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Visualization of two different types of nuclear transcriptional complexes in rat liver cells
JOURNAL OF ULTRASTRUCTURE RESEARCH 58, 1 0 8 - 1 1 7
(1977)
Visualization of Two Different Types of Nuclear Transcriptional Complexes in Rat Liver Cells F. PUVION-DUTILLEUL, A. BERNADAC, E. PUVlON, AND W . BERNHARD Institut de Recherches Scientifiques sur le Cancer, B. P. 8, 94800 Villejuif, France Received July 27, 1976 Transcriptional complexes were visualized by a modification of the method of Miller and Beatty in the chromatin of isolated and cultured r a t liver cells. U n d e r our experimental conditions, spread DNA molecules showing transcriptional activity were extremely rare. Nevertheless, two different p a t t e r n s were observed in most preparations: (i) multiple "Christmas-tree"-like figures, comparable in length and symmetry to the now classical forms and probably related to ribosomal RNA transcription; (ii) twisted fibrils in single units along a DNA molecule, irregular both in the length of the transcriptional complex and particularly in the length of the side branches. These latter are t h o u g h t to represent non-nucleolar transcription.
A considerable number of publications mated to be between 480 and 1100, has demonstrated the existence of tranwhereas the oocyte nucleus of Xenopus scriptional complexes, primarily of ribonulaevis, which has a diameter of 1200 t~m, cleoprotein precursors of rRNA but also of may enclose some 2.106 transcriptional messenger RNA. This work, however, was units of pre-rRNA (26). The moderate depossible only by the use of particularly gree of transcription in rat liver cell nuclei suitable cell systems: oocytes of Amphibia is not the only difficulty which makes its (1, 7, 12, 19) or of insects (27), spermato- electron microscopic visualization probcytes (5, 9) and embryos of Drosophila (8), lematic. In addition, the abundance of conor nuclei of Acetabularia or other algae (2, densed, inactive chromatin, a part of 3, 24, 25, 26, 28). In all these cases, the which is tightly linked to the nucleolar nuclei are very large and can be easily body, is a considerable handicap in obtainseparated from the cytoplasm. Oocytes ing suitable preparations. In spite of these have numerous nucleoli with circular obstacles, we chose liver cells for the prepDNA which is detached from the chromoaration of transcriptional complexes using some carrying the nucleolar organizer. a technique derived from that described by Such nucleoli can be readily isolated with Miller and Beatty (11) because ultrastruca minimum of non-nucleolar chromatin by tural, cytochemical, and autoradiographic simple hypotonic shock. The enormous demethods applied to thin sections of this gree of redundancy of these rRNA genes material have allowed us to gain an imporfacilitates their visualization and quantitant amount of information on RNA syntation (19, 24). thesis in situ (4, 13, 14). Unfortunately, mammalian cells are a Rat liver was dissociated by enzymic much less favorable object for this type of treatment and the isolated hepatocytes investigation. The model employed in this were grown in vitro prior to being submitstudy, for example, presents several techted to hypotonic shock associated with the nical difficulties. The nuclei of rat liver action of a detergent which facilitates the cells (hepatocytes) have a medium diamerapid disintegration of the cytoplasm and ter of only 8 tLm. The number of their the nuclear membrane. On the other redundant rRNA genes has been estihand, the proteins of the nucleohistone ill08 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
109
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
bers are gradually removed from the DNA molecule. The commercial product already used by Miller and Bakken (10), called Joy, which contains both ionic and nonionic detergents, has given us the best results obtained thus far. This paper describes in detail our procedure and our observations on two types of transcriptional units in rat hepatocytes.
