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Experimental Cell Research 111 (1978) 3 17-326
RNA SYNTHESIS
IN FIXED
CELLS
BY ENDOGENOUS
RNA POLYMERASES G. P. M. MOORE School
of Biological Sciences, Macquarie University, North Ryde, NSW 2113, Australia
SUMMARY A method for the detection of endogenous DNA-dependent RNA polymerase activity in eukaryotic cells is described. Incubation of ethanol and acetone-fixed cells with ribonucleoside triphosphates ATP, GTP, CTP and [3H]UTP results in the incorporation of radioactivity into the nuclei. The reaction product is not removed by trichloroacetic acid (TCA). However, labelling is blocked by actinomycin D and RNase indicating that the reaction is dependent on DNA and that RNA is synthesized. Evidence suggests that incorporation occurs as a result of the elongation of RNA chains which were initiated, in vivo. The amount of RNA synthesized is limited and remains complexed within the chromatin. The sites of label incorporation may be localized in the nucleolus and nucleoplasm of the cell by autoradiography. Grain counts give a relative measure of polymerase I and II activities respectively. The use of fixed cells circumvents problems which are associated with labelling of RNA in vivo. Incorporation is not dependent either on the transport of precursors into the cell or their uptake into intracellular pools. The method permits the transcriptional activities of individual cells to be detected and quantitated during differentiation.
This paper describes the characteristics of an assay which permits localization of the activities of endogenous DNA-dependent transcription enzymes, the RNA polymerases, within the cell nucleus. The enzymes are activated, after brief fixation, by incubation with the ribonucleoside triphosphates ATP, GTP, CTP and [3H]UTP. The sites of synthesis of RNA molecules are detected within the nucleus by autoradiography, presumably on DNA which was being actively transcribed in the living state. Two types of polymerase activity have been distinguished in the cell nucleus: one is located in the nucleoplasm which is specifically inhibited by the toxin cu-amanitin; the other occurs in the nucleolus and is inhibited by low concentrations of actino21-771808
mycin D [l-3]. Both activities may be detected simultaneously using the method described here. Polymerases are also present which are not active under usual assay conditions. These appear to be DNA-bound enzymes which are prevented by nuclear proteins from catalyzing the synthesis of RNA [4,51. Incorporation of radioactivity in fixed cells results from the elongation of RNA molecules which were initiated in vivo. The RNAs synthesized remain complexed within the chromatin; no new initiation takes place. Thus, the relative amount of RNA synthesized by a cell may be related to the number of active polymerase molecules present in the nucleolar and nucleoplasmic compartments. Exp
Cd
Res I I I (1978)
318
G. P. M. Moore
Table 1. Incorporation quirements
of [3H]UMP
by HeLa and mouse liver cells; divalent Liver cells
HeLa cells Incubation medium Complete With 12 mM Mg2+ alone With 2.5 mM Mn*+ alone With amm. sulphate alone With no metal ions or salt
cation re-
No. cells counted
Mean grain n f S.E.M.
No. cells counted
Mean grain n 2 S.E.M.
40 60 40
70.2k3.1” 66.5k2.9 47.3k3.6
60
20.850.8 -
20
;.sao.7
70 -
;.3+0.4 -
a Reaction solution contained 4 mM Mg ‘+, 2 mM Mn*+ and 0.04 M ammonium sulphate.
MATERIALS Preparation
AND METHODS
of cells
HeLa cells were grown on glass coverslips using conventional culture techniques [2]. When a suitable cell density was reached, the coverslips were rinsed in fresh culture medium and transferred to the fixative solution. Yoshida ascites tumour cells were propagated in adult female rats of the Wistar strain. Cells were aspirated on the seventh day of tumour growth. Small drops of the cell suspension were smeared on gelatinized slides and immersed successively in (a) isopentane for 30 set at -15°C; (b) isopentane, ethanol and acetone (18 : 1: 1 by vol) for 30 set at - 15°C; and (c) the fixative solution. Small cubes of mouse liver were frozen on to microtome chucks with cold CO,. Sections were cut at a thickness of 8 pm at -2O”C, picked up on gelatinized slides, air-dried and fixed.
Fixation All preparations were fixed for 5 mitt in fresh ethanol and acetone (1 : 1 by vol). The slides were then airdried at room temperature, and stored in the presence of a desiccant at -15°C until required. Cells were usually assayed for RNA polymerase activity within one day of preparation. However, they have been stored for up to one month without apparent loss of activity.
Transcription inhibitors were tested for their effects on RNA polymerase activity by inclusion at various concentrations in the assay solution. A 10 min preincubation was also carried out in the presence of an inhibitor and the assay solution from which the ribonucleoside triphosphates had been omitted. Appropriate control preparations were preincubated in a similar manner in the absence of the inhibitor. Substances tested included DNase, RNase, heparin and actinomycin D (Sigma Chemical Co.), a-amanitin, 3deoxyadenosine (cordycepin) triphosphate, and the rifampicin derivative AF/&13. Rifampicin was dissolved in a small quantity of dimethylsulphoxide (DMSO) before inclusion in the assay solution. DNase and RNase were not preincubated with cells before assay. Cell preparations (2-5 slides/treatment) were incubated with the assay solutions for 30 mitt at 3PC. The reaction was terminated by rinsing slides in distilled water and immersion in ethanol and acetic acid fixative (3 : 1 by vol) for 30 min. Slides were treated with 5% TCA for 5 min at 4°C to remove unincorporated nucleotides and washed in running water for 30 min. Autoradiography was performed with Ilford K2 nuclear emulsion or Kodak AR-IO stripping film using conventional procedures. The incorporation of [3H]UMP into each cell was quantitated by grain counting. Within each experiment, grain counts were performed over cell nuclei of approximately similar sizes [2, 5,7].
RESULTS Assay for RNA polymerase
activity
The procedure has been described in detail previously [1, 2, 5, 61. Except where indicated in the text, the complete reaction medium contained, in 0.5 ml: 50 pmoles Tris-HCI buffer (pH 7.9), 6 pmoles 2-mercaptoethanol, 75 @moles sucrose, 300 nmoles each of ATP, GTP and CTP; 10 nmoles of [3H]UTP (10-15 Cilmmol, Radiochemical Centre, Amersham); 4 pmoles MgCI,, 1 pmole MnCl, and 0.04 M ammonium sulphate. This is referred to as the low salt assay solution. In the high salt assay solution the ammonium sulphate concentration was raised to 0.4 molar. E.rp Cell Ru., 111 (1978)
The RNA polymerases of ethanol and acetone-fixed cells were active only in the presence of a divalent metal cation. Either Mg2+ or MrP was required for incorporation of radioactivity into HeLa cells (table 1). In their absence, the amount of label incorporated was very low. Similarly, omission of one or more of the ribonucleoside tri-
RNA polymerase
Table 2. Incorporation
of [3H]UMP
activity
of fixed cells
by HeLa and mouse liver cells; ribonucleoside
3 19 tri-
phosphate requirements HeLa cells No. cells counted
Incubation medium Complete -ATP -(ATP+CTP) -(ATP+CTP+GTP) High salt -(ATP+CTP)
Liver cells Mean grain n 2~S.E.M.
40
49.3f2.1”
40 40 40
19.Ok2.1
No. cells counted
Mean grain n + S.E.M.
