Effect of amino acids on the α-amanitin-insensitive RNA polymerase activity in the isolated nuclei of ehrlich ascites cells

Biochimica et Biophysica Acta, 331 (1973) 71-80

~) Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 97841

E F F E C T OF A M I N O ACIDS ON T H E c~-AMANITIN-INSENSITIVE RNA POLYMERASE ACTIVITY IN T H E ISOLATED N U C L E I OF E H R L I C H ASCITES CELLS

MARfA T. FRANZE-FERNA/~IDEZ and ANGELINA V. FONTANIVE-SENG~ESA Departamento de Quimica Biol6#ica, Facultad de Farmacia .v Bioqulrnica, Universidad de Buenos Aires, Buenos Aires (Aryentina)

(Received June 14th, 1973)

SUMMARY The effect of amino acids on the ct-amanitin-insensitive R N A polymerase activity in the isolated nuclei of Ehrlich ascites cells was studied. The following results were obtained: 1. The R N A polymerase activity was stimulated by increasing the amino acid concentration in the incubation medium of the cells. 2. The amino acid requirement for RNA polymerase activation differed from the requirement that for the stimulation of protein synthesis. 3. No particular amino acid affected the RNA polymerase activity. The combined effect from the reduction of the concentration of several amino acids was higher than the response given by the deprivation of individual amino acids. 4. The RNA polymerase activation by amino acids has not been obtained in the presence of protein synthesis inhibitors, indicating that protein synthesis is involved in the process. 5. After protein synthesis inhibition by antibiotics there was a rapid decay in the RNA polymerase activity. A similar decay has been obtained after reducing the amino acid concentration. It is postulated that the ~-amanitin-sensitive R N A polymerase activity in the isolated nuclei of Ehrlich ascites cells~,is" regulated by a shortlived protein(s). Amino acids could stimulate the synthesis or decrease the degradation rate of this protein(s).

INTRODUCTION The absence of amino acids in the culture medium of eukaryotic cells affects the RNA metabolism 1- a. It has been shown that although RNA synthesis decreased in HeLa cells cultured in an amino acid-deprived medium compared to cells grown in complete medium, no difference was found in the DNA-dependent RNA polymerase activity in the isolated nuclei 5'9. There was no indication as to which RNA polymerase species was measured.

72

M . T . F R A N Z E - F E R N A / ~ D E Z , A. V. F O N T A N I V E - S A N G O E S A

One of the authors of this paper previously reported that in Ehrlich ascites cells incubated in medium deprived of amino acids, the RNA polymerase activity in isolated nuclei decreased compared to the activity in nuclei from cells incubated with amino acids 1°. The RNA polymerase activity was measured in Mg 2÷ and at low ionic strength. Under these conditions most of the activity was :t-amanitin-insensitive and probably corresponded to the nucleolar RNA polymerase I or A ~1'12 We report in the following a detailed exploration of the effect of amino acids on the ~amanitin-insentisive RNA polymerase activity. METHODS

Ehrlich ascites tumor cells were inoculated into the abdominal cavity of mice and harvested after 4-7 days, washed twice with the incubation medium at room temperature, and then incubated at a concentration of 1-2 • 106 cells/ml of medium.

