Translation of Histone Messenger RNA by Homologous Cell-Free Systems from Synchronized HeLa Cells

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1 Eur. J. Biochem. 42, (1974) Translation of Histone Messenger RNA by Homologous Cell-Free Systems from Synchronized HeLa Cells Fra.nco GABRIELLI and Corrado BAGLIONI Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts (Received October 5, 1973) Cell-free systems have been prepared from HeLa cells synchronized in S phase and from the same cells treated with cytosine arabinoside to inhibit DNA synthesis. Translation of HeLa cell histone mrna by these cell-free systems is equally efficient. The cell-free systems have been characterized for K+ and Mg2f optima which are similar to those of the mouse ascites cell-free system. One major difference between these two cell-free systems is the reduced efficiency of initiation of the HeLa system. This can be overcome by the addition of rabbit reticulocyte initiation factors, which stimulate 4-5-fold translation of histone mrna. The possibility that a translational inhibitor of histone synthesis is lost during the preincubation of the HeLa extracts has been tested by using cell-free systems prepared with non-preincubated extracts. In addition, it has been established that nuclear HeLa cell histones inhibit protein synthesis in a non-specific way; when added to an ascites or HeLa cell-free system they do not inhibit specifically the translation of histone mrna and thus cannot act as specific translational repressors. Histones are synthesized by cells in culture during the S phase of the cell cycle [l-21. A block in the synthesis of DNA causes a rapid decline in the synthesis of histones and the loss of histone mrna [3-51. These events cannot be explained by transcriptional regulation of histone mrna synthesis, since the lifetime of histone mrna is normally a few hours long [6], whereas histone synthesis stops within 30 min of a block in DNA synthesis [3]. It has thus been suggested that histone synthesis is regulated at the translational level [3-61. We have studied the translation of histone mrna from HeLa cells in heterologous cell-free systems prepared from normal cells and cells inactive in DNA synthesis [7]. These cell-free systems translate histone mrna equally well and little information on the regulation of histone synthesis has been obtained, with one exception. It has been clearly established that translation of histone mrna does not require synthesis or activation of a factor for protein synthesis not present in cells which do not synthesize DNA [7]. The possibility that a specific inhibitor blocks translation of histone mrna has also been investigated [7]. An inhibitor of the synthesis of catalase by liver and hepatoma cells has been described [S-9] and it seems thus conceivable that the synthesis of other proteins is regulated in this way at the translational level in eukaryotic cells. We have, however, found no evidence for the presence of a translational inhibitor in the postribosomal supernatant of S- phase HeLa cells blocked in DNA synthesis [7], using a heterologous cell-free system, with histone mrna from HeLa cells and ribosomes from ascites cells. If the hypothetical inhibitor were strictly species-specific, its activity would not be detected in a heterologous cell-free system. For this reason we have studied the translation of HeLa cell histone mrna in cell-free systems prepared from HeLa cells and have also tested the effect of addition of HeLa cell histones on the translation of histone mrna. MATERIALS AND METHODS Cell Growth and Synchronization HeLa cells were grown in minimal Eagle's medium [lo] in spinner flasks to a density of 3--5x lo6 cells/ ml. The procedures used to synchronize the cells by a double thymidine block and to inhibit DNA synthesis with 25 pg/ml of cytosine arabinoside have been described in detail previously [7]. DNA synthesis was measured as thymidine incorporation into trichloroacetic-acid-insoluble material. To 0.5ml of cell suspension were added 40nCi thymidine for 15 min at 37 "C. The incubation was stopped by the addition of cold saline containing 0.1 mm unlabeled