60 IO
20.8kO.8 6.6f0.4
SO
4.6kO.5 -
220.5f7.jb 50.9f3.0
a See footnote, table 1. b Reaction solution contained 4 mM Mg *+, 2 mM Mn*+ and 0.4 M ammonium sulphate.
phosphates from the reaction solution resulted in a marked reduction of radioactive incorporation in HeLa and mouse liver cells (table 2). This indicates that all four RNA precursors are required for the reaction to be completed. Effects of different metal ion and salt concentrations [3H] UMP incorporation
Previous studies have demonstrated that nucleolar polymerases are preferentially ac-
tivated by Mg2+, whereas the nucleoplasmic enzymes require Mn2+ and ammonium sulphate for optimal activity [8-l 11. Using the procedure described here, the effects of particular metal ions and salts on the localization of polymerase activity appeared to depend on the nature of the cell. For example, in human fibroblasts [2] and HeLa cells, Mg2+ alone was sufficient to activate both nucleolar and nucleoplasmic activities. Addition of Mn2+ and 0.04 M ammonium sulphate (low salt assay) or ammonium sul-
Table 3. Effects, of different metal ion and salt concentrations
on incorporation
of [3H]
UMP by HeLa and Yoshida ascites cells Grain counts were made over the whole of the nucleus Yoshida ascites cells
Incubation medium Low salt 12 mM Mg*+ alone 12 mM Mg2++0.04 M amm. sulphate 2.5 mM Mn*+ alone 2.5 mM MnZ++0.04 M amm. sulphate 4 mM Mg*++2 mM Mn*+ High salt
HeLa cells
a-Amanitin absent
cu-Amanitin present
No. cells Mean grain counted nfS.E.M.
No. cells Mean grain counted n f S.E.M.
No. cells Mean grain counted n + S.E.M.
40 60
42.8f 1.8” 40.5kl.8
125 125
24.8f0.8 15.lkO.5
125 125
lO.Of0.5 14.3kO.6
40 40
41.421.3 28.8k2.2
125 125
27.3f0.6 5.5f0.3
125 125
13.2f0.4 5.lkO.3
40 40 40
40.622.3 28.0f1.7 220.5k7.5”
125 125 125
15.2f0.8 7.6kO.3 55.9k2.3
125 125 125
13.lf0.4 6.8f0.3 10.0+0.3
a See footnote, table 1. * See footnote, table 2. Exp Cell Res I I I (1978)
320
G. P. M. Moore
.
lb
RNA polymerase 20( I-
101
4
0
0.1
0.2
03
04
Fig. 6. Abscissa: ammonium sulphate cont. (moles); ordinate: mean arain no. fS.E.M.
Effect of increasing ammonium sulphate concentration on [3H]UMP incorporation by Yoshida ascites cells. x , [3H]UMP incorporation by cells preincubated with 0.4 M ammonium sulohate before incubation with the low salt reaction sol&on (containing 0.04 M ammonium sulphate).
activity
offixed
cells
321
However, addition of 0.04 M ammonium sulphate to either the Mg2+ and/or Mn2+ activated reaction stimulated radioactive incorporation. In the case of Mg2+ (fig. 3 a), the increased activity was approximately equivalent in amount and distribution to that found in the low salt assay (fig. 4a, table 3). On the basis of these results, the metal ion and salt concentrations of the low salt assay solution have been taken as the optimal levels for the simultaneous detection of nucleolar and nucleoplasmic polymerase activities in eukaryotic cells of different origins. The previous finding of low nucleolar incorporation by mouse kidney epithelial cells [5] in the presence of Mg2+, Mn2+ and ammonium sulphate may have been due to the suboptimal concentration of Mg2+ used (see table 3). The effect of the inhibitor cY-amanitin on polymerase activities in Yoshida ascites cells has also been measured (table 3). None was observed on Mg2+ or Mn2+stimulated activities (figs lb, 2b), indicating that these cations individually or together activated enzymes other than the nucleoplasmic type II polymerases [ 121. However, cr-amanitin blocked approximately half of the enzyme activity of ascites cells assayed in the presence of Mg2+ and ammonium sulphate or in the low salt medium (table 3). Residual activity was
phate alone to the reaction solution did not significantly enhance the amount of Mgz+stimulated activity (tables 1 and 3) or alter the distribution of activity within the nuclear compartment. In contrast, the nucleolar polymerases of mouse kidney epithelial cells [5] and Yoshida ascites cells Table 4. Cold chase experiment (fig. 1a and table 3) were activated by Mg2+ Mouse liver cells incubated for 30 min in low salt assay alone. Mn2+ activated these enzymes to a solution (LS) and chased for further 15 min in medium containing x 10 cont. cold UTP much lesser extent (fig. 2a and table 3). Incubation conditions
No. cells counted
Mean grain n + S.E.M.
60
20.8kO.8
70
20.5f0.6
40 50
21.2kO.6 18.8f0.7
Figs Z-5. Localization of [3H]UMP incorporation in
Yoshida ascites cells. Effects of different metal cations and salt concentrations in the absence (figs 1a-5 a) and uresence (figs lb-5b) of 5 ~cafrnl a-amanitin. (I) 12 mM Mg*+/ (2) 2.5 mM Mn*+i (2) 12 mM Mge+, O&t M amm. sulphate; (4) 8 mM Mg2+, 2 mM Mn*+, 0.04 M amm. St&hate; (5) 8 mM Mg*+, 2 mM Mn2+, 0.4 M amm. sulphate. Bar, 10 /,cm.
LS (30 min incubation) LS (30 min); cold UTP chase (15 min) LS (30 min); Tris buffer chase (15 min) LS (45 min incubation)
Exp Cell Rcs 111 (1978)
322
G. P. M. Moore
Table 5. Incorporation transcription inhibitors
of [3H]UMP
by Yoshida ascites and HeLa cells in the presence of Yoshida ascites cells
Incubation medium
No. cells counted
Mean grain n k S.E.M.
Low salt (LS) LS+actinomycin D (30 LLg/ml) LS+a-amanitin (5 &ml) LS+DNase (20 pg/ml) LS+RNase (20 pg/ml) LS+heparin(lOO~g/ml)
150 150 150 150 150 150
19.4f0.5 6.0t0.2 11.8kO.4 13.2f0.4 3.6f0.2 44.1t1.4
LS+rif AF/C13 (5 pg/ml) LS+rif AF/O-13 (50 &ml) LS+DMSO (AF/O-13 control)
150 150 150
16.5kO.7 17.2f0.6 17.7f0.5
High salt (HS) HS+actinomycin D HS+RNase
HeLa cells No. cells counted
Mean grain n f S.E.M.