TABLE I SCHEME FOR SPREADING OF TRANSCRIPTIONAL COMPLEXES OF ISOLATED HEPATOCYTES
Dilution after detaching of the monolayer
Time of treatment
0.05-ml cell suspension + 0.15-ml Joy, 0,40% --) Solution A Solution A + 0.20 ml Joy, 0.43% --) Solution B Solution B + 0.60 ml H20 ~ Solution C
2 min, 15 sec 2 rain, 30 sec 5 rain
MATERIALS AND METHODS Hepatocytes were isolated by the method described by Puvion et al. (17) from livers of 3-monthold Wistar rats. The dissociated cells were incubated at 37°C, in the presence of 5% CO2, for 24 to 48 hr in 30-ml Falcon flasks, with 1.106 cells each in 3 ml of MEM medium containing 10% calf serum. The cells were used before they formed a contiguous monolayer. For the application of the modified Miller-Beatty technique (11), the following solutions were used: (i) the detergent Joy (Procter and Gamble, Cincinnati, Ohio) in different concentrations in bidistilled water (0.02, 0.40, 0.43, and 0.50%) adjusted to pH 8.7-9.0 with 0.1 M sodium borate; (ii) 1% formaldehyde freshly prepared from paraformaldehyde (Merck) in a 0.1 M sucrose solution, also adjusted to pH 8.7-9.0 with 0.1 M borate; (iii) bidistilled water adjusted to pH 9.0 with 0.1 M borate. These three solutions were kept at melting-ice temperature during the entire manipulation. (iv) A wetting agent, Photo-Flo (Kodak) was used at 0.4% at pH 7.4, in borate buffer during drying to improve spreading of the specimens. All these solutions were freshly prepared before each experiment and filtered prior to use on Millipore filters (0.2 ~m). (v! Phosphotungstic acid (PTA) (Prolabo) was prepared from 1 ml of a stock solution of 4% aqueous PTA added to 3 ml of 95% ethanol The supernatant of the cultures was discarded and the cells were rapidly rinsed in Joy solution, 0.02%, then detached with a rubber policeman from their substrate in less t h a n 15 sec under the action of 0.2 ml of 0.50% Joy. After decreasing the concentration of the cells and the detergent progressively (see Table I) a drop of the final suspension was deposited immediately in microcentrifuge chambers as described by Miller and Bakken (10). The surface of the copper grids, coated with a carbon membrane, was rendered hydrophilic by glow discharge in an Edwards coating unit, Type 12E6, for 20 sec, at 1 A and 0.2 Torr. The centrifugation of the chambers was carried out for 8 rain at 2300g and 10°C. The grids were then rinsed for 30 sec in the Photo-Flo solution. After drying in air, the specimens were either
stained with alcoholic PTA for 1 min, then rinsed in ethanol 95% (45 sec), or they were shadow-cast with platinum on a rotating disk at an angle of 6-8 °. Observations were made with a Siemens electron microscope Elmiskop IA or a Philips EM 200 at 40 kV. Measurements were carried out exclusively on the positively stained preparations. RESULTS
The combined hypotonicity of the medium and the presence of the detergent Joy allows an acceptable degree of dispersal of the nucleic acid molecules. The carbon membranes are covered with innumerable d e o x y r i b o n u c l e o p r o t e i n (DNP) fibers, which radiate from a dense opaque clump of unspread chromatin. The fibers have a strongly granular appearance after shadow-casting, which is partially due to the method used, but the positive stain also reveals beaded elements along the DNA axis. An immense majority of the fibers seems to be inactive. The images which we interpret as transcriptional complexes are irregularly dispersed throughout the network of dispersed DNA fibers. As mentioned above, they are extremely rare in our material. Two types of distinct patterns can be observed: on the one hand, the ~'Christmas trees" with the gradient of symmetrically growing, densely packed lateral fibers; on the other hand, loosely clustered lateral fibrils having irregular lengths and irregular distances between the fibrils. These branches have a tendency to be irregularly twisted and their total length is usually longer than that of the classical type.
FIG. 1. Transcriptional complexes, ~Christmas trees," interpreted as nucleolar type visible w i t h i n a network of inactive DNP fibers. The arrows (-*) indicate the limits of a transcriptional u n i t which are of variable length and do not show regular spacers. Beaded aspect of DNP fibers ( ~ ) is probably due to the shadow-casting with p l a t i n u m Cdecoration"). x 20 000, 110
111
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
(i) The Christmas trees occur characteristically in groups ~f 6 t o 14 units, in between a network of inactive DNA fibers and close to the central dense zone of unspread chromatin. Curiously enough, we have not yet observed a tandem repetition of active matrices with regular ~'spacer" regions such as those published in all previous papers (Fig. 1). The length of the active portions of the DNA fibers varies between 4 and 4.5 tLm. The lateral fibrils have a diameter of 80 to 100/~ and over a certain distance their lengths increase gradually and regularly, reaching a maxim u m of about 0.22 tLm. Then, they remain about the same size and acquire a terminal knob of 200 to 300 /~ (Chart 1). These branches are perpendicular to the DNA axis. Granules of 100 to 150/~ in diameter are very frequently observed. They are thought to represent RNA polymerase. The average number of the lateral branches per micrometer is between 20 and 30 fibrils. They succeed each other quite regularly at close intervals, but there also are m a n y patterns where some regions within the transcriptional unit appear interrupted. These ~naked" regions are situated at different positions on the DNA axis within the Christmas tree and may measure up to 0.4 t~m in length (Fig. 2). Although the RNP fibril gradients are usually irregularly clustered, we have also observed totally isolated figures of the same type (Fig. 3). In this case, the density of their succeeding lateral RNP fibrils is only 10 to 15/t~m, but the total length of the transcribing DNA unit may measure up to 6 or 8 t~m. (ii) The second type of image which we also interpret as a transcriptional matrix has a very different aspect (Figs. 4 and 5). First of all, it is much more irregular and, second, it does not occur in clusters. The frequency and the lengths of the lateral fibrils are much more irregular. The initiation point is usually not detectable because the RNA fibrils do not have a gradient of increasing lengths. Granules
40.