2 122 Control of Histone Synthesis thymidine ; the cells were collected by centrifugation and dissolved in 0.5 ml of 0.2 N KOH. 2 ml of loo/, trichloroacetic acid was added and the precipitate collected on Millipore filters and counted as described previously [el. The cells were collected by centrifugation and washed with 46 mm NaC1, 35 mm Tris-HC1 ph 7.6. They were homogenized, fractionated and preincubated as described for mouse ascites cells [12,13]. The preincubated extracts were passed through Sephadex 0-25 columns as previously described [13] and stored frozen at -70 "C in small aliquots. Some extracts were used directly for protein synthesis by omitting preincubation and gel filtration. The composition of the cell-free system was identical to that described for the Krebs ascites cell-free system [12, vol. HeLa extract was used for 10 vol. incubation mixture; aqueous solutions of the other components of the cell-free system were added in the remaining 4 vol. Radioactive amino acids were added as indicated in the legends to the tables and figures; they were purchased from New England Nuclear. HeLa cell transfer RNA (trna or 4-S RNA) was obtained as a byproduct of the preparation of histone mrna [14]. RNA was extracted with phenol from polyribosomes of HeLa cells or from total cellular RNA and fractionated on sucrose gradients and 4-S RNA isolated by preparative polyacrylamide gel electrophoresis [14]. The fractions containing 4-5 RNAwere combined and the RNAprecipitated by the addition of 2 vol. ethanol. Other components used in the cell-free system were histone and globin mrna; these were prepared as previously described [13,14]. A reconstituted cell-free system was prepared by mixing HeLa cell ribosomes with postribosomal supernatant. The preparation of ribosomes and the supernatant has previously been described in detail [7]. Ribosomes were resuspended at a concentration of 4-5mg/ml and used as described in the text. Initiation factors were prepared from rabbit reticulocyte ribosomes according to Shafritz et al. [15]; the preparation used contained 20 mg/ml of protein. The samples were processed for counting as previously described [13]. We are grateful to Dr Yves G. Bauzard for the gift of rabbit reticulocyte initiation factors and to Dr Thaddeus W. Borun for the gift of HeLa cells histone mrna and trna. Analysis of the Products of Protein Synthesis The products of cell-free protein synthesis were analyzed by electrophoresis on 15 polyacrylamide gels according to Panym and Chalkley [ll]. 50 pg/ml deoxyribonuclease was added to each sample and the incubation at 30 "C continued for 30 min; the samples were then dialyzed against 0.2 M acetic acid, 6.25 M urea and 10 mm mercaptoethanol. The dialyzed samples were heated for 2 min at 80 "C and applied immediately to 15O/, polyacrylamide gels [ll]. The gels were fractionated in a Savant Autogeldivider and counted as previously described [14]. An equal amount of a solution of marker histones labeled with l4c-1abeled amino acids was added to each sample before dialysis. RESULTS Characterixation of the HeLa Cell-Free System The preparation of a cell-free system from HeLa cells with little endogenous activity and efficient in translating exogenous mrna has previously been described [ls, 171. Since this system was not characterized in detail for K+ and Mg2+ optima, we have preliminarily established the best conditions for translation of added mrna. Three different cell-free systems were prepared, one from unsynchronized cells, one from cells synchronized in S phase by a double thymidine block (see Methods) and one from the same cells treated for 30 min with cytosine arabinoside to inhibit DNA synthesis. The postmitochondrial fraction obtained from these cells was preincubated following the procedure used for the preparation of the mouse ascites cell-free system [12,13], to reduce endogenous protein synthesis. The K+ and Mg2+ optima were determined for all these extracts using histone and globin mrna. The results were essentially identical for all the extracts and the K+ optimum was between 90 and 100 mm KC1 (Fig.1). The Mg2+ optimum was at 2.5 mm. This suggested that the HeLa cell-free system did not differ significantly from the ascites cell-free system, which has been shown previously to have a Mg2+ optimum between 2 and 3 mm [13]. Some of the HeLa extracts gave rather poor incorporation with histone mrna. Since some authors [18,19] have previously