40 40
49.3f2.1” 10.8kO.8
40 40 25
220.5f7.5’ 28.0+ 1.5 3.9f0.4
0 See footnote, table 1. b See footnote, table 2.
predominantly localized in the nucleoli (figs 3 b, 4 b). The persistence of some extranucleolar label in the presence of (Yamanitin (figs 3 b, 4 b) suggests that type III polymerases may be active [8, 10, 131. It has been shown previously [2, 51 as well as in numerous biochemical studies [9, 10, 11, 14, 151, that nucleoplasmic polymerase activity may be enhanced by increasing the salt concentration of the assay solution. Chambon et al. [ 161found that this only occurs in systems in which the enzymes have already begun to catalyse the synthesis of RNA. Table 3 shows the effect of raising the ammonium sulphate concentration from 0.04 to 0.4 M in the assay medium. Total nuclear RNA polymerase activity increased four-fold in HeLa cells and about two-fold in Yoshida ascites cells (fig. 5a). The increase in radioactive incorporation was solely due to the activities of type II polymerases. Inclusion of o-amanitin in the assay solution reduced the grain numbers to levels found in the a-amanitin inhibited low salt assay (table 3 and ref. [2]) Exp Cell Res I II (1978)
and label was almost exclusively located in the nucleolus (fig. 5b and ref. [2]). The effects of increasing concentrations of ammonium sulphate between 0.04 M and 0.4 M on RNA polymerase activity of Yoshida ascites cells are shown in fig. 6. A peak of incorporation was obtained with 0.2 M ammonium sulphate. The cause of the high salt stimulation of RNA polymerase activity was investigated. Ascites cells were treated to a 10 min preincubation in the presence of 0.4 M ammonium sulphate (see Methods). The cells were then rinsed thoroughly in assay buffer containing 0.04 M ammonium sulphate and finally assayed in a low salt solution. The polymerase activity of these cells was not significantly different from that assayed in the presence of a high salt concentration (fig. 6). This appears to rule out the possibility that ammonium sulphate affects the activity of the polymerase molecules alone. Rather, the increase in label incorporation in the presence of 0.4 M ammonium sulphate is interpreted as an artifactual situation resulting from the ex-
RNA polymerase activity offixed cells
323
pletion. It follows that the activities of the poiymerases of fixed cells are limited and the reaction product is bound within the nucleus. This was confirmed with a cold chase experiment. Liver cells were incubated in the low salt assay solution for 30 min, rinsed in buffer and then treated for a further 15 min with the complete reaction solution in which [3H]UTP had been replaced with a 15 25 5 x IO concentration of unlabelled UTP. The Fig. 7. Abscissa: time (mm); ordinate: mean gram no. results are shown in table 4. Chase with fS.E.M. O-O, 12 mM Mg*+; A-A, 4 mM Mg2+, cold UTP failed to alter the amount of label 2 mM Mn*+; O-O, 4 mM Mg*+, 2 mM Mn*+, 0.04 M ammonium sulphate; Cm, 4 mM Mg*+, 2 mM Mn*+, incorporated into the nuclei. There were no 0.4 M ammonium sulphate. significant differences in the grain numbers Time course of [3H]UMP incorporation by HeLa cells. Effects of different metal ion and salt concentraof cells in any of the experimental groups tions on the rate at which the reaction goes to com- when compared with the control incubapletion tion. This supports the view outlined above that all of the RNA chains synthesized retraction of nuclear proteins. Either more main within the nucleus after the reaction templates have been exposed for transcriphas ceased. tion [14, 151 possibly leading to the synthesis of longer RNA chains [17], or more Inhibitors of RNA synthesis DNA-bound enzymes have been released DNase and RNase both inhibited the in[5,18] (see also the effect of heparin,table 5). corporation of radioactivity into Yoshida ascites cells (table 5). The effects of difKinetics of label incorporation ferent concentrations of actinomycin D and The amount of label incorporated by HeLa a-amanitin have been previously described. cell nuclei relative to incubation time is shown in fig. 7. The reaction was completed rapidly in the presence of 12 mM 100 Mg2+; grain numbers reached a plateau within 5 min. With the low salt assay the reaction proceeded more slowly but reached the level of the Mg2+-stimulated reaction within 30 min. At a high salt concentration, the amount of label incor1I / 10 O 0 20 porated was greatly increased, but the rate Fig. 8. Abscissa: cordycepin triphosphate cont. of formation of the reaction product rapidly (nmoles/0.5 ml reaction solution); odinare: grain no. .-, declined after 5 min incubation. The evi- (normalized). Inhibitory effect of cordycepin triphosphate on indence of fig. 7, together with that of table 3, corporation of [3H]UMP or [3H]AMP by Yoshida cells. Reaction solutions were prepared as desuggests that different amounts or combina- ascites scribed in Methods except that ATP was included, tions of Mg2+, Mn2+ and ammonium sul- either labelled or unlabefied, at a concentration of 5 ml. O-O, [3H]ATP, spec. act. 15.4 Ci/ phate at low concentrations merely affect nmoles/O.S mmol; Radiochemical Centre, Amersham; O-O, the rate at which the reaction goes to com- [3H]UTP.
324
G. P. M. Moore
cu-Amanitin at concentrations of 0.05-5 pug/ ml blocked r3H]UMP incorporation into the nucleoplasm almost exclusively (figs 1b5b, tables 3 and 5 and refs [l, 2, 3, 5, 191). Actinomycin D at low concentrations (0.5 b&g/ml)preferentially inhibited nucleolar labelling [ 1,2, 19,201 and at higher concentrations also blocked incorporation into the whole nucleus (table 5). These observations are consistent with the existence of distinct DNA-bound nucleolar and nucleoplasmic polymerases. It has been reported that the rifampicin derivative AF/O-13 blocks initiation of RNA synthesis in mammalian cells in vitro but does not affect the elongation of RNA molecules [21]. Inclusion of 5 or 50 pg/ml of AF/O-13 in the reaction solution had no effect on the incorporation of radioactivity into ascites cells (table 5). Heparin has also been shown to inhibit initiation of RNA synthesis in vitro [22]. However, once synthesis of an RNA molecule has begun, label incorporation is stimulated, apparently due to the removal of nuclear proteins [22]. In ascites cells, a two-fold stimulation of label incorporation over control levels was evident (table 5), approximating that found after high salt assay (table 3). The lack of any inhibitory activity by rifampicin AF/ O-13 and heparin suggests that the endogenous RNA polymerase activity of fixed cells is solely concerned with the elongation of RNA chains which were’ initiated in vivo. No new RNA molecules are initiated in vitro. The effects of the nucleotide analogue cordycepin triphosphate were also investigated using either [3H]UTP or r3H]ATP as a labelled precursor. Cordycepin is reported to inhibit transcription by substituting for a ribonucleotide in a growing RNA chain [23]. At increasing concentrations in the reaction solution, cordycepin triphosphate Exp Cell Res 111 (1978)