2 c
E 3o-
i
E
~
2o-
l
I
E Z
10i
o11 Length
0,2 [pro]
CHART 1. Distribution p a t t e r n of lengths of the lateral fibrils of preribosomal RNP.
which might represent RNA polymerase molecules on the DNA axes are present, but are m u c h rarer than in Christmas trees (Fig. 4). In addition, the lateral fibrils do not have a terminal knob (Figs. 46); they are longer and more irregularly spread and may display lateral loops, probably due to incomplete spreading. The total length of these branches may reach 1.7 t~m but 70% of them measure less t h a n 0.55 t~m (Chart 2). The length of the active DNA reaches on the average 2.0 t~m but may go up to 4 tLm. The medium density of the lateral fibrils per micrometer is only between 6 and 8 units. With the technique described above and under our experimental conditions, we have observed per well-spread single nucleus an average of about 20 complexes of the Christmas tree type and only two of the second type. It is possible, of course, that, with further improvements in the method, more patterns of both types might be revealed. On the other hand, it is important to point out the fact that when the same technique described above was applied to oocyte nuclei of amphibians which were kindly offered to our laboratory by Prof. J. C. Lacroix (Ivry) and Dr. M. F. Trendelenburg (Heidelberg), we were also able to visualize an abundance of regular repeats of the classical rRNA transcrip-
FIG. 2. Matrix with a gradient of transcripts of nueleolar type of a total length of 3.8 /xrn. Two areas within the complex do not show lateral fibers ( ~ ). The arrow ( ~ ) indicates the direction of the transcription. Shadow-cast with Pt. x 43 000. FIG. 3. Isolated transcriptional unit. The terminal portion forms a loop, probably a spreading artifact. Direction of transcription (~). The average density of the lateral RNP branches is 15 fibrils/ttrh of DNP. Shadow-cast with Pt. x 43 000.
FIG. 4. Two transcriptional figures on a single DNP axis of the non-nucleolar type. This p a t t e r n usually appears as a single unit only. Rare, twisted lateral branches. The arrowheads ( ~ ) may indicate the rare presence of RNA polymerase molecules fixed on the DNP fiber (~). Shadow-cast with Pt. x 60 000. Fio. 5. Non-nucleolar transcriptional complex. Loosely spaced, highly twisted lateral fibers of irregular length, on two different axes of DNP ( ~ ). Probable direction of the transcription on m a i n figure (~). Positive stain with PTA. × 30 000. 113
FIG. 6. Transcriptional complexes of the non-nucleolar type. Very irregular and loose spacing of the highly twisted, lateral fibers along the faintly stained DNP axes (-->). The longest of these RNP branches measures 1.7 t*m. Many other "silent" DNP fibers with granular appearance ( ) ). Positive stain with PTA, × 43 000. 114
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
2c 2o-
E
.D
E
IO-
z
0,5 Length
1 ( iJm )
1,5
CHART 2. Distribution p a t t e r n of l e n g t h s of the lateral fibrils of non-nucleolar RNP.
tional units with regular spacers between them. DISCUSSION
The relatively moderate transcriptional activity of rat liver cell nuclei compared with that of other experimental systems represents a primary difficulty for visualizing transcriptional patterns in the electron microscope. A second handicap is certainly the fact that in our material, contrary to amphibian oocytes, the nucleolar genes are not separated from the carrier chromosome and the separation of active from nonactive components cannot be achieved with the method applied above. Finally, the extreme difficulty in demonstrating transcription in our material might be due to an inadequate technique which chopped off the nascent RNA molecules by shearing forces or by the action of an endogenous ribonuclease. This possibility has to be envisaged very seriously. However, if so, it is rather amazing that the same technique allowed us to visualize classical transcriptional complexes in cellular systems used by Miller and Franke and their collaborators and by Angelier and Lacroix (1). The granules on the inactive DNA fibers in rat hepatocytes differ from the RNA polymerase molecules by exhibiting weaker contrast after PTA staining. Their heterogeneous size and irregular distribu-
115
tion along the DNP fiber make their identification with the "nucleosomes" unlikely (15, 16, 23). We suggest that these granules are artifacts induced by the specimen preparation. Rat liver transcriptional patterns of the Christmas tree type, when clustered, very likely correspond to preribosomal RNA in spite of the absence of regular spacing. The length of the active DNA is in accordance with the molecular weight of 45S rRNA of mammals (4.5 × 106 daltons) (21). This RNA corresponds to a chain of 12,800 nucleotides. However, there is the usual but even more pronounced discrepancy between the length of the active part of the DNA axis and the longest RNA fibers which measure only up to 0.22 t~m. This value is slightly inferior to that obtained by Miller and Bakken (10) with HeLa cells (0.25 tLm), but the difference is greater when compared to the length of the transcribed rRNA in amphibian oocytes (0.5 t~m) (10). This short transcript in our material is always characterized by a "terminal knob" which may have wrapped up a large portion of the molecule and does not allow unravelling under the given spreading conditions. The absence of lateral fibrils in some areas within the complex probably translates a local degradation of the transcripts or a discontinuous initiation of the transcription. We can hardly make the action of the detergent responsible for this irregularity because Joy, applied under the same conditions, neither alters the aspect of the matrix of preribosomal RNP in Salamandra, nor does it change their regular repeats (unpublished observation). Our findings are in good agreement with those of Scheer et al. (20) showing the presence of incomplete transcription units in Triton oocytes with naturally reduced transcriptional activity. As mentioned above, we have also found single, isolated transcriptional matrices measuring 6 to 8 t~m. Although these are not clustered, we believe that they represent unusual transcripts of rRNA. Indeed, a certain degree of heterogeneity
116
PUVION-DUTILLEUL E T A L .
