomal and a nonribosomal RNP array in a cortisol-treated hepatocyte. (Fig. 1) x 45 000; (Fig. 2) x 30 000. The RNA polymerase density is in this case 37 molecules per micrometer of transcribed DNP in the ribosomal m a t r i x unit; the axis is easily observed in regions free of lateral fibrils or ~:gaps" which are also granule free (Fig. 1, arrows). In a transcriptional complex of nonnucleolar type (Fig. 2), a very irregular spacing of the lateral RNP fibrils is observed. Some RNA polymerase molecules are free of transcripts (double arrows). Platinum-shadowed preparation. 121

FIGs. 3-5. Various aspects of the nonribosomal RNP fibrils. Positive staining with PTA (Fig. 3); platinum-shadowed preparations (Figs. 4,5). (Fig. 3) x 25 000; (Fig. 4) x 60 000; (Fig. 5) x 240 000. Short RNP fibrils are located at both extremities of the array (Fig. 3, arrows). The DNP fibers carrying few transcripts display a beaded appearance (Fig. 4, arrow). The RNP fibrils are considerably branched, or twisted (Fig. 4), or linear (Fig. 5). The transcripts have a nodular appearance with particles of 280/~ in diameter; thus they are larger t h a n nucleosomes. 122

123

TRANSCRIPTIONAL COMPLEXES IN RAT LIVER CELLS

extending from the shortest fibril to all following ones. The regression line obtained was further analyzed if a signifi30

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CHART 1. Length distribution of isolated RNP fibrils. Controls: hatched blocks; cortisol stimulation: open blocks. Abscissa: fiber lengths in micrometers; ordinate: number of fibrils. The difference in the length distribution pattern observed between these histograms (longer RNP fibrils with cortisol) is not significant (X~ test). In untreated and stimulated preparations, the mean lengths are, respectively, 0.4 + 0.2 and (1.6 ± 0.5 fxm.

cant (10%) correlation was demonstrated by the r correlation coefficient. The correlation was estimated by a t test. If the correlation coefficient was satisfactory, the intersection of the regression line with the abscissa was considered as the best estimation of the initiation site. Two types of nonnucleolar arrays were obtained. Some arrays did not show a regular increase of the fibril lengths (Figs. 6-8), while the others displayed a linear gradient of the transcripts (Figs. 8-12). Both types were similar in their general aspect; in both cases, short RNP fibrils were seen among long fibrils; they were sometimes located at both extremities of the RNP transcript arrays (Fig. 3). This might be due either to stronger packing of the RNP fibrils insufficiently modified by the spreading procedure or to post-transcriptional processing of the nascent RNP fibrils. When regular growth of the fibril length was observed (Figs. 9-12) the analysis indicated a considerable variation in the slope of the regression lines from one array to another within the same area of observation. The base of the smallest RNP

TABLE I NONNUCLEAR RNP FIBRIL LENGTHS IN UNTREATED AND STIMULATED LIVER CELLS Number of individual measurements

Longest fibril (~m)

Mean length (tLm)

SD

Isolated RNP fibrils

Controls, stimulated cells

32 125

0.8 2.4

0.4 0.6

0.2 0.4

RNP fibrils in arrays

Controls, stimulated cells

102 438

1.6 1.7

0.5 0.3

0.3 0.2

TABLE II LENGTHS OF NONNUCLEOLAR DNP FIBERS CARRYING RNP ARRAYS IN UNTREATED AND STIMULATED CELLSa Number of Extreme length Mean length individual measureDirect meaData from Direct meaData from ments surement regression surement regression Controls Stimulated cells

7

0.7-4.5

1.1-5.2

2.4 +_ 1.3 a

3.7 +_ 2.1 b

10

2.0-9.2

2.3-11.3

3.5 x 2.1

4.8 _+ 2.5

a Transcribed chromatin lengths. b Standard deviation.

124

PUVION-DUTTILLEUL, PUVION, AND BERNHARD

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CHART 2. Length distribution of RNP fibrils associated in arrays. (A) Controls; (B) cortisol stimulation. Abscissa: fiber lengths in micrometers; ordinate: n u m b e r of fibrils. The difference in the length distribution pattern observed between controls and treated cells is statistically significant ( ~ test). In untreated and stimulated preparations, the m e a n lengths are, respectively, 0.4 -+ 0.2 and 0.3 _+ 0.2 tim. TABLE III NONNUCLEOLAR R N P FIBRIL DENSITIES IN ARRAYS OF UNTREATED AND STIMULATED LIVER CELLS a

Number of individual measurements Controls

9

Stimulated 32 cells a RNP Fibril density.