progressively inhibited the incorporation of [3H]UMP and [3H]AMP into ascites cells (fig. 8). The inhibitory effect of the nucleotide analogue was more pronounced at low concentrations in AMP-labelled cells than in those labelled with UMP. It is possible that this represented a selective inhibition of the synthesis of adenylic acidrich sequences within the nuclei. DISCUSSION In this study we have demonstrated that treatment of cells or tissues with ethanol and acetone permits the passage of nucleotides into the nuclei and their utilization without significantly affecting some of the functions of the transcription machinery (see also Zetterberg et al. [28]). The incorporation of ribonucleotide precursors of RNA into a TCA-insoluble, RNase digestible product by cell nuclei is a feature characteristic of RNA polymerase activity. The requirements for the synthesis of RNA shown here are similar to those reported for a DNA primed reaction [24, 251. The presence of multiple forms of the enzyme are indicated by their responses to Mg2+, Mn2+ and ammonium sulphate, the localization of actinomycin D sensitive activity in the nucleolus [ 1, 2, 191and cw-amanitin sensitive activity in the nucleoplasm. These data are consistent with their classification as types I and II [8, 12, 261 or forms AI and B [27] respectively. Form I is involved in the synthesis of ribosomal RNA (rRNA), form II in the synthesis of heterogeneous nuclear RNA (HnRNA) [lo]. In fixed cells, the RNA polymerase reaction is completed relatively rapidly and the label is not removed by cold chase. The two compounds rifampicin AF/CL13 and hepar-in, which have been reported to inhibit binding of the enzyme to the template [2 1,
RNA polymerase activity
291or initiation [22], do not reduce label incorporation. This suggests that RNA molecules which have begun to be synthesized in vivo undergo further elongation in the presence of the assay solution but no new chains are initiated. When RNA synthesis ceases, the transcription complex remains in a stable form within the nucleus. It follows that the amount of label incorporated by a cell is a function of the number of active polymerase molecules which are present and bound to DNA. Thus by performing grain counts over the nucleolus and nucleoplasm of a cell, a relative measure of polymerase I and II activities may be obtained. A simple shift in the rate of polymerization by existing active polymerases would not be detected except in experiments where the reaction was stopped before completion (fig. 7). Increases in label incorporation by cells undergoing growth reactivation [3, 4, 5, 30, 361 are attributed to the activation of more polymerase molecules, either on existing DNA, or on new templates which have become available for transcription. Either DNA bound [4, 51 or free [31] enzymes present in the cell could facilitate this [32]. No RNA polymerase activity has been detected in highly differentiated [30], meiotic [6, 71 or mitotic cells [2] which have been shown to contain polymerases [33, 34, 351 but which are not active in RNA synthesis. The use of fixed cells to study transcriptional processes has some advantages over other cytological and biochemical methods. It has become increasingly apparent that the specific functions of RNA polymerases during transcription may only be evaluated biochemically by using enzymes and DNA from the same source [8, 361. Even where these conditions are satisfied, the binding of homologous RNA polymerases may not occur only at specific initiation sites on the
offixed
cells
325
DNA or chromatin, in vitro [22]. The assay system described here utilises the transcription machinery already present and functioning within the cell. Both nucleolar and nucleoplasmic polymerases are active in ‘low salt’ assay conditions, indicating that no significant redistribution of proteins occurs in the nucleus as a result of fixation [ 151.In addition, the labelling of RNA is not subject either to fluctuations in rates of precursor transport into the cell which occur in vivo, or to their final concentrations in the intracellular pools [28]. Methods similar to the one described here have been developed for plant [37, 381 and animal [39] cells. However, the ghost monolayer technique of Tsai & Green [39, 401 is restricted to cell cultures growing on glass. The present procedure is directly applicable to individual cells [3, 71, and to tissues which, for various reasons, do not lend themselves to biochemical methods of analysis. For example, we have been able to study the early events of genome activation in individual embryos of the macropod marsupial Macropus eugenii at the end of a developmentally quiescent period of approx. 10 months duration [36]. This work was supported, in part, by the Wellcome Trust, The European Molecular Biology Organisation, The Danish Medical Research Council, The Nordisk Insulinfond and by a Queen Elizabeth II Fellowship to the author. I wish to thank Professor N. R. Ringertz of the Karolinska Institute, Stockholm, Professor M. Faber and Dr H. Peters of the Finsen Institute, Copenhagen, and Professor S. A. Bamett of the Zoology Department ANU, Canberra, for their support and hospitality. The following gifts are gratefully acknowledged: cr-amanitin from Professor Th. Wieland, Max-PlanckInstitut, Heidelberg; 3-deoxyadenosine triphosphate from Professor H. Klenow, Fibiger Laboratory, Copenhagen; rifampicin AF/O-13 from Professor L. G. Silvestri, Gruppo Lepetit, Milan. Jette Christiansen performed some of the grain counts.
REFERENCES 1. Moore, G P M, Proc int symp on the genetics of the spermatozoon (ed R A Beatty & S Gluecksohn-Waelsch) p. 90. Forum Press, Copenhagen (1972). EXP Cd Res I II (1978)
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2. Moore, G P M & Ringertz, N R, Exp cell res 76 (1973) 223. 3. Moore, G P M, J embryo1 exp morph01 34 (1975) 291. 4. Auer, G, Moore, G P M, Ringertz, N R & Zetterberg, A, Exp cell res 76 (1973) 229. 5. Moore, G P M, Auer, G & Zetterberg, A, Exp cell res 88 (1974) 375. 6. Moore; G P’M, Exp cell res 68 (1971) 462. 7. Moore, G P M, Lintem-Moore, S M, Peters, H & Faber, M, J cell biol60 (1974) 416. 8. Jacob, S T. Prog nucleic acid res mol biol 13(1973) 93. ‘9. Poao, A 0, Littau, V C, Allfrev, V G & Mirsky, A E,?roc natl acad sci US 57 (1967) 743. 10. Zvlber. E A & Penman, S. Proc natl acad sci US 68 (1971) 2861. 11. Widnell, C C & Tata, J R, Biochim biophys acta 123(1%6) 478. 12. Lindell, T J. Weinberg, F, Morris, P W, Roeder, P G & Rutter,’ W J, Sci&ce 170(1970) 447. 13. Weinmann. R & Roeder, R G, Proc natl acad sci us 71(1974) 1790. 14. Butterworth, P H W, Cox, R F & Chesterton, C J, Eur j biochem 23 (1971) 229. 15. Chambon, P, Ramuz, M, Mandel, P & Doly, J, Biochim biophys acta 157(1968) 504. 16. Chambon, P, Karon, H, Ramuz, M & Mandel, P, Biochim biophys acta 157(1968) 520. 17. Petersen. E E & Kriiger, H, Z Natmforsch 25B (1970) 1042. 18. Goldberg, M L, Fed proc 29 (1970) 1261. 19. Moore, G P M, Gamete competition in plants and animals (ed D L Mulcahy) p. 69. North-Holland. Amsterdam (1975). 20. Perrv. R P. Exn cell res 29 (1%3) 400. 21. Meiihac, M, Typser, Z & dhambon, P, Eur j biothem 28 (1972) 291.
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22. Cox, R F, Eur j biochem 39 (1973) 49. 23. Penman, S, Rosbash, M & Penman, M, Proc natl acad sci US 67 (1970) 1878. 24. Hurwitz. J & Aueust. J T. Proe nucleic acid res 1 (1%3) 59. 25. Fox. C F&Weiss. S B. J biol them 239 (1964) 175. 26. Roeder, R G & Rutter; W J, Proc natl acad sci US 65 (1970) 675. 27. Kedinger, C, Nuret, P & Chambon, P, FEBS lett 15 (1971) 169. 28. Zetterberg, A, Auer, G & Moore, G P M, Exp cell res 88 (1975) 382. 29. Riva, S, Fietta, A & Silvestri, L G, Biochem biophys res commun 49 (1972) 1263. 30. Carlsson, S-A, Moore, G P M & Ringertz, N R, Exp cell res 76 (1973) 234. 31. Fu-Li, Y, Nature 251 (1974) 344. 32. Tocchini-Valentini, G P & Crippa, M, Nature 228 (1970) 993. 33. Wassarman, P M, Hollinger, T G & Smith, L D, Nature new biol240 (1972) 208. 34. Schechter. N M, Biochim bionhvs acta 308 (1973) 129. 35. Hotta, Y & Stem, H, Nature 210 (1%8) 1043. 36. Moore, G P M, J cell physiol. In press. 37. Fisher, D B, J cell biol39 (1%8) 745. 38. Payne, J F Br Bal. A K, Phytochemistry 11 (1972) 3105. 39. Tsai, R L & Green, H, Nature new biol 243 (1973) 168. 40. ~owkn, P D, Meek, R L & Daniel, C W, Exp cell res 101 (1976) 434. L
Received June 22, 1977 Accepted August 5, 1977
.