in the length of rDNA has been found in
Acetabularia by Berger and Schweiger (2, 3). Concerning the second type of transcriptional pattern, we assume that this may represent non-nucleolar RNA synthesis. With regard to the length of the lateral fibrils, these RNAs could belong to the class of heterogeneous RNA (HnRNA) giving rise to mRNA (22). The longest fibers we have measured are not very long: 1.7 t~m, which corresponds to a molecular weight of the order of 1.7 × l0 s daltons containing about 5000 nucleotides. Although premessenger RNA may be 10 times longer, so far we have not observed the corresponding figures. It is quite possible that fibers of such length are detached or degraded during the preparation procedures. This phenomenon was observed with RNP in spermatocyte nuclei of Drosophila (5). As mentioned above, such patterns are extremely rare in the hepatocytes we used. They are always single, never clustered. This might be an argument in favor of the hypothesis that they represent structural genes which are said to occur as single units in the chromosomes, except for histone genes, and are not redundant as are ribosomal genes (6). There is also an important difference compared with the transcripts of lampbrush chromosomes. In the latter the numerous RNA polymerase molecules succeed each other regularly. In our electron micrographs they are rare and the intervals between the lateral branches are extremely irregular. They are comparable to those shown by Miller and Bakken (10) in HeLa cells and seem to indicate that, in mammalian cells, the extranucleolar transcription may not take place clustered or in regular repeats, as has been observed in other cells studied so far. In conclusion, in spite of the very unfavorable material we have used compared to cell systems which are much more suitable for this type of investigation, it is possi-
ble to visualize two types of transcriptional patterns in rat liver cell chromatin which probably represent two different modes of gene activity. This work was carried out with the financial aid of INSERM. We are very m u c h indebted to Mrs. N. Angelier and Prof. J. L. Lacroix as well as to Prof. W. W. F r a n k e and his collaborators, Dr. H. Spring a n d Dr. M. F. Trendelenburg, for demonstration of the basic steps of the spreading method applied above. We also wish to t h a n k Prof. Elizabeth Leduc for critical discussion and Mrs. Christiane Taligault for the preparation of the manuscript.
Note added in proof. In a recent paper [Amabis, J. M., and Naip, K. K., Z. Naturforsch. 31, 186 (1976)] on transcription in Diptera spermatocytes, nonribosomal transcription was also observed as irregularly spaced RNP fibrils, not as closely packed as in the active ribosomal DNP. REFERENCES
1. ANGELIER,N., AND LACROIX,J. C., Chromosoma (Berlin) 51, 323 (1975). 2. BERGER, S., AND SCHWEIGER, H. G., Planta (Berlin) 127, 49 (1975). 3. BERGER, S., AND SCHWEIGER, H. G., Mol. Gen. Genet. 139, 269 (1975). 4. FAKAN, S., PUVION, E., AND SPOHR, G., Exp. Cell Res. 99, 155 (1976). 5. GLATZER, K. H., Chromosoma (Berlin) 53, 371 (1975). 6. GREENBERG,J. R., J. Cell Biol. 64, 269 (1975). 7. HAMKALO,B. A., AND MILLER, O. L., J R . , A n n u . Rev. Biochem. 42, 379 (1973). 8. MAC KNIGHT, S. L., AND MILLER, 0. L., JR., Cell 8, 305 (1976). 9. MEYER, G. F., AND HENNIG, W., Chromosoma (Berlin) 46, 121 (1974). 10. MILLER, O. L., JR., AND BAKKEN, A. H., In DICZFALUSY E., (Ed.), Gene Transcription in Reproductive Tissue, p. 155. Karolinska Institute, Stockholm, 1972. 11. MILLER, O. L., JR., AND BEATTY, B. R., Science 164, 955 (1969). 12. MILLER, O. L., JR., AND HAMKALO, B. A., Int. Rev. Cytol. 33, 1 (1972). 13. MONNERON, A., AND BERNHARD, W., J. Ultrastruct. Res. 27, 266 (1969). 14. NASH, R. E., PUVION, E., AND BERNHARD, W., J . Ultrastruct. Res. 53, 395 (1975). 15. OLINS, A. L., AND OLINS, D. E., Science 183, 330 (1974). 16. OUDET, P., GROSS-BELLARD, M., AND CHAMBON,
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI P., Cell 4, 281 (1975). 17. PUVION, E., GARRIDO,J., AND VIRON, A., C. R. Acad. Sci. (Paris) 279, 509 (1974). 18. PUVION-DUTILLEUL,F., AND BERNADAC, A., J. Microsc. Biol. Cell. 26, 23a (1976). 19. SOHSF.R, U., TRENDELENBURG, M. F., AND FRANKS, W. W., Exp. Cell Res. 80, 175 (1973). 20. SCHEER, U., TRENDELENBURG, M. F., AND FRANKS, W. W., J. Cell Biol. 69, 465 (1976). 21. SCHERRER, K., LATHAM, H., AND DARNELL, J. E., Proc. Nat. Acad. Sci. USA 49, 240 (1963). 22. SCHERRER, K., AND MARCAUD, L., Bull. Soc. Chim. Biol. 47, 1967 (1965). 23. SCHWARZACHER,H. G.,Pathol. Eur. 11, 5 (1976).