Incubation media All of them had in common the following constituents per 100 ml of final volume: 10 ml of 10-fold concentrated phosphate-buffered saline (Dulbecco's medium) 4 ml of nondialyzed bovine serum, 1 ml of 100-fold concentrated Eagle's basal medium vitamins, 0.20 g glucose, 5000 units of penicillin and 5 mg of streptomycin. The complete medium received (per 100 ml) the addition of 2 ml of 50-fold concentrated Minimum Essential Medium (Eagle) amino acids and 1 ml of 100-fold concentrated Minimum Essential Medium non-essential amino acids. The incomplete media had no amino acids or fraction or multiples of the amino acid concentration in the complete medium as indicated in each case. The p H of the media was adjusted to 7.3 with 1 M NaOH. Preparation of nuclei Nuclei were prepared starting with about 7 • 10 7 cells as previously described 10 with the following modifications: the crude nuclear pellet was resuspended in 10 ml of a solution containing 0.3 M sucrose, 4 mM MgCI2, 10 mM Tris-HCl buffer (pH 7.90) and 1 mM dithioerythreitol, sedimented by centrifugation, washed with 10 ml of the same solution containing 100/tg/ml polyvinylsulfate and finally resuspended in the medium to be used for the RNA polymerase assay. RNA polymerase assay RNA polymerase was assayed by measurement of the incorporation of [ 3 H ] GTP into RNA. Unless otherwise indicated the assay mixture contained in a final volume of 0.25 ml: 7.5/~moles Tris-HCl buffer pH 8.0; 1 kLmole MgC12; 15/tmoles NaCI; 0.25/~mole dithioerythreitol; 0.08/~mole each of ATP, CTP and UTP; 0.025 #mole [3H]GTP (spec. act. 20-50 Ci/mole); 1 /zmole phosphoenolpyruvate; l ltg pyruvate kinase; 1 #g ~-amanitin and nuclear suspension containing 80-90 pg DNA. Unless otherwise indicated incubation was for 8 min at 37 °C. The reaction was stopped by the addition of 7 ml of 5 ~ cold trichloroacetic acid containing 40 mM sodium pyrophosphate. The precipitate was collected and washed and the radioactivity was counted as previously described ~°. All the assays were performed in triplicate.

E F F E C T O F A M I N O A C I D S ON R N A P O L Y M E R A S E

73

Incorporation of radioactive amino acids into proteins About 1.5 • 106 cells labeled with [~4C]leucine were poured into 10 ml of cold Dulbecco's saline containing 0.4 #mole of leucine, and sedimented by centrifugation. The cells pellet was dissolved in 2 ml of 1 m KOH, allowed to stand for 10 min at room temperature, and precipitated with 1.5 ml of 50 % trichloroacetic acid. The precipitate was collected by centrifugation and washed three times with 7 ml of 5 trichloroacetic acid. The final precipitate was dissolved and the radioactivity counted as described elsewhere ~o. All the assays were performed in triplicate. In the experiments where protein synthesis has to be measured the cells were incubated in media containing variable concentrations of amino acids and a fixed amount of [a4C]Leucine. It is possible that at higher concentrations of the nonlabeled amino acids the transport of the labeled amino acid into the cells and, therefore, the isotope incorporation could be inhibited. In this case, the inhibition would be reverted by higher concentrations of the labeled amino acid. With this in mind the experiment illustrated in Fig. 1 was performed. The cells were incubated in media containing two different concentrations of non-labeled amino acids and the kinetics of the [a4C]leucine incorporation was determined in each medium at two different

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Fig. 1. Effect o f variable concentrations o f cold a m i n o acids on the incorporation o f [14C]leucine into protein. Ehrlich ascites cells were incubated in m e d i u m containing 1.5 times the a m i n o acid concentration in complete m e d i u m ( © ) or in a m e d i u m that contains the a m i n o acids o f group 1 (see Table I) at 1.5-fold the concentration in complete m e d i u m a n d the a m i n o acids o f groups 2 a n d 3 at 0.10 o f the concentration in the complete m e d i u m ( A ) . The m e d i a contained 0.15 m M ( --) or 0.60 m M (- - -) o f [14C]leucin e with a specific activity o f 0.30 Ci/mole. At the times indicated the isotope incorporation was stopped a n d the cells were processed as described in Methods. Fig. 2. Kinetics o f the R N A polymerase activation by a m i n o acids. Ehrlich ascites cells were preincubated for 2 h in m e d i u m with no a m i n o acids.'After this time the nuclei were isolated in an aliquot o f the cell suspension a n d the R N A polymerase activities were m e a s u r e d . These are the zero time values. T h e cells o f the r e m a i n d e r o f the incubate were collected by centrifugation a n d resuspended in complete m e d i u m ( ) or in m e d i u m without a m i n o acids ( - - - ) . After incubation the cells for the times indicated the R N A polymerase activity in the isolated nuclei was determined. ( A ) , the assay m e d i u m was the one as described under M e t h o d s ; ( A ) , the assay m e d i u m o f high ionic strength contained in a final v o l u m e o f 0.25 ml: 0.37 # m o l e MnClz; 1.5 # m o l e s KCI; 100 # m o l e s (NH4)2SO4 adjusted to p H 7.9 with Nlff3; the rest o f the constituents were the s a m e as indicated in M e t h o d s with the omission o f MgCI2 and NaCI.