Extreme value

Mean

SD

2-10

6.0

2.5

1.7-25

7.3

5.2

fibrils was sometimes near the estimated initiation site; however, it was frequently located so far from it that this distance was longer than the part of the DNP strand covered with transcripts. In this latter case, the length of the first RNP fibril was more than 0.2 tzm. RNA polymerase molecules without RNP fibrils were found on the DNP strand between adjacent transcripts or in front of the first RNP

fiber. Indeed, polymerase molecules can be found without the corresponding RNP transcripts (Fig. 2). RNA polymerase molecules located even before the smallest transcript were never found on the presumed initiation site. The granular configuration described above for isolated RNP fibrils appeared to be a common feature of the nonnucleolar RNP transcripts. It was visualized as well by positive staining with PTA (Fig. 8) as by shadow casting with platinum (Fig. 9). Contrary to the ribosomal RNP transcript, the diameter of the nonribosomal RNP fibril did not exhibit an abrupt enlargement; the terminal knob was absent. The length of the longest RNP fibril from each array was always shorter than the length of the corresponding DNA as measured from the shortest transcript to the largest. From the RNP length/DNA length ratio it was possible to estimate the degree of

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FIos. 6,7. Interpretation of the ultrastructural aspect ofa nonribosomal RNP fibril array in a stimulated l~,epatocyte. Figure 7 is an interpretative drawing of Fig. 6 similar to those used by FOE et al. (1976). Chromatin: dotted line; transcript: solid line. Platinum-shadowed preparation (Fig. 6), x 65 000. The transcript frequency is 6.5 fibrils/tLm of DNP fiber. The length of the DNP-carrying transcripts is 2.5 t~m. Note its ~'smooth" aspect (arrow) while the other DNP fibers have a ~beaded" configuration (arrowheads). 125

FIGS. 8-10. Various aspects of probably nonribosomal transcription units in stimulated hepatocytes with unusually high fiber density. Figure 8 is a micrograph of a PTA-stained unit. Figures 9 and 10 are directly shadow cast. x 40 000. The granular configuration of the RNP fibrils is visible after positive staining and after shadow casting. The array of Fig. 9 shows a regular increase of the fibril length; the equation of its regression line is: y = 0.10x + 0.17 (r = 0.35). The RNP fibers of the array of Fig. 10 look, at first sight, like a plateau, but careful measurement revealed a regular gradient of length of the RNP fibril (y = 0.17x + 0.31; r = 0.57). 126

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FIGs. 11-14. Interpretation of the ultrastructure of a nonribosomal RNP fibril array in stimulated hepatocytes. Platinum-shadowed preparation (Fig. 11), x 20 000. Figure 12 is an interpretative drawing of Fig. 11; chromatin: dotted line, transcript: solid line. Figure 13 is the regression line; its equation is y = 0.05x + 0.16 (r = 0.62); it intercepts the abscissa 3.1 /~m from the shortest transcript (arrow). The DNP fiber carrying the transcripts is 8.3 t~m long; the regression dasta yield a length of 11.4 t~m. The transcript density is four fibrils per micrometer of DNP fiber. Figure 14 is the length distribution of RNP fibrils; their m e a n length is 0.4 +_ 0.2 t~m. In the alternative hypothesis of two initiation sites, the two regression lines have the equationsy = 0.10x + 0.11, r = 0.87 (left), a n d y = 0.19x + 0.05, r = 0.83 (right). 127

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128

PUVION-DUTTILLEUL, PUVION, AND BERNHARD

compaction of the RNP, assuming a negligible compaction of the extended DNA and no cleavage of the RNP. Under our experimental conditions, the length of the largest RNP fibrils represented only 21 _+ 7.5% of the transcribed regions of the DNP fiber. This value decreased to 14 _+ 5.0% when the length of the DNP axis was estimated from the regression line. Indeed, the site of transcription initiation inferred from the analysis of each array was located in front of the smallest R N P transcript; the ~active"DNP fibers were consequently longer than the chromatin strand lengths extending from the shortest RNP fibril to the last transcript in each array. This is illustrated by Figs. 13 and 14 where our estimation of the transcribed DNP fiber length varies from 8.3 to 11.4 tLm according to the location of the presumed initiation site. Active nonribosomal fibril arrays occurred usually alone along the DNP strand. The array illustrated in Fig. 11 is exceptional in length. However, it displays a discontinuity in fiber length. It was consequently analyzed by the leastsquare method assuming one or two initiation sites. In the latter case the discontinuity localized the second initiation site. One or two groups of RNP fibrils being accordingly defined, the results of the analysis showed that the likelihood of two initiation sites was not different from that of one site only. We cannot exclude either that heterogeneities in thickness and packing of the nascent RNP fibrils resulted from their incomplete spreading or from the partial dissociation of proteins. We are aware that neither the true length of the RNA in nascent RNP nor that of the ~active" DNA was correctly evaluated. The nonribosomal DNA packing ratio is unknown in our system. The above-mentioned evaluations are based on the assumption that DNA of the nonribosomal transcription units is completely extended without nucleosomes and, also, that the length of the ~active" DNP is identical to that of the extended chromatin. According