Experimental Cell Research 111 (1978) 3 17-326
RNA SYNTHESIS
IN FIXED
CELLS
BY ENDOGENOUS
RNA POLYMERASES G. P. M. MOORE School
of Biological Sciences, Macquarie University, North Ryde, NSW 2113, Australia
SUMMARY A method for the detection of endogenous DNA-dependent RNA polymerase activity in eukaryotic cells is described. Incubation of ethanol and acetone-fixed cells with ribonucleoside triphosphates ATP, GTP, CTP and [3H]UTP results in the incorporation of radioactivity into the nuclei. The reaction product is not removed by trichloroacetic acid (TCA). However, labelling is blocked by actinomycin D and RNase indicating that the reaction is dependent on DNA and that RNA is synthesized. Evidence suggests that incorporation occurs as a result of the elongation of RNA chains which were initiated, in vivo. The amount of RNA synthesized is limited and remains complexed within the chromatin. The sites of label incorporation may be localized in the nucleolus and nucleoplasm of the cell by autoradiography. Grain counts give a relative measure of polymerase I and II activities respectively. The use of fixed cells circumvents problems which are associated with labelling of RNA in vivo. Incorporation is not dependent either on the transport of precursors into the cell or their uptake into intracellular pools. The method permits the transcriptional activities of individual cells to be detected and quantitated during differentiation.
This paper describes the characteristics of an assay which permits localization of the activities of endogenous DNA-dependent transcription enzymes, the RNA polymerases, within the cell nucleus. The enzymes are activated, after brief fixation, by incubation with the ribonucleoside triphosphates ATP, GTP, CTP and [3H]UTP. The sites of synthesis of RNA molecules are detected within the nucleus by autoradiography, presumably on DNA which was being actively transcribed in the living state. Two types of polymerase activity have been distinguished in the cell nucleus: one is located in the nucleoplasm which is specifically inhibited by the toxin cu-amanitin; the other occurs in the nucleolus and is inhibited by low concentrations of actino21-771808
mycin D [l-3]. Both activities may be detected simultaneously using the method described here. Polymerases are also present which are not active under usual assay conditions. These appear to be DNA-bound enzymes which are prevented by nuclear proteins from catalyzing the synthesis of RNA [4,51. Incorporation of radioactivity in fixed cells results from the elongation of RNA molecules which were initiated in vivo. The RNAs synthesized remain complexed within the chromatin; no new initiation takes place. Thus, the relative amount of RNA synthesized by a cell may be related to the number of active polymerase molecules present in the nucleolar and nucleoplasmic compartments. Exp
Cd
Res I I I (1978)
318
G. P. M. Moore
Table 1. Incorporation quirements
of [3H]UMP
by HeLa and mouse liver cells; divalent Liver cells
HeLa cells Incubation medium Complete With 12 mM Mg2+ alone With 2.5 mM Mn*+ alone With amm. sulphate alone With no metal ions or salt
cation re-
No. cells counted
Mean grain n f S.E.M.
No. cells counted
Mean grain n 2 S.E.M.
40 60 40
70.2k3.1” 66.5k2.9 47.3k3.6
60
20.850.8 -
20
;.sao.7
70 -
;.3+0.4 -
a Reaction solution contained 4 mM Mg ‘+, 2 mM Mn*+ and 0.04 M ammonium sulphate.
MATERIALS Preparation
AND METHODS
of cells
HeLa cells were grown on glass coverslips using conventional culture techniques [2]. When a suitable cell density was reached, the coverslips were rinsed in fresh culture medium and transferred to the fixative solution. Yoshida ascites tumour cells were propagated in adult female rats of the Wistar strain. Cells were aspirated on the seventh day of tumour growth. Small drops of the cell suspension were smeared on gelatinized slides and immersed successively in (a) isopentane for 30 set at -15°C; (b) isopentane, ethanol and acetone (18 : 1: 1 by vol) for 30 set at - 15°C; and (c) the fixative solution. Small cubes of mouse liver were frozen on to microtome chucks with cold CO,. Sections were cut at a thickness of 8 pm at -2O”C, picked up on gelatinized slides, air-dried and fixed.
Fixation All preparations were fixed for 5 mitt in fresh ethanol and acetone (1 : 1 by vol). The slides were then airdried at room temperature, and stored in the presence of a desiccant at -15°C until required. Cells were usually assayed for RNA polymerase activity within one day of preparation. However, they have been stored for up to one month without apparent loss of activity.
Transcription inhibitors were tested for their effects on RNA polymerase activity by inclusion at various concentrations in the assay solution. A 10 min preincubation was also carried out in the presence of an inhibitor and the assay solution from which the ribonucleoside triphosphates had been omitted. Appropriate control preparations were preincubated in a similar manner in the absence of the inhibitor. Substances tested included DNase, RNase, heparin and actinomycin D (Sigma Chemical Co.), a-amanitin, 3deoxyadenosine (cordycepin) triphosphate, and the rifampicin derivative AF/&13. Rifampicin was dissolved in a small quantity of dimethylsulphoxide (DMSO) before inclusion in the assay solution. DNase and RNase were not preincubated with cells before assay. Cell preparations (2-5 slides/treatment) were incubated with the assay solutions for 30 mitt at 3PC. The reaction was terminated by rinsing slides in distilled water and immersion in ethanol and acetic acid fixative (3 : 1 by vol) for 30 min. Slides were treated with 5% TCA for 5 min at 4°C to remove unincorporated nucleotides and washed in running water for 30 min. Autoradiography was performed with Ilford K2 nuclear emulsion or Kodak AR-IO stripping film using conventional procedures. The incorporation of [3H]UMP into each cell was quantitated by grain counting. Within each experiment, grain counts were performed over cell nuclei of approximately similar sizes [2, 5,7].
RESULTS Assay for RNA polymerase
activity
The procedure has been described in detail previously [1, 2, 5, 61. Except where indicated in the text, the complete reaction medium contained, in 0.5 ml: 50 pmoles Tris-HCI buffer (pH 7.9), 6 pmoles 2-mercaptoethanol, 75 @moles sucrose, 300 nmoles each of ATP, GTP and CTP; 10 nmoles of [3H]UTP (10-15 Cilmmol, Radiochemical Centre, Amersham); 4 pmoles MgCI,, 1 pmole MnCl, and 0.04 M ammonium sulphate. This is referred to as the low salt assay solution. In the high salt assay solution the ammonium sulphate concentration was raised to 0.4 molar. E.rp Cell Ru., 111 (1978)
The RNA polymerases of ethanol and acetone-fixed cells were active only in the presence of a divalent metal cation. Either Mg2+ or MrP was required for incorporation of radioactivity into HeLa cells (table 1). In their absence, the amount of label incorporated was very low. Similarly, omission of one or more of the ribonucleoside tri-
RNA polymerase
Table 2. Incorporation
of [3H]UMP
activity
of fixed cells
by HeLa and mouse liver cells; ribonucleoside
3 19 tri-
phosphate requirements HeLa cells No. cells counted
Incubation medium Complete -ATP -(ATP+CTP) -(ATP+CTP+GTP) High salt -(ATP+CTP)
Liver cells Mean grain n 2~S.E.M.
40
49.3f2.1”
40 40 40
19.Ok2.1
No. cells counted
Mean grain n + S.E.M.