74

M.T. FRANZE-FERNA/~IDEZ, A. V. FONTANIVE-SANGI~ESA

concentrations of [t4C]ieucine. It can be seen that there is a slight increase in the isotope incorporated at the higher concentration of [14C]leucine. But the same increase was observed at the two different concentrations of the cold amino acids. Similar results were obtained when the incorporation of [14C]methionine was measured at concentrations of 0.04 mM or 0.16 mM of this amino acids. These results indicate that an inhibitory effect by the cold amino acids, if any, is the same at the two different concentrations of the non-labeled amino acids. At these amino acid concentrations a variation was found in the RNA polymerase activity (see Table I, Figs 7 and 8). In the experiments where [14C]leucine incorporation was measured the concentration of leucine in all the incubation media was 0.15 mM.

Chemicals ~-Amanitin was a generous gift from Professor Th. Wieland of the Max Planck Institute, Heidelberg (Germany); pactamycin was kindly supplied by the Upjohn Co.; cold amino acids were purchased from Sigma Chemical Co.; [14C]leucine (spec. act 170Ci/mole) from SchwarzMann; [3H]GTP (spec. act 1 Ci/ 0.12 g) from New England Nuclear; Vitamins Minimal Eagle was purchased from Difco and cycloheximide from Calbiochem. RESULTS AND D[SCUSSION

Effect of amino acids on the cl-amanitin-insensitive RNA polymerase activity in Ehrlich ascites cells Following the addition of amino acids to the incubation medium of Ehrlich ascites cells there is a rapid increase in the ~-amanitin-insensitive RNA polymerase activity in the isolated nuclei when assayed at low salt or at 0.4 M (NH4)2SO 4 (Fig. 2.). Under the latter condition there is a substantial dissociation of chromosomal proteins and the activity is probably a reflection of the amount of RNA polymerase present 13' 14. As shown in Fig. 3 the kinetics of the RNA polymerase reaction is similar in nuclei isolated from ceils incubated in media with or without amino acids. However, the rate of G M P incorporation is considerably enhanced by amino acids. The amount of [3H]GMP incorporated by a mixture of equal parts of nuclei from cells incubated in complete medium or in an amino acid-deprived medium corresponds to the mean values of the incorporations observed when the nuclei are assayed independently. As the assays were performed at a salt concentration where the nuclei are disrupted this result argues against the presence of diffusing inhibitors or activators in the nuclei from cells incubated both with or without amino acids. Similar results to those in Fig. 3 were obtained when the RNA polymerase assays were carried out in whole cell homogenates (Unpublished results). Effect of protein synthesis inhibitors and amino acids on RNA polymerase activity and protein synthesis The data in Fig. 4 show that inhibition of protein synthesis by pactamycin prevents the activation of the R N A polymerase by amino acids. The inhibitor, however, does not affect the RNA polymerase activity in the nuclei from cells incubated