reported a partial dependence of the ascites cell-free system on the addition of exogenous trna, we have tested the effect of addition of HeLa cell trna on the translation of histone and globin mrna (Table 1). No stimulation of the translation of globin mrna was observed by adding trna to the HeLa cell-free system. Stimulation of histone mrna translation was instead observed with some of the HeLa extracts tested, whereas little stimulation was observed with other extracts (Table 1). We have attributed this variability of the stimulation by added trna to slight differences in the preparation of the extracts. In particular, some trna may be lost in the elution of preincubated extracts from the Sephadex G-25 column when only the first fractions eluted with the void colume are combined [13]. Alternatively, trna species necessary for the translation of histone

3 ~~ F. Gabrielli and C. Baglioni 123 Table 1. Effect of addition of trna 012 protein synthesis by the HeLa cell-free system Each incubation contained 1.4 yci [3H]lysine (26 Ci/mmol), 6 yl extract, 1 pl of the 10-fold concentrated components of the cell-free system (see Methods), 1 yl unsynchronized HeLa cell trna solution (1.1 mg/ml), 2 PI histone mrna solution (1.1 mg/ml) or 1 p1 globin mrna solution (0.45mg/ ml) and water to a final vol. of 10 pl. After 60 min at 30 "C duplicate 4-y1 samplings were processed for counting as described in Methods Extract mrna trna 3H-labeled protein S-phase cells - S-phase cells histone histone treated with Cyt-Aran histone histone Unsynchronized cells histone histone globin globin a Cytosine arabinoside counts/min mrna and present in HeLa cells in small amount, may become limiting if partially removed by gel filtration or inactivated during the preincubation. To avoid this problem, we have used in all subsequent experiments extracts which showed little stimulation by added trna or otherwise we have supplemented the incubations with HeLa cell trna. Translation of Histone mrna by HeLa Cell-Free Xystems The cell-free system prepared from HeLa cells synchronized in S phase and that prepared from the same cells treated with cytosine arabinoside have been used to translate histone mrna. The first cellfree system is prepared from cells actively synthesizing histones, whereas the latter is prepared from cells which do not synthesize histones [3-61. The results obtained (Table 1) have indicated that both cell-free systems are equally active in translating histone mrna. In the cell-free system from cells treated with cytosine arabinoside we have also tested the translation of histone mrna in the presence of trna from S-phase cells and from the same cells treated with cytosine arabinoside (Table 2) ; no difference in translation of histone mrna was seen. The cell-free systems described above employed HeLa cell extracts which had been preincubated for Fig.1. Dependence on Kf concentration of the translation of histone mrna (Y-9-8 RNA) by the HeLa cell-free system. Each incubation contained in 10 p1 final vol: 1.4 yci [3H]- lysine (29 Ci/mmol), 2 y1 of an aqueous solution of histone mrna (1.1 mg/ml) or no RNA, 6 yl of HeLa cell extract, 1 yl of the 10-fold concentrated components of the cell-free system described under Methods minus KCI and 1 y1 KCI solutions, to obtain the Kf concentration indicated. 4-yl samples were taken in duplicate for counting after 60 min at 30 "C. Stimulation is defined as the radioactivity of the protein obtained in the incubation plus 7-9-S RNA divided by that in the incubation minus added RNA. (0) 3H-labeled protein in system-rna and (0) in system RNA; (0) stimulation Table 2. Translation of histone mrna by cell-free system from 8-phase HeLa cells treated with cytosine arabinoside The incubations are identical to those described in the legend of Table 1, with the only expection that 1.45 yci [3H]lysine (29 Ci/mmol) was used for each incubation. This batch of [SHIlysine was found to give better incorporation than other batches previously used; this may be explained by the previous observation [7] that some [3H]lysine preparations contain some compound inhibitory for the ascites cell-free system mrna trna 3H-labeled protein None Histone Histone S phase 2646 Histone Cyt -ha% 2863 Globin counts/min * From cells treated with cytosine arabinoside.