60 IO
20.8kO.8 6.6f0.4
SO
4.6kO.5 -
220.5f7.jb 50.9f3.0
a See footnote, table 1. b Reaction solution contained 4 mM Mg *+, 2 mM Mn*+ and 0.4 M ammonium sulphate.
phosphates from the reaction solution resulted in a marked reduction of radioactive incorporation in HeLa and mouse liver cells (table 2). This indicates that all four RNA precursors are required for the reaction to be completed. Effects of different metal ion and salt concentrations [3H] UMP incorporation
Previous studies have demonstrated that nucleolar polymerases are preferentially ac-
tivated by Mg2+, whereas the nucleoplasmic enzymes require Mn2+ and ammonium sulphate for optimal activity [8-l 11. Using the procedure described here, the effects of particular metal ions and salts on the localization of polymerase activity appeared to depend on the nature of the cell. For example, in human fibroblasts [2] and HeLa cells, Mg2+ alone was sufficient to activate both nucleolar and nucleoplasmic activities. Addition of Mn2+ and 0.04 M ammonium sulphate (low salt assay) or ammonium sul-
Table 3. Effects, of different metal ion and salt concentrations
on incorporation
of [3H]
UMP by HeLa and Yoshida ascites cells Grain counts were made over the whole of the nucleus Yoshida ascites cells
Incubation medium Low salt 12 mM Mg*+ alone 12 mM Mg2++0.04 M amm. sulphate 2.5 mM Mn*+ alone 2.5 mM MnZ++0.04 M amm. sulphate 4 mM Mg*++2 mM Mn*+ High salt
HeLa cells
a-Amanitin absent
cu-Amanitin present
No. cells Mean grain counted nfS.E.M.
No. cells Mean grain counted n f S.E.M.
No. cells Mean grain counted n + S.E.M.
40 60
42.8f 1.8” 40.5kl.8
125 125
24.8f0.8 15.lkO.5
125 125
lO.Of0.5 14.3kO.6
40 40
41.421.3 28.8k2.2
125 125
27.3f0.6 5.5f0.3
125 125
13.2f0.4 5.lkO.3
40 40 40
40.622.3 28.0f1.7 220.5k7.5”
125 125 125
15.2f0.8 7.6kO.3 55.9k2.3
125 125 125
13.lf0.4 6.8f0.3 10.0+0.3
a See footnote, table 1. * See footnote, table 2. Exp Cell Res I I I (1978)
320
G. P. M. Moore
.
lb
RNA polymerase 20( I-
101
4
0
0.1
0.2
03
04
Fig. 6. Abscissa: ammonium sulphate cont. (moles); ordinate: mean arain no. fS.E.M.
Effect of increasing ammonium sulphate concentration on [3H]UMP incorporation by Yoshida ascites cells. x , [3H]UMP incorporation by cells preincubated with 0.4 M ammonium sulohate before incubation with the low salt reaction sol&on (containing 0.04 M ammonium sulphate).
activity
offixed
cells
321
However, addition of 0.04 M ammonium sulphate to either the Mg2+ and/or Mn2+ activated reaction stimulated radioactive incorporation. In the case of Mg2+ (fig. 3 a), the increased activity was approximately equivalent in amount and distribution to that found in the low salt assay (fig. 4a, table 3). On the basis of these results, the metal ion and salt concentrations of the low salt assay solution have been taken as the optimal levels for the simultaneous detection of nucleolar and nucleoplasmic polymerase activities in eukaryotic cells of different origins. The previous finding of low nucleolar incorporation by mouse kidney epithelial cells [5] in the presence of Mg2+, Mn2+ and ammonium sulphate may have been due to the suboptimal concentration of Mg2+ used (see table 3). The effect of the inhibitor cY-amanitin on polymerase activities in Yoshida ascites cells has also been measured (table 3). None was observed on Mg2+ or Mn2+stimulated activities (figs lb, 2b), indicating that these cations individually or together activated enzymes other than the nucleoplasmic type II polymerases [ 121. However, cr-amanitin blocked approximately half of the enzyme activity of ascites cells assayed in the presence of Mg2+ and ammonium sulphate or in the low salt medium (table 3). Residual activity was
phate alone to the reaction solution did not significantly enhance the amount of Mgz+stimulated activity (tables 1 and 3) or alter the distribution of activity within the nuclear compartment. In contrast, the nucleolar polymerases of mouse kidney epithelial cells [5] and Yoshida ascites cells Table 4. Cold chase experiment (fig. 1a and table 3) were activated by Mg2+ Mouse liver cells incubated for 30 min in low salt assay alone. Mn2+ activated these enzymes to a solution (LS) and chased for further 15 min in medium containing x 10 cont. cold UTP much lesser extent (fig. 2a and table 3). Incubation conditions
No. cells counted
Mean grain n + S.E.M.
60
20.8kO.8
70
20.5f0.6
40 50
21.2kO.6 18.8f0.7
Figs Z-5. Localization of [3H]UMP incorporation in
Yoshida ascites cells. Effects of different metal cations and salt concentrations in the absence (figs 1a-5 a) and uresence (figs lb-5b) of 5 ~cafrnl a-amanitin. (I) 12 mM Mg*+/ (2) 2.5 mM Mn*+i (2) 12 mM Mge+, O&t M amm. sulphate; (4) 8 mM Mg2+, 2 mM Mn*+, 0.04 M amm. St&hate; (5) 8 mM Mg*+, 2 mM Mn2+, 0.4 M amm. sulphate. Bar, 10 /,cm.
LS (30 min incubation) LS (30 min); cold UTP chase (15 min) LS (30 min); Tris buffer chase (15 min) LS (45 min incubation)
Exp Cell Rcs 111 (1978)
322
G. P. M. Moore
Table 5. Incorporation transcription inhibitors
of [3H]UMP
by Yoshida ascites and HeLa cells in the presence of Yoshida ascites cells
Incubation medium
No. cells counted
Mean grain n k S.E.M.
Low salt (LS) LS+actinomycin D (30 LLg/ml) LS+a-amanitin (5 &ml) LS+DNase (20 pg/ml) LS+RNase (20 pg/ml) LS+heparin(lOO~g/ml)
150 150 150 150 150 150
19.4f0.5 6.0t0.2 11.8kO.4 13.2f0.4 3.6f0.2 44.1t1.4
LS+rif AF/C13 (5 pg/ml) LS+rif AF/O-13 (50 &ml) LS+DMSO (AF/O-13 control)
150 150 150
16.5kO.7 17.2f0.6 17.7f0.5
High salt (HS) HS+actinomycin D HS+RNase
HeLa cells No. cells counted
Mean grain n f S.E.M.