EFFECT OF AMINO ACIDS ON RNA POLYMERASE

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Fig. 3. Kinetics of the RNA polymerase reaction in isolated nuclei of Ehrlich ascites cells. The cells were incubated in complete medium or in medium with no amino acids for 2 h. At that time the nuclei were isolated and the RNA polymerase activity was estimated in the assay medium described in Methods, with the addition (for 0.25 ml) of 100 ffmoles (NH4)2SO4. Nuclei from cells incubated in complete medium ( ~ ) ; nuclei from cells incubated in the amino acids deprived medium (O); equal parts of nuclei from cells incubated in complete medium or in medium without amino acids (O). Fig. 4. Effect of inhibition of protein synthesis on the RNA polymerase activation by amino acids. Ehrlich ascites cells were incubated in medium deprived of amino acids. After 20 min the cells were collected by centrifugation and resuspended in the following media: medium with no amino acids ( x ) ; the same, containing 0.2#g/ml pactamycin (A); complete medium (~l); complete medium plus 0.2 ffg/ml pactamycin (O). After incubating for the times indicated nuclei were isolated and the RNA polymerase activity determined as described in Methods. The inhibition of [14C]leucine incorporation into proteins by 0.2 ffg/ml pactamycin was more than 95 %. i

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Fig. 5. Protein synthesis and RNA polymerase activity in Ehrlich cells during incubation at different amino acid concentrations. Cells were incubated in media containing the fractions or multiples of the amino acid concentrations in the complete medium indicated. After 1 h (O) or 2.5 h ( 0 ) the nuclei were isolated and the RNA polymerase activity was assayed as described under Methods. The inset shows the kinetics of the [14C]leucine incorporation into proteins in cells incubated in medium with no amino acids ( x ) , with 0.1 of the amino acid concentrations in the complete medium (A); with 0.2, 0.5, 1.0 or 3.0 of the amino acid concentrations in the complete medium (11). All the media had 0.05 ffCi/ml ([14C]leucine. At the times indicated the isotope incorporation was determined as described in Methods. It can be seen that the incorporation is linear during the 3 h of incubation. The data o f the [14C]leucine incorporation expressed in percentage, were plotted in the main figure against the amino acid concentration (A).

76

M.T. FRANZE-FERNA/~IDEZ, A. V. FONTAN1VE-SANGOESA

without amino acids. Similar results are obtained when protein synthesis is inhibited by cycloheximide. These results indicate that protein synthesis is involved in the activation of the R N A polymerase by amino acids. When Ehrlich cells are incubated in media without amino acids, protein synthesis is reduced to 20-30 % o f the control values (Fig. 5). It m a y be possible that the increase in R N A polymerase activity observed when the cells are transferred to a medium enriched in amino acids is a consequence of the general stimulus to protein synthesis. To test this possibility the experiments described in Figs 5 and 6 were performed. In the experiment illustrated in Fig. 5 the ceils were incubated in media with amino acid concentrations varying from 0-3-fold that o f the concentration in the complete medium. The R N A polymerase activity and the [t 4C]leucin e incorporation into proteins were estimated and the values plotted against the amino acid concentration in the incubation media. It can be seen that while the m a x i m u m [14C]leucine incorporation is attained at an amino acid concentration o f approx. 0.2 that o f the complete medium, a higher concentration is required for full activation of the R N A polymerase, either after incubating the cells for 1 or 2.5 h in the different media. This result shows that the increase in R N A polymerase activity by amino acids is not simply correlated with the stimulus to protein synthesis. 100

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Fig. 6. Comparison of the effects of low amounts of protein synthesis inhibitors and reduced amino acid concentrations on RNA polymerase activity and protein synthesis. After 50 min of incubation of Ehrlich ascites cells in the media described below, nuclei were isolated and the RNA polymerase activity assayed as indicated in Methods. Aliquots of the cell suspensions were labeled for 50 min with 0.05 ffCi/ml [t 4C]leucine and the isotope incorporation was determined as described in Methods. The data of the RNA polymerase activity were plotted as a function of the [t 4C]leucine incorporated. The values when the cells are incubated in complete medium are 100 % (~,); cells incubated in complete medium plus cycloheximide at the concentrations indicated, (A); in complete medium plus pactamycin at the concentrations indicated (S); cells incubated in medium without amino acids (11); or in medium with 0.05 ( × ) or 0.10 (IS]) the amino acid concentration of the complete medium. If protein synthesis is partially inhibited by antibiotics there is a corresponding reduction in the R N A polymerase activity. In Fig. 6 the relative effects of submaximal concentrations o f cycloheximide and pactamycin, and of increasing amino acid deprivation, on R N A polymerase activity and protein synthesis are compared.