4 124 Control of Histone Synthesis 1 h to depress endogenous protein synthesis. It seemed possible that during this preincubation some factor involved in the regulation of histone synthesis could be inactivated. Friedman et al. [21] have recently reported that an extract of interferontreated L cells infected with vaccinia virus translates poorly encephalomyocarditis virus RNA ; preincubation of the extract at 37 "C reverses this effect, presumably by destroying an inhibitor of translation. In order to exclude the possibility that an unstable inhibitor is involved in the regulation of histone synthesis, we have used cell-free systems reconstituted with postribosomal supernatant of non-preincubated HeLa cell extracts and ribosomes from the extracts described above. No difference could be detected in the efficiency of translation of histone mrna by a cell-free system supplemented with postribosomal supernatant from S-phase cells or from cells blocked in DNA synthesis (Table 3). This allowed us to exclude that a hypothetical translational inhibitor of histone synthesis present in the postribosomal supernatant is inactivated during the preincubation. It does not eliminate the possibility that an inhibitor may be bound to ribosomes and may be inactivated in the preincubation. Xtimulation of the HeLa Cell-Free System by Rabbit-Reticulocyte Initiation Factors In comparing the efficiency of translation of histone or globin mrna in the HeLa cell-free system with that obtained in the ascites cell-free system [7], we noticed that the HeLa cell-free system is less active. It has previously been reported that this is true also for the translation of viral RNA [I61 and Table 3. Translation of histone mrna by reconstituted cellfree systems Each incubation contained in a final vol. of 10 pl: 1.4 pci [SH]lysine (29 Ci/mmol), 1 pl of ribosome suspension (see Methods), 5 pl postribosomal supernatant, 1 p1 of the 10- fold concentrated components of the cell-free system, 1 pl histone mrna solution (0.6 mg/ml) and 2 pl water. After 1 h at 30 "C, 4-pl aliquots were sampled in duplicate for counting. Stimulation is defined as the radioactivity of the protein of an incubation with added mrna, divided by that of the incubation minus added RNA Ribosomes Postribo- mrna 3H-labeled Stimulation soma1 protein supernatant counts/min Sphase Sphase Sphase Sphase Sphase Cyt-Ara Cyt-Araa Cyt-Araa Cyt-Araa Cyt-Araa Cyt-Araa Sphase a From cells treated with cytosine arabinoside. that addition of a crude preparation of reticulocyte initiation factors stimulates translation of immunoglobulin light chain mrna by the HeLa cell-free system [17]. We have thus investigated the effect of addition of reticulocyte initiation factors on the translation of histone mrna. The initiation factors were prepared by washing reticulocyte ribosomes with 0.5 M KC1 and treating this preparation with DEAE-cellulose to eliminate globin mrna as described by Shafritz et al. [15]. The ribosomal wash obtained is enriched in initiation factors and has been shown to stimulate the translation of rabbit globin mrna by an ascites cell-free system [19]. We found that addition of reticulocyte factors stimulated the translation of histone mrna 4-5-fold (Table 4). In this experiment we also tested the effect of added trna alone or together with the reticulocyte factors and found that it had no effect on the translation of histone mrna. These results suggest that the preincubated HeLa cell-free system is rather poor in initiation factors, compared to the ascites cell-free system. The reticulocyte factors appear to stimulate protein synthesis in a non-specific way, since they promote translation of the mrna for histones, which is not present in reticulocytes. It has already been shown that a reticulocyte cell-free system translates HeLa cell histone mrna [22]. The reasons for the relative inefficiency of the preincubated HeLa cell-free system are not known; we have noticed that a somewhat better incorporation is obtained with celbfree systems reconstituted Table 4. Stimulation of histone mrna translation by reticulocyte initiation factors Each incubation contained 1.5 pci [SH]lysine(28.7 Ci/mmol), 6 p1 HeLa cell extract, 1 p1 of the concentrated components of the cell-free system minus KCl (see Methods), 1 p1 histone mrna solution (1.1 mg/ml), 1 p1 HeLa trna solution (1.1 mg/ml), 0.5 or 1.0 pl reticulocyte factors in 0.3 IW KCl, 0.5 p1 or 1 p10.3 M KCl in incubations containing 0.5 or 0 pl reticulocyte factors, respectively, and water to the final vol. of 10 pl. After 1 h at 30 "C duplicate 4-p1 samplings were processed for counting. Stimulation is defined in the legend of Table 3 Histone Reticulo- trna SH-labeled Stimulation mrna cyte protein factors Pl counts/min

