H I S T O N E - H I S T O N E I N T E R A C T I O N S AS R E V E A L E D BY F O R M A L D E H Y D E T R E A T M E N T OF C H R O M A T I N
Yu. v. ILYIN and A. A. BAYEV,Jr. Institute of Molecular Biology, Academy of Sciences of the U.S.S.R., Moscow, U.S.S.R.
(Received 25 March, 1975) Abstract. Histone oligomers produced by formaldehyde treatment of chromatin were studied. It was shown that these histone oligomers could be converted into monomers by boiling in 0.1 N H2 SO4. Dimers H2B-H4 and H2B-H2A were identified by two-dimensional polyacrylamide gel electrophoresis. Abbreviations. Histones Nomenclature: H1 (formerly histone F 1); H2B (formerly histone F2b); H2A (formerly histone F2a2); H3 (formerly histone F3); H4 (formerly histone F2al). This nomenclature has been proposed at the recent Symposium on the Structure and Function of Chromatin. Ciba Foundation. London. April 1974. I. INTRODUCTION Bifunctional reagents are being widely used to reveal mutual arrangements of proteins in ribosomes and chromatin [1-10]. Diimidoesters [ 1-5], thioimidoesters and dithioimidoesters [6, 7] which cross-link adjacent amino groups in proteins were used for this purpose. Resulting oligomers can be converted into monomers by selective cleavage of the cross-links by ammonolysis or by 2-mercaptoethanol. We used recently bifunctional reagents to study neighbouring histones in chromatin [4-6] and discovered that all five histones take part in oligomer formation. However, the composition of histone dimers obtained was difficult to determine because a large number of different histone dimers was formed upon diimidoester treatment of the chromatin. Therefore, it was difficult to isolate individual dimers by means of polyacrylamide gel electrophoresis. On the other hand, treatment of chromatin with formaldehyde (HCHO), the simplest bifunctional reagent, produced only a few dimers [4] which could be completely resolved by acetic acid-urea gel electrophoresis. The only difficulty in this case was the determination dimer composition since HCHO is considered to be an irreversible cross-linking reagent. In the present work we have found that HCHO-induced histone dimers can be converted into initial monomers by boiling in 0.1 N H2 SO4. We report here the identification of two histone dimers (H2B-H2A and H2B-H4) in the formaldehyde-treated chromatin by means of the above-mentioned approach. 159 Molecular Biology Reports 2 (1975) 159-165. All Rights Reserved Copyright 9 1975 by D. Reidel Publishing Company, Dordrecht-Holland.
II. MATERIALS ANDMETHODS Chromatin simultaneously labelled in vivo with [aH]-thymidine (labelled in CH3) and with a hydrolysate of chlorella 14C-proteins was isolated from mouse Ehrlich ascites tumor cells as previously described [ 10, 12]. Chromatin isolation included the following steps: isolation of nuclei, extraction of the nuclei with 0.14 M NaCI, 0.01 M Na-EDTA; thereafter with 0.35 M NaC1 and additional purification of the chromatin by centrifugation through a layer of 1.7 M sucrose; 5 mM triethanolamine-HC1 buffer (TEA-HCI), pH 7.5 was present in all solutions. The specific radioactivity of DNA was about 2000 [3H] cpmgg -1 and that of histones approximately 500 [14C] cpm#g -1 histone contained about 50% of the total 14C-counts in the chromatin). The use of the labelled material facilitated calculations of yields after various procedures. The initial chromatin gel (from 300 to 500/ag of DNA per ml) was suspended in 5 mM TEA-HC1, pH 7.5, by 3 0 - 5 0 strokes with a tightly fitted Dounce homogenizer. Sheared chromatin (DNP) was obtained after passing through the water-cooled French press under a pressure drop of 1 kbar. The DNA in the sheared chromatin had a molecular weight of approximately 5• 10 s daltons [12]. Urea-treated DNP preparations were obtained by incubation of the DNP in 4 M deionized urea followed by dialysis against 5 mM TEA-HC1, pH 7.5 to remove urea [10, 12]. DNP preparations devoid of histone H1 were obtained according to Johns and