EFFECT OF AMINO ACIDS ON RNA POLYMERASE

77

F o r a given decrease in p r o t e i n synthesis, b o t h inhibitors show a similar decrease in R N A p o l y m e r a s e activity thus arguing against a side effect o f the antibiotics. By c o m p a r i s o n , a m i n o acid d e p r i v a t i o n results in a greater effect on the R N A p o l y m e r a s e activity t h a n w o u l d be expected solely f r o m its effect on protein synthesis. Amino acid requirement f o r activation o f the R N A polymerase To d e t e r m i n e whether all or only some o f the a m i n o acids affect the ~-amanitin-insensitive R N A p o l y m e r a s e activity the ceils were i n c u b a t e d in m e d i a c o n t a i n i n g variable a m o u n t s o f each o f three different g r o u p s o f a m i n o acids. I n T a b l e I it can be seen that the enzyme is affected by reducing the c o n c e n t r a t i o n o f any o f the tested groups, a l t h o u g h u n d e r certain c o n d i t i o n s there is no change in protein synthesis. F r o m these results a n d those o f Figs 5 a n d 6 it is clear that the a m i n o acid r e q u i r e m e n t for R N A p o l y m e r a s e activation differs f r o m that for the r e q u i r e m e n t for the stimulation o f p r o t e i n synthesis.

TABLE I EFFECTS OF DIFFERENT AMINO ACID GROUPS ON RNA POLYMERASE ACTIVITY AND PROTEIN SYNTHESIS IN EHRLICI-[ ASCITES CELLS Cells were incubated in media containing the amino acids at the concentrations indicated. After 1 h the nuclei were isolated and the RNA l:olymerase activity assayed as described in Methods. Aliquots of the cell suspension were incubated with 0.05/~Ci/ml [14Clleucine for l h and the incorporation into proteins estimated as described in Methods. Amino acids in each group, group l : Arg, Ileu, Cys, Thr, His, Tyr; group 2: Lys, Val, Met, Phe, Trp, Gin; group 3: Ala, Asp, Pro, Asn, Glu, Ser, Gly. Expt. no.

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M. T. FRANZE-FERNA~4DEZ, A. V. FONTANIVE-SANGOESA