5 B. Gabrielli and C. Baglioni 125 with ribosomes from preincubated extracts and postribosomal supernatant from non-preincubated extracts (Table 3). It thus seems possible that some factor present in the supernatant is partially inactivated during the preincubation. Translation of Histone mrna by Non-Preincubated Extracts As discussed above, one possible explanation for the failure to detect translational control of histone synthesis in cell-free systems is the inactivation of some component during the preincubation. For this reason we have studied the translation of histone mrna by HeLa cell extracts that were neither preincubated nor passed through Sephadex G-25. These crude extracts are homologous to the mrna added and have a high endogenous background of protein synthesis. It is thus necessary to separate histone from other proteins and we have chosen to use electrophoresis in 15 polyacrylamide gels at acid ph [ll]. Four different extracts were prepared from synchronized HeLa cells, two from cells treated with cytosine arabinoside for 10 min and 1 h, respectively, and two from the control untreated cells. Fig Time (h) Fig.2. DNA synthesis by synchronized HeLa cells and inhibition by cytosine arabinoside. The cells were synchronized by a double thymidine block [7]. After release from the second block aliquots of cell culture were used to measure DNA synthesis using [14C]thymidine(see Materials and Methods). To half the culture 25 pg/ml cytosine arabinoside was added after 4.5 h. (0-0) Control cells; (&--a) cells treated with cytosine arabinoside. The arrows indicate the times at which aliquots of the cultures were taken for preparation of the extracts used in the incubations described in Fig. 3 and 4 +Histone mrna +Histone mrna Fig.3. Analysis by electrophoresis on plyacrylamide gels of the proteins synthesized by the nun-preincubated Heh cell-free system. In the four incubations extracts obtained from untreated cells (A and B) or from cells treated for 10 min with cytosine arabinoside (C and D) were used. The composi- Gel fractions tion of the incubation mixtures is identical to tha described in Fig. 1, with the exception of mrna added. Two incubations (A and C) had no mrna added; the other two (B and D) contained 118 pg/ml histone mrna. The samples were analyzed as described in Methods. (0) SH; (0) I4C

6 126 Control of Histone Synthesis C - 20c.- c E. In c ; 1oc v U 0) c L 0 F c 8._ I 30C (1 U m 0 O 20c + Histone mrna 1oc C Gel fractions Fig.4. Analysis by electrophoresis on polyacrylamide gels of the proteins synthesized by the non-preincubated HeLa cell-free system. The experiment is identical to that described in Fig.3, except that extracts used in B and D were obtained from cells treated for 1 h with cytosine arabinoside shows the synthesis of DNA by synchronized HeLa cells and the inhibition of DNA synthesis by cytosine arabinoside. As shown by the arrows in Fig.2, the extracts were prepared from cells in which DNA synthesis was inhibited by either about goo/, (10 min after cytosine arabinoside addition) or by close to 990/, (1 h after cytosine arabinoside). The product of each cell-free system with or without added histone mrna was mixed with the same amount of marker HeLa cell I*C-labeled histones, dialyzed and analyzed by electrophoresis (Fig. 3 and 4). The pattern obtained with the control cellfree systems (Fig.3A and 4A) showed several poorly resolved peaks and some radioactive protein in the same position as the main peak of marker histones; a similar pattern, but with a decrease in the radioactive protein running in the position of histones, was observed with the cell-free systems obtained from cells treated with cytosine arabinoside (Fig.3C and 4C). This was expected, since histone synthesis is tightly coupled to DNA synthesis [l to 51. Addition of histone mrna to these cell-free systems results in a selective stimulation of histone synthesis, in the cell-free systems from cells not treated with cytosine arabinoside (Fig.3B and 4B) as well as in the cell-free systems from cells treated 10 min or 1 h (Fig.3D and 4D). The stimulation of