40 40
49.3f2.1” 10.8kO.8
40 40 25
220.5f7.5’ 28.0+ 1.5 3.9f0.4
0 See footnote, table 1. b See footnote, table 2.
predominantly localized in the nucleoli (figs 3 b, 4 b). The persistence of some extranucleolar label in the presence of (Yamanitin (figs 3 b, 4 b) suggests that type III polymerases may be active [8, 10, 131. It has been shown previously [2, 51 as well as in numerous biochemical studies [9, 10, 11, 14, 151, that nucleoplasmic polymerase activity may be enhanced by increasing the salt concentration of the assay solution. Chambon et al. [ 161found that this only occurs in systems in which the enzymes have already begun to catalyse the synthesis of RNA. Table 3 shows the effect of raising the ammonium sulphate concentration from 0.04 to 0.4 M in the assay medium. Total nuclear RNA polymerase activity increased four-fold in HeLa cells and about two-fold in Yoshida ascites cells (fig. 5a). The increase in radioactive incorporation was solely due to the activities of type II polymerases. Inclusion of o-amanitin in the assay solution reduced the grain numbers to levels found in the a-amanitin inhibited low salt assay (table 3 and ref. [2]) Exp Cell Res I II (1978)
and label was almost exclusively located in the nucleolus (fig. 5b and ref. [2]). The effects of increasing concentrations of ammonium sulphate between 0.04 M and 0.4 M on RNA polymerase activity of Yoshida ascites cells are shown in fig. 6. A peak of incorporation was obtained with 0.2 M ammonium sulphate. The cause of the high salt stimulation of RNA polymerase activity was investigated. Ascites cells were treated to a 10 min preincubation in the presence of 0.4 M ammonium sulphate (see Methods). The cells were then rinsed thoroughly in assay buffer containing 0.04 M ammonium sulphate and finally assayed in a low salt solution. The polymerase activity of these cells was not significantly different from that assayed in the presence of a high salt concentration (fig. 6). This appears to rule out the possibility that ammonium sulphate affects the activity of the polymerase molecules alone. Rather, the increase in label incorporation in the presence of 0.4 M ammonium sulphate is interpreted as an artifactual situation resulting from the ex-
RNA polymerase activity offixed cells
323
pletion. It follows that the activities of the poiymerases of fixed cells are limited and the reaction product is bound within the nucleus. This was confirmed with a cold chase experiment. Liver cells were incubated in the low salt assay solution for 30 min, rinsed in buffer and then treated for a further 15 min with the complete reaction solution in which [3H]UTP had been replaced with a 15 25 5 x IO concentration of unlabelled UTP. The Fig. 7. Abscissa: time (mm); ordinate: mean gram no. results are shown in table 4. Chase with fS.E.M. O-O, 12 mM Mg*+; A-A, 4 mM Mg2+, cold UTP failed to alter the amount of label 2 mM Mn*+; O-O, 4 mM Mg*+, 2 mM Mn*+, 0.04 M ammonium sulphate; Cm, 4 mM Mg*+, 2 mM Mn*+, incorporated into the nuclei. There were no 0.4 M ammonium sulphate. significant differences in the grain numbers Time course of [3H]UMP incorporation by HeLa cells. Effects of different metal ion and salt concentraof cells in any of the experimental groups tions on the rate at which the reaction goes to com- when compared with the control incubapletion tion. This supports the view outlined above that all of the RNA chains synthesized retraction of nuclear proteins. Either more main within the nucleus after the reaction templates have been exposed for transcriphas ceased. tion [14, 151 possibly leading to the synthesis of longer RNA chains [17], or more Inhibitors of RNA synthesis DNA-bound enzymes have been released DNase and RNase both inhibited the in[5,18] (see also the effect of heparin,table 5). corporation of radioactivity into Yoshida ascites cells (table 5). The effects of difKinetics of label incorporation ferent concentrations of actinomycin D and The amount of label incorporated by HeLa a-amanitin have been previously described. cell nuclei relative to incubation time is shown in fig. 7. The reaction was completed rapidly in the presence of 12 mM 100 Mg2+; grain numbers reached a plateau within 5 min. With the low salt assay the reaction proceeded more slowly but reached the level of the Mg2+-stimulated reaction within 30 min. At a high salt concentration, the amount of label incor1I / 10 O 0 20 porated was greatly increased, but the rate Fig. 8. Abscissa: cordycepin triphosphate cont. of formation of the reaction product rapidly (nmoles/0.5 ml reaction solution); odinare: grain no. .-, declined after 5 min incubation. The evi- (normalized). Inhibitory effect of cordycepin triphosphate on indence of fig. 7, together with that of table 3, corporation of [3H]UMP or [3H]AMP by Yoshida cells. Reaction solutions were prepared as desuggests that different amounts or combina- ascites scribed in Methods except that ATP was included, tions of Mg2+, Mn2+ and ammonium sul- either labelled or unlabefied, at a concentration of 5 ml. O-O, [3H]ATP, spec. act. 15.4 Ci/ phate at low concentrations merely affect nmoles/O.S mmol; Radiochemical Centre, Amersham; O-O, the rate at which the reaction goes to com- [3H]UTP.
324
G. P. M. Moore
cu-Amanitin at concentrations of 0.05-5 pug/ ml blocked r3H]UMP incorporation into the nucleoplasm almost exclusively (figs 1b5b, tables 3 and 5 and refs [l, 2, 3, 5, 191). Actinomycin D at low concentrations (0.5 b&g/ml)preferentially inhibited nucleolar labelling [ 1,2, 19,201 and at higher concentrations also blocked incorporation into the whole nucleus (table 5). These observations are consistent with the existence of distinct DNA-bound nucleolar and nucleoplasmic polymerases. It has been reported that the rifampicin derivative AF/O-13 blocks initiation of RNA synthesis in mammalian cells in vitro but does not affect the elongation of RNA molecules [21]. Inclusion of 5 or 50 pg/ml of AF/O-13 in the reaction solution had no effect on the incorporation of radioactivity into ascites cells (table 5). Heparin has also been shown to inhibit initiation of RNA synthesis in vitro [22]. However, once synthesis of an RNA molecule has begun, label incorporation is stimulated, apparently due to the removal of nuclear proteins [22]. In ascites cells, a two-fold stimulation of label incorporation over control levels was evident (table 5), approximating that found after high salt assay (table 3). The lack of any inhibitory activity by rifampicin AF/ O-13 and heparin suggests that the endogenous RNA polymerase activity of fixed cells is solely concerned with the elongation of RNA chains which were’ initiated in vivo. No new RNA molecules are initiated in vitro. The effects of the nucleotide analogue cordycepin triphosphate were also investigated using either [3H]UTP or r3H]ATP as a labelled precursor. Cordycepin is reported to inhibit transcription by substituting for a ribonucleotide in a growing RNA chain [23]. At increasing concentrations in the reaction solution, cordycepin triphosphate Exp Cell Res 111 (1978)