to these criteria, the longest ~active" DNA we have found was 11.3 tLm long (Figs. 1113). Thus it should have led to the transcription of an RNA of 11.3 x 106 dalton containing about 34 000 nucleotides. But, when we took into account the usual case of regression data, the mean length of the ~active" chromatin was 4.3 _+ 2.1 ~m theoretically representing 13 000 nucleotides. In this case, the lengths of the transcripts were only about 16% of the ~active" chromatin (Fig. 14). In the present study, when DNP fibers carried few isolated RNP transcripts, chromatin frequently had a ~beaded-necklace" configuration (Figs. 4,5). However, when RNP fibrils were grouped in nontandemly repeated clusters, the DNP axis underlying the transcripts exhibited a ~smooth" aspect (Figs. 6,7). The other DNP fibers deprived of transcript showed a ~beaded" aspect. DISCUSSION

The spreading procedure used in this study does not yield total chromatin dispersion. As stated in our previous work (Puvion-Dutilleul et al., 1977c), ~innumerable DNP fibers radiate from a dense opaque clump of unspread chromatin." Consequently, it is not possible to evaluate, even approximately, the proportion of well-dispersed chromatin. This constatation is constant when the spreading technique is performed with entire cells as described by McKnight and Miller (1976). With respect to nucleolar transcription, it can be argued that our preparative procedure applied to whole cells is not favorable for a detailed study of nucleolar transcription. This is certainly true for our cell system with only one or two nucleoli per cell, which are in addition surrounded by a thick border of nucleolus-associated chromatin. In other mammalian cells, we have obtained more satisfactory results for the visualization of ribosomal transcription using purified nucleolar fractions (Puvion-Dutilleul et al., 1977a,b). Spreading

TRANSCRIPTIONAL COMPLEXES IN RAT LIVER CELLS of the coiled ~Christmas-tree" figures and detection of the "spacer" regions should indeed be improved by prior fractionation of stimulated hepatocytes, but this rather complex manipulation may lead to a considerable loss of RNP fibrils due to the shearing forces or the use of various centrifugation media. After spreading of entire cells, the tandem repetition of active matrices with regular RNP fibril-free regions was never obtained such as visualized in nonmammalian materials (Miller and Hamkalo, 1972; Spring et al., 1974; Angelier and Lacroix, 1975; Berger and Schweiger, 1975a,b; Scheer et al., 1977). With respect to the nonribosomal transcription figures, solitary RNP fibrils are thought to represent scarce transcriptional events. Laird and Chooi (1976) have obtained similar results in Drosophila melanogaster. After the action of cortisol, the transcriptional arrays were more frequent and the fiber density of the transcripts was increased; it could exceptionally reach the value classically obtained in the nucleolar matrix units. Between the RNP fibrils of some arrays, some granules attached to the DNP axis were free of RNP fibril. Their morphology and staining properties were similar to those of the basal granules of the lateral RNP fibrils. Consequently, we considered them as RNA polymerase molecules. The absence of transcript could indicate either that RNA polymerase molecules are very numerous and that only some of them are involved in transcriptional events, or that some transcripts were detached by artifactual or physiological events before the end of RNA synthesis. Some nonnucleolar RNP arrays displayed a regular increase of the fibril lengths such as visualized in nonmammalian materials (Foe et al., 1976; Laird et al., 1976; Laird and Chooi, 1976). In this case, the slope of the regression line displayed a considerable variability (0.06-0.53). Nascent RNP fibrils were shorter than the corresponding transcribed DNA and the