117
24. SPRING, H., KROHNE, G., FRANKE, W. W., SCHEER, U., AND TRENDELENBURG,M. F., J. Microsc. Biol. Cell. 25, 107 (1976). 25. SPRING, H., SCHEER, U., FRANKE~ W. W., AND TRENDELENBURG, M. F., Chromosoma (Berlin) 50, 25 (1975). 26. SPRING, H., TRENDELENBURG, M. F., SCHEER, U., FRANKE, W. W., AND HERTH~W., Cytobiologie 10, 1 (1974). 27. TRENDELENBURG,M. F., Chromosoma (Berlin) 48, 119 (1974). 28. TRENDELENBURG, M. F., SPRING, H., SCHEER, U., AND FRANKE, W. W., Proc. Nat. Acad. Sci. USA 71, 3626 (1974).
(1977)
Visualization of Two Different Types of Nuclear Transcriptional Complexes in Rat Liver Cells F. PUVION-DUTILLEUL, A. BERNADAC, E. PUVlON, AND W . BERNHARD Institut de Recherches Scientifiques sur le Cancer, B. P. 8, 94800 Villejuif, France Received July 27, 1976 Transcriptional complexes were visualized by a modification of the method of Miller and Beatty in the chromatin of isolated and cultured r a t liver cells. U n d e r our experimental conditions, spread DNA molecules showing transcriptional activity were extremely rare. Nevertheless, two different p a t t e r n s were observed in most preparations: (i) multiple "Christmas-tree"-like figures, comparable in length and symmetry to the now classical forms and probably related to ribosomal RNA transcription; (ii) twisted fibrils in single units along a DNA molecule, irregular both in the length of the transcriptional complex and particularly in the length of the side branches. These latter are t h o u g h t to represent non-nucleolar transcription.
A considerable number of publications mated to be between 480 and 1100, has demonstrated the existence of tranwhereas the oocyte nucleus of Xenopus scriptional complexes, primarily of ribonulaevis, which has a diameter of 1200 t~m, cleoprotein precursors of rRNA but also of may enclose some 2.106 transcriptional messenger RNA. This work, however, was units of pre-rRNA (26). The moderate depossible only by the use of particularly gree of transcription in rat liver cell nuclei suitable cell systems: oocytes of Amphibia is not the only difficulty which makes its (1, 7, 12, 19) or of insects (27), spermato- electron microscopic visualization probcytes (5, 9) and embryos of Drosophila (8), lematic. In addition, the abundance of conor nuclei of Acetabularia or other algae (2, densed, inactive chromatin, a part of 3, 24, 25, 26, 28). In all these cases, the which is tightly linked to the nucleolar nuclei are very large and can be easily body, is a considerable handicap in obtainseparated from the cytoplasm. Oocytes ing suitable preparations. In spite of these have numerous nucleoli with circular obstacles, we chose liver cells for the prepDNA which is detached from the chromoaration of transcriptional complexes using some carrying the nucleolar organizer. a technique derived from that described by Such nucleoli can be readily isolated with Miller and Beatty (11) because ultrastruca minimum of non-nucleolar chromatin by tural, cytochemical, and autoradiographic simple hypotonic shock. The enormous demethods applied to thin sections of this gree of redundancy of these rRNA genes material have allowed us to gain an imporfacilitates their visualization and quantitant amount of information on RNA syntation (19, 24). thesis in situ (4, 13, 14). Unfortunately, mammalian cells are a Rat liver was dissociated by enzymic much less favorable object for this type of treatment and the isolated hepatocytes investigation. The model employed in this were grown in vitro prior to being submitstudy, for example, presents several techted to hypotonic shock associated with the nical difficulties. The nuclei of rat liver action of a detergent which facilitates the cells (hepatocytes) have a medium diamerapid disintegration of the cytoplasm and ter of only 8 tLm. The number of their the nuclear membrane. On the other redundant rRNA genes has been estihand, the proteins of the nucleohistone ill08 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
109
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
bers are gradually removed from the DNA molecule. The commercial product already used by Miller and Bakken (10), called Joy, which contains both ionic and nonionic detergents, has given us the best results obtained thus far. This paper describes in detail our procedure and our observations on two types of transcriptional units in rat hepatocytes.