The effects of single essential amino acid deprivation on RNA polymerase activity are shown in Fig. 4. The decrease in enzyme activity when individual amino acids are supressed from the complete medium is correlated with the reduction in protein synthesis. But the RNA polymerase activity is more affected in cells incubated in media deprived of more than one amino acid or with a reduced concentration of several amino acids than in cells starved of a single amino acid. Upon suppression of tyrosine and histidine an increase in the RNA polymerase activity over control levels was found in most experiments. This effect cannot be explained at present. One of the possibilities is that in the absence of certain amino acids there is an increased transport of others into the ceils. From these experiments it is not possible to assess the relative effects of all the amino acids on the RNA polymerase activity. As shown in Fig. 6 protein synthesis inhibition affects the enzyme activity, however, the results are difficult to interpret when protein synthesis is reduced Nevertheless the data availabe suggests that no amino acid in particular affects the RNA polymerse activity and indicates that the combined effect of the reduction of the concentration of several amino acids is higher than the response given by the deprivation of individual amino acids. Decay of R N A polymerase activity after shiftin9 Ehrlich cells to media with a lower amino acid concentration or with protein synthesis inhibitors When Ehrlich cells are transferred from the complete medium to a medium without amino acids or containing a reduced amino acids concentration than in the complete medium, or upon inhibition of protein synthesis there is a rapid decay in the RNA polymerase activity (Fig. 8). It is remarkable that the R N A polymerase activity decays to similar values in all cases although protein synthesis varies from less than 2 ~ of the control in the ceils treated with pactamycin to more than 90 in the cells incubated in media with reduced concentrations of several amino acids. The results of Fig. 4 and 8 indicate that a fraction of the :~-amanitin-insensitive RNA polymerase activity is not regulated by amino acids, nor is it affected by protein synthesis inhibition. Whether this activity corresponds to another enzyme remains to be seen. It is of importance to note, however, that the 45-S rRNA precursor is being synthesized although at a reduced rate in Ehrlich cells deprived of amino acids 1° and in Ehrlich L and HeLa cells after inhibition of protein synthesis ~5-17 The findings that, when protein synthesis is partially inhibited by antibiotics there is a corresponding decrease in the RNA polymerase activity (Fig. 6), and the rapid decay of the enzyme activity after complete inhibition of protein synthesis (Fig. 8) strongly suggest that a short-lived protein(s) regulates the c~-amanitin-insensitive RNA polymerase activity. This conclusion is in agreement with the suggesttion that a short-lived protein(s)is required for the transcription of the nucleolar genes in rat liver ~s and with observations that in HeLa cells the rate of synthesis of the 45-S rRNA precursor is regulated by the availability of a particular protein. One possible interpretated of our results is that the amino acids either stimulate the synthesis or activation or decrease the degradation rate of a short-lived protein(s) that regulates the ~-amanitin-insensitive R N A polymerase activity in the isolated nuclei. This short-lived protein(s) may alter the availability of the DNA template or may be a subunit or factor of the RNA polymerase that controls the amount of active enzyme. In this regard it has to be pointed out that

E F F E C T OF A M I N O ACIDS ON R N A POLYMERASE

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Fig. 7. E•ects•fdeprivati•n•fsing•eamin•acids•nRNAp••ymeraseactivityandpr•teinsynthesis. Ehrlich cells were incubated in the following media: complete medium ( x ) ; complete medium deprived of the single amino acid indicated (A); complete medium deprived of: Arg, His, Thr, and Tyr (A); or Arg, Thr, Tyr, Phe, Val and T r p ( O ) ; medium that contains the amino acids of group 1 (see Table I) at 1.5 the concentrations in complete medium and the amino acids of groups 2 and 3 at 0.10 the concentration in complete medium (O). After 1 h of incubation at 37 °C the nuclei were isolated and the R N A polymerase activity estimated as indicated in Methods. Aliquots of the incubates were labeled for 1 h with 0.05 pCi/ml of [~4C]leucine and the incorporation into protein determined as described in Methods. The data from the R N A polymerase activity were plotted as a function of the [14C]leucineincorporated into protein. The values obtained in cells incubated in complete medium are 100 %. The results of three different experiments are shown and they are indicated by numbers. Fig. 8. R N A polymerase activity and protein synthesis after shifting Ehrlich cells to media with lower amino acid concentrations or with protein synthesis inhibitors. Cells were preincubated 90 min in complete medium. After this time the nuclei were isolated in an aliquot of the cell suspension, and the RNA polymerase activity was assayed as described in Methods. This was the zero time value in A and was considered 100 % (O). Another aliquot was pulsed with 0.05 pCi/ml of [~4C]leucine the last 20 min of the preincubation and the isotope incorporation was determined as described in Methods. The value obtained was considered as 100 % and corresponded to the zero time in B (O). The cells of the remainder of the incubate were collected by centrifugation and resuspended in the following media: complete medium (O, 0 ) ; medium without amino acids (F1, I ) ; medium containing the amino acids of group 1 (see Table [) at 1.5 times and of groups 2 and 3 at 0.10 times the concentrations in the complete medium (,~, A); complete medium plus 0.2 #g/ml pactamycin ( × ) . After incubation of the cells for the times indicated the nuclei were isolated and the R N A polymerase activity asssayed as described in Methods. (A) To aliquots of the cell suspension 0.05/zCi/ml [L4C]leucine was added 20 min before the indicated times. At these times the isotope incorporation was stopped and the cells were processed as described in Methods. (B) The black and the white symbols ( x ) correspond to independent experiments. t h e a m o u n t s o f t h e d i f f e r e n t R N A p o l y m e r a s e species i s o l a t e d f r o m r a t l i v e r a r e n o t a f f e c t e d b y p r o t e i n s y n t h e s i s i n h i b i t i o n o v e r p r o l o n g e d p e r i o d s o f t i m e 19. W e a r e in t h e p r o c e s s o f p u r i f y i n g t h e R N A p o l y m e r a s e s f r o m E h r l i c h a s c i t e s cells t o d e t e r m i n e whether the isolated enzymes are affected by the amino acid concentrations in the medium.