progressively inhibited the incorporation of [3H]UMP and [3H]AMP into ascites cells (fig. 8). The inhibitory effect of the nucleotide analogue was more pronounced at low concentrations in AMP-labelled cells than in those labelled with UMP. It is possible that this represented a selective inhibition of the synthesis of adenylic acidrich sequences within the nuclei. DISCUSSION In this study we have demonstrated that treatment of cells or tissues with ethanol and acetone permits the passage of nucleotides into the nuclei and their utilization without significantly affecting some of the functions of the transcription machinery (see also Zetterberg et al. [28]). The incorporation of ribonucleotide precursors of RNA into a TCA-insoluble, RNase digestible product by cell nuclei is a feature characteristic of RNA polymerase activity. The requirements for the synthesis of RNA shown here are similar to those reported for a DNA primed reaction [24, 251. The presence of multiple forms of the enzyme are indicated by their responses to Mg2+, Mn2+ and ammonium sulphate, the localization of actinomycin D sensitive activity in the nucleolus [ 1, 2, 191and cw-amanitin sensitive activity in the nucleoplasm. These data are consistent with their classification as types I and II [8, 12, 261 or forms AI and B [27] respectively. Form I is involved in the synthesis of ribosomal RNA (rRNA), form II in the synthesis of heterogeneous nuclear RNA (HnRNA) [lo]. In fixed cells, the RNA polymerase reaction is completed relatively rapidly and the label is not removed by cold chase. The two compounds rifampicin AF/CL13 and hepar-in, which have been reported to inhibit binding of the enzyme to the template [2 1,
RNA polymerase activity
291or initiation [22], do not reduce label incorporation. This suggests that RNA molecules which have begun to be synthesized in vivo undergo further elongation in the presence of the assay solution but no new chains are initiated. When RNA synthesis ceases, the transcription complex remains in a stable form within the nucleus. It follows that the amount of label incorporated by a cell is a function of the number of active polymerase molecules which are present and bound to DNA. Thus by performing grain counts over the nucleolus and nucleoplasm of a cell, a relative measure of polymerase I and II activities may be obtained. A simple shift in the rate of polymerization by existing active polymerases would not be detected except in experiments where the reaction was stopped before completion (fig. 7). Increases in label incorporation by cells undergoing growth reactivation [3, 4, 5, 30, 361 are attributed to the activation of more polymerase molecules, either on existing DNA, or on new templates which have become available for transcription. Either DNA bound [4, 51 or free [31] enzymes present in the cell could facilitate this [32]. No RNA polymerase activity has been detected in highly differentiated [30], meiotic [6, 71 or mitotic cells [2] which have been shown to contain polymerases [33, 34, 351 but which are not active in RNA synthesis. The use of fixed cells to study transcriptional processes has some advantages over other cytological and biochemical methods. It has become increasingly apparent that the specific functions of RNA polymerases during transcription may only be evaluated biochemically by using enzymes and DNA from the same source [8, 361. Even where these conditions are satisfied, the binding of homologous RNA polymerases may not occur only at specific initiation sites on the
offixed
cells
325
DNA or chromatin, in vitro [22]. The assay system described here utilises the transcription machinery already present and functioning within the cell. Both nucleolar and nucleoplasmic polymerases are active in ‘low salt’ assay conditions, indicating that no significant redistribution of proteins occurs in the nucleus as a result of fixation [ 151.In addition, the labelling of RNA is not subject either to fluctuations in rates of precursor transport into the cell which occur in vivo, or to their final concentrations in the intracellular pools [28]. Methods similar to the one described here have been developed for plant [37, 381 and animal [39] cells. However, the ghost monolayer technique of Tsai & Green [39, 401 is restricted to cell cultures growing on glass. The present procedure is directly applicable to individual cells [3, 71, and to tissues which, for various reasons, do not lend themselves to biochemical methods of analysis. For example, we have been able to study the early events of genome activation in individual embryos of the macropod marsupial Macropus eugenii at the end of a developmentally quiescent period of approx. 10 months duration [36]. This work was supported, in part, by the Wellcome Trust, The European Molecular Biology Organisation, The Danish Medical Research Council, The Nordisk Insulinfond and by a Queen Elizabeth II Fellowship to the author. I wish to thank Professor N. R. Ringertz of the Karolinska Institute, Stockholm, Professor M. Faber and Dr H. Peters of the Finsen Institute, Copenhagen, and Professor S. A. Bamett of the Zoology Department ANU, Canberra, for their support and hospitality. The following gifts are gratefully acknowledged: cr-amanitin from Professor Th. Wieland, Max-PlanckInstitut, Heidelberg; 3-deoxyadenosine triphosphate from Professor H. Klenow, Fibiger Laboratory, Copenhagen; rifampicin AF/O-13 from Professor L. G. Silvestri, Gruppo Lepetit, Milan. Jette Christiansen performed some of the grain counts.
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2. Moore, G P M & Ringertz, N R, Exp cell res 76 (1973) 223. 3. Moore, G P M, J embryo1 exp morph01 34 (1975) 291. 4. Auer, G, Moore, G P M, Ringertz, N R & Zetterberg, A, Exp cell res 76 (1973) 229. 5. Moore, G P M, Auer, G & Zetterberg, A, Exp cell res 88 (1974) 375. 6. Moore; G P’M, Exp cell res 68 (1971) 462. 7. Moore, G P M, Lintem-Moore, S M, Peters, H & Faber, M, J cell biol60 (1974) 416. 8. Jacob, S T. Prog nucleic acid res mol biol 13(1973) 93. ‘9. Poao, A 0, Littau, V C, Allfrev, V G & Mirsky, A E,?roc natl acad sci US 57 (1967) 743. 10. Zvlber. E A & Penman, S. Proc natl acad sci US 68 (1971) 2861. 11. Widnell, C C & Tata, J R, Biochim biophys acta 123(1%6) 478. 12. Lindell, T J. Weinberg, F, Morris, P W, Roeder, P G & Rutter,’ W J, Sci&ce 170(1970) 447. 13. Weinmann. R & Roeder, R G, Proc natl acad sci us 71(1974) 1790. 14. Butterworth, P H W, Cox, R F & Chesterton, C J, Eur j biochem 23 (1971) 229. 15. Chambon, P, Ramuz, M, Mandel, P & Doly, J, Biochim biophys acta 157(1968) 504. 16. Chambon, P, Karon, H, Ramuz, M & Mandel, P, Biochim biophys acta 157(1968) 520. 17. Petersen. E E & Kriiger, H, Z Natmforsch 25B (1970) 1042. 18. Goldberg, M L, Fed proc 29 (1970) 1261. 19. Moore, G P M, Gamete competition in plants and animals (ed D L Mulcahy) p. 69. North-Holland. Amsterdam (1975). 20. Perrv. R P. Exn cell res 29 (1%3) 400. 21. Meiihac, M, Typser, Z & dhambon, P, Eur j biothem 28 (1972) 291.
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22. Cox, R F, Eur j biochem 39 (1973) 49. 23. Penman, S, Rosbash, M & Penman, M, Proc natl acad sci US 67 (1970) 1878. 24. Hurwitz. J & Aueust. J T. Proe nucleic acid res 1 (1%3) 59. 25. Fox. C F&Weiss. S B. J biol them 239 (1964) 175. 26. Roeder, R G & Rutter; W J, Proc natl acad sci US 65 (1970) 675. 27. Kedinger, C, Nuret, P & Chambon, P, FEBS lett 15 (1971) 169. 28. Zetterberg, A, Auer, G & Moore, G P M, Exp cell res 88 (1975) 382. 29. Riva, S, Fietta, A & Silvestri, L G, Biochem biophys res commun 49 (1972) 1263. 30. Carlsson, S-A, Moore, G P M & Ringertz, N R, Exp cell res 76 (1973) 234. 31. Fu-Li, Y, Nature 251 (1974) 344. 32. Tocchini-Valentini, G P & Crippa, M, Nature 228 (1970) 993. 33. Wassarman, P M, Hollinger, T G & Smith, L D, Nature new biol240 (1972) 208. 34. Schechter. N M, Biochim bionhvs acta 308 (1973) 129. 35. Hotta, Y & Stem, H, Nature 210 (1%8) 1043. 36. Moore, G P M, J cell physiol. In press. 37. Fisher, D B, J cell biol39 (1%8) 745. 38. Payne, J F Br Bal. A K, Phytochemistry 11 (1972) 3105. 39. Tsai, R L & Green, H, Nature new biol 243 (1973) 168. 40. ~owkn, P D, Meek, R L & Daniel, C W, Exp cell res 101 (1976) 434. L
Received June 22, 1977 Accepted August 5, 1977
.