129

RNP/DNP ratios were variable (0.070.30). The pronounced shortness of the nascent RNP fibrils, as compared to the length of the DNP strand underlying the transcripts of the array, might be correlated with the various ultrastructural aspects of the RNP fibrils (linear or branched, constituted by granules of various size). Under our preparative procedure in this study of nonnucleolar transcription, the fibril-free chromatin exhibited both ~smooth" and "beaded" configurations. The diameter of the ~%eaded" elements was about 130 /~ in platinum-shadowed structures. The length of the connecting filament located between the ~%eads" was very variable. Biochemical and electron microscopic studies have demonstrated the regular pattern of nucleosomal structures (Kornberg, 1974; Olins and Olins, 1974; Olins et al., 1975, 1976; Oudet et al.. 1975). Thus, our observation seems to indicate that the spreading procedure has induced an irregular decompaction of the DNA fiber. This is in good agreement with the results of Thoma and Koller (1977) and Worcel and Benyajati (1977) on the morphological changes of chromatin upon removal of the histones. In our study, it appeared that the "smooth" configuration of the chromatin strand coincided with a rather high transcriptional activity. Indeed, when the transcript frequency was very low (four RNP fibrils per micrometer of DNP), "beaded" chromatin also carried transcription. Are both aspects of the "active" chromatin related to physiological events or do they result from the spreading procedure? The method we used was not applied specifically to the study of nucleosomes: The shearing forces, the pH near 9.0, and the low ionic strength are known to disorganize these structures. In our experiments, "beaded" DNP fibers could be related to moderate transcription while strong transcriptional activity was carried by "smooth" DNP fiber. This fact is to be compared with the "smooth" configuration

130

PUVION-DUTTILLEUL, PUVION, AND BERNHARD

of the DNP axis of the nucleolar matrix unit easily visible in "gap" segments (Puvion-Dutilleul et al., 1977b). In a very different system with a very high transcriptional activity, Franke et al. (1976b) did not observe nucleosomes on DNP fibers of either lampbrush chromosomes or nucleolar chromatin in amphibian oocytes. The discontinuity observed in the gradients of RNP fibril lengths could result from two types of RNP packing. One kind would be particularly fragile and suppressed easily by a detergent or by stretching forces. It would result in an extended RNP transcript. The other kind of packing could be related to the granular RNP fibrils. "Branched" or "twisted" RNP structures could represent intermediate stages between the short but thick transcript and the linear, fully extended RNP fibrils. Another interpretation could be that abnormally short RNP fibrils resulted from artifactual breaking or cleavage of the transcript. Indeed, a local degradation of the RNP fibrils by the spreading procedure could be responsible for the abrupt discontinuity in the RNP length gradient as well as physiological processing of the transcript. A possible release of RNP during the transcriptional event was suggested by Laird and Chooi (1976). We consider artifactual degradation as unlikely because our spreading procedure applied to oocytes of Salamandra did not alter at all the typical pre ribosomal RNP fibrils (unpublished observations). Other authors who used low concentrations of detergent did not observe a modification of the transcriptional figure (Glatzer, 1975; Scheer et al., 1977). Moreover, biochemical control experiments designed to test the effect of Joy at the same concentration as used under our conditions have shown the absence of change in length of purified RNA (Dr. R. Lindigkeit, personal communication). This control study supports our assumption that, at least under our preparative conditions, Joy did not modify the length of the RNP fibrils. Yet, if alterations of the transcriptional complexes by the deter-

gent can be eliminated, some degradations during the spreading procedure by the endogenous liver RNases cannot be excluded. However, the action of these enzymes on RNP structures should be minimized by the low temperature and the short duration of the treatment before fixation. The least-square analysis of the peculiar array shown in Fig. 11 suggests that it contains a short untranscribed DNP fiber. But, as suggested by Laird and Chooi (1976), such discontinuity might result from a specific cleavage of RNP fibrils before the termination of transcription. Unfortunately, this dramatic picture is single in our material. Cortisol is well known to stimulate transcriptional activity. It has been directly visualized by means of high-resolution radioautography in electron micrographs (Nash et al., 1975; Moyne et al., 1977). Our observations of the transcriptional complexes in treated cells confirm, in a spectacular way, the considerable increase of transcriptional units after administration of the hormone for both ribosomal and nonribosomal RNA. In addition, the length distribution of the nonnucleolar RNP fibrils associated in arrays showed a significant difference (X2 test). The increased number of these units after hormone treatment was not sufficient to explain this difference. At present, we do not have a physiological explanation for this observation. Note added in proof. In a recent paper on nonribosomal transcription in Drosophila spermatocytes, RNP fibrils were also observed as a "bead"-like structure (ODA, T., NAKAMURA,T., AND WATANABE, S. J. (1977)Electr. Microsc. 26, 203-207). Moreover, biochemical data (KARN, J., VIDALI, G., BOFFA, L. C., AND ALLFRE¥, V. G. (1977) J. Biol. Chem. 252, 7307-7322) support the hypothesis t h a t nascent chains of HnRNA are constituted by "repeated globular structures connected by ribonuclease-sensitive strands." This work was supported in part by a g r a n t from the CNRS (ATP 2883) and by the Institut National de la Sant~ et de la Recherche M~dicale (INSERM). Detergents were kindly provided by Procter and Gamble. The authors wish to express their gratitude

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