Bolund [ 13]. Chromatin (2/ag of DNA per ml) was mixed with three volumes of 0.6 M NaC1; 0.05 M phosphate buffer, pH 7.0 and thereafter incubated with the resin AGW50• [13]. The soluble DNP devoid of histone H1 was separated from the resin by low-speed centrifugation and thereafter dialysed against 5 mM TEA-HCI, pH 7.5. HCHO treatment of the DNP was carried out as follows. The chromatin suspension or a DNP solution (200-300/ag of DNA per ml) in 5 mM TEA-HC1, pH 7.5 or in 0.12 M NaCI, 5 mM TEAHCI, pH 7.5 was mixed with a 5% HCHO solution (pH 7.5) to a final HCHO concentration of 0.2% or 1%. The mixture was incubated at 0 ~ After incubation the histones were extracted with 0.25 m HC1 [10], dialysed against 0.1 M CH3COOH and lyophilized. These samples were dissolved in 0.9 N CH3 COOH, 7 M urea, 0.1 M 2-mercaptoethanol and subjected to acetic acid-urea-polyacrylamide gel electrophoresis [14]. Twenty to fourty/ag of histone was applied to each gel. Two-dimensional gel electrophoresis was carried out in the apparatus of Studier [ 16], in the same buffer as was used in one-dimensional electrophoresis. The acrylamide concentration in the slab gel was 15%. III. RESULTS AND DISCUSSION An increase in the time of treatment of chromatin with HCHO results in a decrease of the percentage of total histone which can be extracted with 0.25 N HCI (Figure 1). With sufficiently long incubation with HCHO all histones become unextractable with 0.25 N HC1 (from the chromatin). The results of acetic acid-urea gel electrophoresis of histones isolated from chromatin treated with 1% HCHO in 0.12 M NaC1 are shown in Figure 2. One can see five dimer histone bands, the second and the third of which are the most prominent. Band 5 is the S - S dimer of histone H3 (dH3) [ 10, 14], because a similar dimer with the same electrophoretic mobility could be 160
100
90 80 70 13 ,,-I--,'
u cI
60
X
9
50
cO ffl
Z- 40 r
,_ 30 O_
20 10 I 10
I 20
I I 30 40 Time (min.)
I 50
I 60
I 70
Fig. 1. Time dependence of histone extraction after treatment of chromatin with 1% HCHO in 0.12 M NaC1, 5 mM TEA-HC1, pH 7.5.
161
Fig. 2. Acetic acid-urea gel electrophoresis of histones from chromatin treated with 1% HCHO in 0.12 M NaCI, 5 mM TEA-HCI, pH 7.5. Histones from untreated chromatin (A) and from chromatin treated with HCHO for 5 min (B), 15 min (C), 30 min (D) and 60 min (E).
observed in the chromatin not treated with HCHO (Figure 2A; Figure 3, O) and since dithiotreitol treatment of histone samples before electrophoresis completely eliminates this dimer. The dimer band 1 is the weakest one, it can be seen only after prolonged chromatin treatment with HCHO (Figure 2E). Figure 3 represents the acetic acid-urea gel electrophoresis pattern of histones which were extracted from the chromatin and from various DNP preparations previously subjected to HCHO treatment at an ionic strength of 0.005 and 0.12, respectively. The removal of histone H 1 does not influence the formation of histone dimers upon HCHO treatment of chromatin [4, 6]. On the HCHO-treated DNP lacking histone H 1 the same band of histone dimers were observed as in the initial chromatin. The yield of dimers was less at low ionic strength than at high ionic strength (Figure 3). This yield was almost negligible upon the HCHO-treatment of the urea-treated chromatin (Figure 3, III A). The absence of dimers in this case can be explained in two ways: Firstly, the observed dimers may reflect side-by-side interactions between neughbouring DNP fibrils. In the urea-treated chromatin which is a true DNP solution [ 12] these interactions can be negligible. Secondly, if the formation of histone dimers is due to histone-histone interaction within DNP fibrils one may suggest that urea treatment of the chromatin results in a strong 162
weakening of such interactions. Furthermore, it should be noted that the virtual absence of histone dimers in the chromatin which was treated with HCHO at low ionic strength also can be explained by the same line of reasoning.