histone synthesis appeared to be of the same order of magnitude in all these extracts, although it could not be precisely measured because of the high background of endogenous protein synthesis. This high background prevented us from making any useful observation about the translation of the F1 histones which separate from the main histone peak and migrate more slowly in electrophoresis [ll]. Inhibition of Protein Synthesis by Histones A block in DNA synthesis during the S phase of the cell cycle leads to the accumulation of histones in the cytoplasm [23]. Histones would normally migrate to the nucleus in cells active in DNA synthesis and combine with DNA. The cytoplasmic concentration of histones may presumably regulate histone synthesis by a mechanism which has not yet been elucidated [7,23]. We have tested whether HeLa cell histones are directly involved in translational regulation by adding them to the ascites cell-free system or to the reconstituted HeLa cell-free system together with histone mrna. The results of the experiment in the ascites cellfree system are shown in Fig.5. Increasing concentrations of histones are inhibitory for protein synthesis, but translation of histone or globin mrna

7 F. Gabrielli and C. Baglioni 127 are equally affected. This suggests that histones are non-specific inhibitors of protein synthesis, presumably because of their basic charge. Identical results were obtained in the experiment utilizing HeLa cell ribosomes from a preincubated extract and postribosomal supernatant from a non-preincubated extract (not shown). DISCUSSION We have used homologous cell-free systems to study the translational regulation of HeLa cell histone synthesis. The preincubated cell-free system has been characterized for K+ and Mg2+ optima and the effect of added trna on translation of histone or globin mrna has been determined. In most cases addition of trna is not necessary to obtain translation significantly better than that without added trna. This cell-free system is rather inefficient in translating added mrna, when compared to the ascites cell-free system. The efficiency can be improved by using a reconstituted cell-free system containing postribosomal supernatant obtained from non-preincubated extracts or by adding initiation factors obtained from reticulocyte ribosomes. We have found that all the cell-free systems prepared from HeLa cells synchronized in S phase or from the same cells blocked in DNA synthesis by cytosine arabinoside translate histone mrna equally well. The translational control, which operates in vivo to stop histone synthesis when DNA synthesis is inhibited does not operate in the cell-free systems studied. Otherwise, extracts from cells blocked in DNA synthesis while in S phase should be less active in translating histone mrna than extracts from S-phase cells. There are several possible explanations for our failure to observe translational control in a cellfree system. The rather limited efficiency of the preincubated HeLa cell-free system in initiating protein synthesis may prevent us from observing any difference in the translation of histone mrna. We have attempted to improve the efficiency of initiation by using cell-free systems reconstituted from preincubated ribosomes and non-preincubated postribosomal supernatants, but no difference in the efficiency of translation of histone mrna has been observed between S-phase and cytosine arabinoside treated cell extracts (Table 3). Similar results have been obtained by translating histone mrna in HeLa cell-free systems that were not preincubated (Fig. 3 and 4). These experiments have ruled out the possibility that an unstable inhibitor of translation present in the postribosomal supernatant is inactivated in the preincubation step, as it has been reported for other translational inhibitors [21]. We have not eliminated, however, the possibility that an inhibitor is produced in an amount Big.5. Effect of addition of nuclear HeLa cell histones on the translation. of histone and globin mrna by the ascites cell-free system. The incubations were identical to those described in the legend of Fig. 1, with 86 mm KCl and 3 mm MgCI,; 1 pl histone mrna (0.43 mg/ml) or 1 p1 globin mrna (0.45 mg/ ml) was added together with an aqueous solution of HeLa histones at the concentrations indicated. Theifinal vol. of the incubations was lop1 and the aliquots were counted as described above. The inhibition of