TABLE I SCHEME FOR SPREADING OF TRANSCRIPTIONAL COMPLEXES OF ISOLATED HEPATOCYTES
Dilution after detaching of the monolayer
Time of treatment
0.05-ml cell suspension + 0.15-ml Joy, 0,40% --) Solution A Solution A + 0.20 ml Joy, 0.43% --) Solution B Solution B + 0.60 ml H20 ~ Solution C
2 min, 15 sec 2 rain, 30 sec 5 rain
MATERIALS AND METHODS Hepatocytes were isolated by the method described by Puvion et al. (17) from livers of 3-monthold Wistar rats. The dissociated cells were incubated at 37°C, in the presence of 5% CO2, for 24 to 48 hr in 30-ml Falcon flasks, with 1.106 cells each in 3 ml of MEM medium containing 10% calf serum. The cells were used before they formed a contiguous monolayer. For the application of the modified Miller-Beatty technique (11), the following solutions were used: (i) the detergent Joy (Procter and Gamble, Cincinnati, Ohio) in different concentrations in bidistilled water (0.02, 0.40, 0.43, and 0.50%) adjusted to pH 8.7-9.0 with 0.1 M sodium borate; (ii) 1% formaldehyde freshly prepared from paraformaldehyde (Merck) in a 0.1 M sucrose solution, also adjusted to pH 8.7-9.0 with 0.1 M borate; (iii) bidistilled water adjusted to pH 9.0 with 0.1 M borate. These three solutions were kept at melting-ice temperature during the entire manipulation. (iv) A wetting agent, Photo-Flo (Kodak) was used at 0.4% at pH 7.4, in borate buffer during drying to improve spreading of the specimens. All these solutions were freshly prepared before each experiment and filtered prior to use on Millipore filters (0.2 ~m). (v! Phosphotungstic acid (PTA) (Prolabo) was prepared from 1 ml of a stock solution of 4% aqueous PTA added to 3 ml of 95% ethanol The supernatant of the cultures was discarded and the cells were rapidly rinsed in Joy solution, 0.02%, then detached with a rubber policeman from their substrate in less t h a n 15 sec under the action of 0.2 ml of 0.50% Joy. After decreasing the concentration of the cells and the detergent progressively (see Table I) a drop of the final suspension was deposited immediately in microcentrifuge chambers as described by Miller and Bakken (10). The surface of the copper grids, coated with a carbon membrane, was rendered hydrophilic by glow discharge in an Edwards coating unit, Type 12E6, for 20 sec, at 1 A and 0.2 Torr. The centrifugation of the chambers was carried out for 8 rain at 2300g and 10°C. The grids were then rinsed for 30 sec in the Photo-Flo solution. After drying in air, the specimens were either
stained with alcoholic PTA for 1 min, then rinsed in ethanol 95% (45 sec), or they were shadow-cast with platinum on a rotating disk at an angle of 6-8 °. Observations were made with a Siemens electron microscope Elmiskop IA or a Philips EM 200 at 40 kV. Measurements were carried out exclusively on the positively stained preparations. RESULTS
The combined hypotonicity of the medium and the presence of the detergent Joy allows an acceptable degree of dispersal of the nucleic acid molecules. The carbon membranes are covered with innumerable d e o x y r i b o n u c l e o p r o t e i n (DNP) fibers, which radiate from a dense opaque clump of unspread chromatin. The fibers have a strongly granular appearance after shadow-casting, which is partially due to the method used, but the positive stain also reveals beaded elements along the DNA axis. An immense majority of the fibers seems to be inactive. The images which we interpret as transcriptional complexes are irregularly dispersed throughout the network of dispersed DNA fibers. As mentioned above, they are extremely rare in our material. Two types of distinct patterns can be observed: on the one hand, the ~'Christmas trees" with the gradient of symmetrically growing, densely packed lateral fibers; on the other hand, loosely clustered lateral fibrils having irregular lengths and irregular distances between the fibrils. These branches have a tendency to be irregularly twisted and their total length is usually longer than that of the classical type.
FIG. 1. Transcriptional complexes, ~Christmas trees," interpreted as nucleolar type visible w i t h i n a network of inactive DNP fibers. The arrows (-*) indicate the limits of a transcriptional u n i t which are of variable length and do not show regular spacers. Beaded aspect of DNP fibers ( ~ ) is probably due to the shadow-casting with p l a t i n u m Cdecoration"). x 20 000, 110
111
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
(i) The Christmas trees occur characteristically in groups ~f 6 t o 14 units, in between a network of inactive DNA fibers and close to the central dense zone of unspread chromatin. Curiously enough, we have not yet observed a tandem repetition of active matrices with regular ~'spacer" regions such as those published in all previous papers (Fig. 1). The length of the active portions of the DNA fibers varies between 4 and 4.5 tLm. The lateral fibrils have a diameter of 80 to 100/~ and over a certain distance their lengths increase gradually and regularly, reaching a maxim u m of about 0.22 tLm. Then, they remain about the same size and acquire a terminal knob of 200 to 300 /~ (Chart 1). These branches are perpendicular to the DNA axis. Granules of 100 to 150/~ in diameter are very frequently observed. They are thought to represent RNA polymerase. The average number of the lateral branches per micrometer is between 20 and 30 fibrils. They succeed each other quite regularly at close intervals, but there also are m a n y patterns where some regions within the transcriptional unit appear interrupted. These ~naked" regions are situated at different positions on the DNA axis within the Christmas tree and may measure up to 0.4 t~m in length (Fig. 2). Although the RNP fibril gradients are usually irregularly clustered, we have also observed totally isolated figures of the same type (Fig. 3). In this case, the density of their succeeding lateral RNP fibrils is only 10 to 15/t~m, but the total length of the transcribing DNA unit may measure up to 6 or 8 t~m. (ii) The second type of image which we also interpret as a transcriptional matrix has a very different aspect (Figs. 4 and 5). First of all, it is much more irregular and, second, it does not occur in clusters. The frequency and the lengths of the lateral fibrils are much more irregular. The initiation point is usually not detectable because the RNA fibrils do not have a gradient of increasing lengths. Granules
40.