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M . T . F R A N Z E - F E R N A N D E Z , A. V. F O N T A N I V E - S A N G ~ E S A

ACKNOWLEDGEMENTS

We thank Dr A. C. Paladini (Department of Biochemistry, Schoo! of Pharmacy and Biochemistry, University of Buenos Aires) for his support and Drs O. A. Scornik (Department of Biochemistry, Darmouth Medical School) and O. A. Pogo (Mew York Blood Center) for the critical revision of the manuscript. M. T. Franze-Fern~indez is member of the Career Investigator of the Consejo Nacional de Investigaciones Cientificas y T~cnicas de la Reptlblica Argentina. This work was supported, in part, by grant from the same Institution. REFERENCES I Vaughan, M. H., Soeiro, R., Warner, J. R. and Darnell, J. E. (1967) Proc. Natl. Acad. Sci. U.S. 58, 1527-1534 2 Maden, B. E. H., Vaughan, M. H., Warner, J. R. and Darnell, J. E. (1969) J. MoL Biol. 45, 265-275 3 Maden, B. (1971) Biochem. J. 123, 36P 4 Shields, R. and Korner, A. (1970) Bioehim. Biophys. Aeta 204, 521-530 5 Smulson, M. and Thomas, J. (1969) J. Biol. Chem. 244, 5309-5312. 6 Ellem, K. A. O., Fahrizio, A. M. and Jackson, L. G. (1970) Bioehim. Biophys. Acta 209, 425--444 7 B01csfOldi, G., Lambert, P. and Elliasson, E. (1971) Bioehim. Biophys. Aeta 228, 664-675 8 Btilcsf/51di, G. and Elliasson, E. (1972) Bioehim. Biophys. Aeta 272, 67-74 9 Smulson, M. E. (1970) Biochim. Biophys. Aeta 199, 537-541) 10 Franze-Fern~-ndez, M. T. and Pogo, A. O. (1971) Proc. Natl. Aead. Sci. U.S. 68, 3040-3044 1! Roeder, R. G. and Rutter, W. J. (1969) Nature 224, 234-237 12 Kedinger, C., Gniazdowski, M., Mandel, Jr, J. L., Gissinger, F. and Chambon, P. (1970) Biochem. Biophys. Res. Commun. 38, 165-171 13 Butterworth, P. H. W., Cox, R. F. and Chesterton, C. J. (197l) Eur. J. Biochem. 23,229-241 14 Spelsberg, T. C. and Hnilica, L. S. (1971) Bioehim. Biophys. Acta 228,202-211 15 Mandal, R. K. (1969) Bioehim. Biophys. Aeta 182, 375-381 16 Craig, N. C. and Perry, R. P. (1970)J. CelIBiol. 45, 554-564 17 Willems, M., Penman, M. and Penman, S. (1969) J. Cell Biol. 41, 177-187 18 Muramatsu, M., Shimada, N. and Higashinakagawa, T. (1970) J. Mol. Biol. 53, 91-106 19 Benecke, B. J., Ferencz, A. and Seifart, K. H. (1973) FEBS Lett. 31, 53-58