translation has been calculated by taking the incorporation in the incubation without added histones to be equal to 100 stoichiometric with that of the histone mrna present in HeLa cells at the time of the block in DNA synthesis. If this hypothetical inhibitor binds to histone mrna and then looses its activity, it may be rather difficult to detect it. It is rather important then to know whether mrna is added in our cellfree system in physiological concentrations or in large excess. We have calculated that in the experiments with non-preincubated extracts the ratio histone mrna/ribosomes was 111. In experiments with the preincubated extracts this ratio was These figures have been obtained by measuring the amount of ribosomes present in an incubation and assuming that all the RNA added is mrna; since this is probably an overestimate of the mrna added, the actual ratio mrna/ribosomes is probably even lower. Whether under these conditions it is possible to detect the activity of an inhibitor with the characteristics discussed above is questionable. The cytoplasmic concentration of histones increases after a block in DNA synthesis [23]. It thus

8 128 F. Gabrielli and C. Baglioni: Control of Histone Synthesis seems worthwhile to test the possibility that histones may act as translational repressors of their own synthesis. While nuclear histones inhibit protein synthesis (Fig. 5), they do not inhibit specifically translation of histone mrna. However, since nuclear histones undergo many modifications of their aminoacid residues [24], it is still possible that only newly made cytoplasmic histones have an effect on translation of histone mrna, which the nuclear histones no longer show. For these reasons, further experiment with unfractionated, non-preincubated HeLa cellfree systems are presently being carried out to investigate the translational control of histone synthesis in a system as close as possible to that of the intact cell. This work was supported by Grant AI from the National Institute of Health, by Grant GB from the National Science Foundation to C.B., and by Grant CA from the National Institute of Health to Dr T. W. Borun, Fels Research Institute and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pa. (U. S. A.). REFERENCES 1. Robbins, E. & Borun, T. W. (1967) Proc. Natl. Acad. xci. U. S. A. 57, Spalding, J., Kajuwara, J. & Mueller, G. C. (1966) Proc. Natl. Acad. Sci. U. S. A. 56, Borun, T. W., Scharff, M. D. & Robbins, E. (1967) Proc. Natl. Ad. Sci. U. S. A. 58, Gallwitz, D. & Mueller, G. C; (1969) Science (Wash. D. C.) 163, Gallwitz. D. & Mueller. G. C. (1969) J. Biol. Chem \ I Liberti, P., Festucci, A. & Baglioni, C. (1973) Mol. Biol. Reports 1, Jacobs-Lorena, M., Gabrielli, 3. & Baglioni, C. (1973) Biochim. Biophys. Acta, in press. 8. Uenoyama, K. & Ono, T. (1973) J. Xol. Biol. 74, Uenoyama, K. & Ono, T. (1973) J. Mol. Biol. 74, Eagle, H. (1959) Science (Wash. D. C.) 130, Panym, S. & Chalkley, R. (1969) Arch. Biochem. Biovhqs. 130, Mitgews, M. B. & Koerner, A. (1970) Eur. J. Biochem. 17, Jacobs-Lorena, M. & Baglioni, C. (1972) Biochemistry, 11, Jacobs-Lorena. M.. Baelioni. C. & Borun. T. W. (1972) \ I Proc. Natl. Ad. Scx U. k. A. 69, Shafritz, D. A., Pritchard, P. M. & Anderson, F. W. (1972) in Methods in Molecular Biology (Last, J. A. & Laskin, A. I., eds) Vol. 2, pp , M. Dekker, New York. 16. McDowell, M. J., Joklik, W. K., Villa-Komaroff, L. & Lodish, H. F. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, Delovitch, T. & Baglioni, C. (1973) Proc. Natl. Ad. Sci. U. S. A. 70, Aviv, H., Boime, I. & Leder, P. (1971) Prov. Natl. Ad. Sci. U. S. A. 68, Metafora. S.. Terada. M.. Dow. L.. Marks. P. A. & Bank, A. (1972) Proc. Natl. Ad: Sci. U:S. A. 69, Gross, K., Ruderman, J., Jacobs-Lorena, M., Baglioni,C. & Gross,P. R. (1973) Nut. New Biol. 241, Friedman, R. M., Esteban, R. M., Metz, D. H., Tovell, D. R. & Williamson, R. (1972) FEBS Lett. 24, Gallwitz, D. & Breindl, M. (1972) Biochim. Biophys. Res. Commun. 47, Butler, W. B. & Mueller, G. C. (1973) Biochdm. Biophys. Acta, 294, Bradbury, E. M. & Crane-Robinson, C. (1971) in Histone and Nucleohistones, (Phillips, D. N. P., ed.) Plenum Press, New York. F. Gabrielli, Department of Biology, Massachussetts Institute of Technology, Cambridge, Massachusetts, U. S. A C. Baglioni s present address: Department of Biological Sciences, State University of New York at Albany, 1400 Washington Avenue, Albany, New York, U. S. A