2 c
E 3o-
i
E
~
2o-
l
I
E Z
10i
o11 Length
0,2 [pro]
CHART 1. Distribution p a t t e r n of lengths of the lateral fibrils of preribosomal RNP.
which might represent RNA polymerase molecules on the DNA axes are present, but are m u c h rarer than in Christmas trees (Fig. 4). In addition, the lateral fibrils do not have a terminal knob (Figs. 46); they are longer and more irregularly spread and may display lateral loops, probably due to incomplete spreading. The total length of these branches may reach 1.7 t~m but 70% of them measure less t h a n 0.55 t~m (Chart 2). The length of the active DNA reaches on the average 2.0 t~m but may go up to 4 tLm. The medium density of the lateral fibrils per micrometer is only between 6 and 8 units. With the technique described above and under our experimental conditions, we have observed per well-spread single nucleus an average of about 20 complexes of the Christmas tree type and only two of the second type. It is possible, of course, that, with further improvements in the method, more patterns of both types might be revealed. On the other hand, it is important to point out the fact that when the same technique described above was applied to oocyte nuclei of amphibians which were kindly offered to our laboratory by Prof. J. C. Lacroix (Ivry) and Dr. M. F. Trendelenburg (Heidelberg), we were also able to visualize an abundance of regular repeats of the classical rRNA transcrip-
FIG. 2. Matrix with a gradient of transcripts of nueleolar type of a total length of 3.8 /xrn. Two areas within the complex do not show lateral fibers ( ~ ). The arrow ( ~ ) indicates the direction of the transcription. Shadow-cast with Pt. x 43 000. FIG. 3. Isolated transcriptional unit. The terminal portion forms a loop, probably a spreading artifact. Direction of transcription (~). The average density of the lateral RNP branches is 15 fibrils/ttrh of DNP. Shadow-cast with Pt. x 43 000.
FIG. 4. Two transcriptional figures on a single DNP axis of the non-nucleolar type. This p a t t e r n usually appears as a single unit only. Rare, twisted lateral branches. The arrowheads ( ~ ) may indicate the rare presence of RNA polymerase molecules fixed on the DNP fiber (~). Shadow-cast with Pt. x 60 000. Fio. 5. Non-nucleolar transcriptional complex. Loosely spaced, highly twisted lateral fibers of irregular length, on two different axes of DNP ( ~ ). Probable direction of the transcription on m a i n figure (~). Positive stain with PTA. × 30 000. 113
FIG. 6. Transcriptional complexes of the non-nucleolar type. Very irregular and loose spacing of the highly twisted, lateral fibers along the faintly stained DNP axes (-->). The longest of these RNP branches measures 1.7 t*m. Many other "silent" DNP fibers with granular appearance ( ) ). Positive stain with PTA, × 43 000. 114
TRANSCRIPTIONAL COMPLEXES IN LIVER NUCLEI
2c 2o-
E
.D
E
IO-
z
0,5 Length
1 ( iJm )
1,5
CHART 2. Distribution p a t t e r n of l e n g t h s of the lateral fibrils of non-nucleolar RNP.
tional units with regular spacers between them. DISCUSSION
The relatively moderate transcriptional activity of rat liver cell nuclei compared with that of other experimental systems represents a primary difficulty for visualizing transcriptional patterns in the electron microscope. A second handicap is certainly the fact that in our material, contrary to amphibian oocytes, the nucleolar genes are not separated from the carrier chromosome and the separation of active from nonactive components cannot be achieved with the method applied above. Finally, the extreme difficulty in demonstrating transcription in our material might be due to an inadequate technique which chopped off the nascent RNA molecules by shearing forces or by the action of an endogenous ribonuclease. This possibility has to be envisaged very seriously. However, if so, it is rather amazing that the same technique allowed us to visualize classical transcriptional complexes in cellular systems used by Miller and Franke and their collaborators and by Angelier and Lacroix (1). The granules on the inactive DNA fibers in rat hepatocytes differ from the RNA polymerase molecules by exhibiting weaker contrast after PTA staining. Their heterogeneous size and irregular distribu-
115
tion along the DNP fiber make their identification with the "nucleosomes" unlikely (15, 16, 23). We suggest that these granules are artifacts induced by the specimen preparation. Rat liver transcriptional patterns of the Christmas tree type, when clustered, very likely correspond to preribosomal RNA in spite of the absence of regular spacing. The length of the active DNA is in accordance with the molecular weight of 45S rRNA of mammals (4.5 × 106 daltons) (21). This RNA corresponds to a chain of 12,800 nucleotides. However, there is the usual but even more pronounced discrepancy between the length of the active part of the DNA axis and the longest RNA fibers which measure only up to 0.22 t~m. This value is slightly inferior to that obtained by Miller and Bakken (10) with HeLa cells (0.25 tLm), but the difference is greater when compared to the length of the transcribed rRNA in amphibian oocytes (0.5 t~m) (10). This short transcript in our material is always characterized by a "terminal knob" which may have wrapped up a large portion of the molecule and does not allow unravelling under the given spreading conditions. The absence of lateral fibrils in some areas within the complex probably translates a local degradation of the transcripts or a discontinuous initiation of the transcription. We can hardly make the action of the detergent responsible for this irregularity because Joy, applied under the same conditions, neither alters the aspect of the matrix of preribosomal RNP in Salamandra, nor does it change their regular repeats (unpublished observation). Our findings are in good agreement with those of Scheer et al. (20) showing the presence of incomplete transcription units in Triton oocytes with naturally reduced transcriptional activity. As mentioned above, we have also found single, isolated transcriptional matrices measuring 6 to 8 t~m. Although these are not clustered, we believe that they represent unusual transcripts of rRNA. Indeed, a certain degree of heterogeneity
116
PUVION-DUTILLEUL E T A L .
in the length of rDNA has been found in
Acetabularia by Berger and Schweiger (2, 3). Concerning the second type of transcriptional pattern, we assume that this may represent non-nucleolar RNA synthesis. With regard to the length of the lateral fibrils, these RNAs could belong to the class of heterogeneous RNA (HnRNA) giving rise to mRNA (22). The longest fibers we have measured are not very long: 1.7 t~m, which corresponds to a molecular weight of the order of 1.7 × l0 s daltons containing about 5000 nucleotides. Although premessenger RNA may be 10 times longer, so far we have not observed the corresponding figures. It is quite possible that fibers of such length are detached or degraded during the preparation procedures. This phenomenon was observed with RNP in spermatocyte nuclei of Drosophila (5). As mentioned above, such patterns are extremely rare in the hepatocytes we used. They are always single, never clustered. This might be an argument in favor of the hypothesis that they represent structural genes which are said to occur as single units in the chromosomes, except for histone genes, and are not redundant as are ribosomal genes (6). There is also an important difference compared with the transcripts of lampbrush chromosomes. In the latter the numerous RNA polymerase molecules succeed each other regularly. In our electron micrographs they are rare and the intervals between the lateral branches are extremely irregular. They are comparable to those shown by Miller and Bakken (10) in HeLa cells and seem to indicate that, in mammalian cells, the extranucleolar transcription may not take place clustered or in regular repeats, as has been observed in other cells studied so far. In conclusion, in spite of the very unfavorable material we have used compared to cell systems which are much more suitable for this type of investigation, it is possi-
ble to visualize two types of transcriptional patterns in rat liver cell chromatin which probably represent two different modes of gene activity. This work was carried out with the financial aid of INSERM. We are very m u c h indebted to Mrs. N. Angelier and Prof. J. L. Lacroix as well as to Prof. W. W. F r a n k e and his collaborators, Dr. H. Spring a n d Dr. M. F. Trendelenburg, for demonstration of the basic steps of the spreading method applied above. We also wish to t h a n k Prof. Elizabeth Leduc for critical discussion and Mrs. Christiane Taligault for the preparation of the manuscript.
Note added in proof. In a recent paper [Amabis, J. M., and Naip, K. K., Z. Naturforsch. 31, 186 (1976)] on transcription in Diptera spermatocytes, nonribosomal transcription was also observed as irregularly spaced RNP fibrils, not as closely packed as in the active ribosomal DNP. REFERENCES
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24. SPRING, H., KROHNE, G., FRANKE, W. W., SCHEER, U., AND TRENDELENBURG,M. F., J. Microsc. Biol. Cell. 25, 107 (1976). 25. SPRING, H., SCHEER, U., FRANKE~ W. W., AND TRENDELENBURG, M. F., Chromosoma (Berlin) 50, 25 (1975). 26. SPRING, H., TRENDELENBURG, M. F., SCHEER, U., FRANKE, W. W., AND HERTH~W., Cytobiologie 10, 1 (1974). 27. TRENDELENBURG,M. F., Chromosoma (Berlin) 48, 119 (1974). 28. TRENDELENBURG, M. F., SPRING, H., SCHEER, U., AND FRANKE, W. W., Proc. Nat. Acad. Sci. USA 71, 3626 (1974).