Fig. 3. Acetic acid-ureagel electrophoresisof histones from chromatin and DNP treated with 0.2%HCHO for 2 hr in 5 mM TEA-HCI,pH 7.5 (A), or in 0.12 M NaCI,5 mM TEA-HC1,pH 7.5 (B). O -Histones from untreated chromatin. I - chromatin. II - DNP (sheared chromatin). III - DNP obtained by urea treatment of sheared chromatin. IV - DNP lackinghistones H1.
The selective hydrolysis of methylene bridges [ 16] in histone dimers was carried out by boiling in 0.1 N H2 SO4. If histones isolated from the HCHO-treated chromatin were boiled for 5-10 min in 0.1 N H2 SO4, all dimers were converted into monomers as could be seen by gel electrophoresis. Identification of dimers was performed by two-dimensional gel electrophoresis. After electrophoresis in the first direction [ 15] the gel was removed from the tube and put into 0.1 N H2 SO4 at 100 ~ for 5-7 min. Thereafter H2 SO4 was removed from the gel by soaking it in a large volume of 0.9 N CH3COOH, 2.5 M urea and 1% 2-mercaptoethanol. The gel was cut longitudinally using a thin wire and thereafter one of the halves was put onto a slab-gel. As can be seen in Figure 4 the bands 1,2, 3 correspond to the dimers which were formed by the same pair of histones H2B-H4. The presence of three conformers of the same dimer H2B-H4 can be explained by the close proximity of histone H2B and H4 in chromatin. Therefore HCHO can probably form bonds between different sites of the two histones. Band 4 is a histone dimer H2B-H2A. Our data on the pattern of histone dimers produced by the formaldehyde treatment of the chromatin can be compared with those obtained by Olins and Wright [8]. In the latter work glutaraldehyde treatment of erythrocyte nuclei has been studied. It was shown that besides histone dimers a significant proportion of higher histone oligomers have been formed upon glutaraldehyde treatment. The authors did not identify obtained oligomers but showed that many of them included histones H1 and H5. Pairs similar to those discovered by us in chromatin have been obtained also upon mixing of solutions of different histone fractions [ 17, 18, 19]. The 163
pair H2B-H4 was also observed in chromatin after tetranitromethane treatment [20]. When this manuscript was in preparation we became alware of the investigation of Weintraub et al. who obtained analogous histone dimers after treating chromatin with HCHO [21 ]. The method of selective cleavage of methylene cross-links in HCHO-produced histone oligomers which was developed in the present work, probably, can be used in many other cases, for example, to reveal the protein topography of the ribosome. It is not yet clear whether the formation of histone dimers is due to histone-histone interaction along a single deoxyribonucleoprotein fibril or due to side-by-side interactions between different DNP fibrils in the chromatin. The first possibility appears to be more probable since the same histone dimers are formed upon HCHO treatment either in the original insoluble chromatin gel or in the soluble histone HI-depleted chromatin [22]. This question requires further study.
Fig. 4. Two-dimensionalacetic acid-urea gel electrophoresis of histone dirners treated with 0.1 N H2SO4. (A) original gel. (B) schematic drawing.
ACKNOWLEDGEMENTS We would like to thank Prof. G. P. Georgiev for helpful discussions and Dr. A. J. Varshavsky for critical reading of the manuscript. REFERENCES 1. Slobin, L. J.,J. Mol. Biol. 64, 297 (1972). 2. Lutter, L. S., Zeichhardt, H., Kurland, C. G., and St6ffler, G.,Mol. Gen. Genet. 119,357 (1972)o 164
3. Bickle, T. A., Hershey, J. W. B., and Traut, R. R.,Proc. Nat. Acad. ScL U.S. 69, 1327 (1972). 4. Ilyin, Yu. V., Bayev, A. A., Jr., Zhuze, A. L., and Varshavsky, A. J., Mol. Biol. Reports 1, 343 (1974). 5. Kornberg, R. D. and Thomas, J. O.,Science 184, 865 (1974). 6. Georgiev, G. P., in D. W. Fitzsimons and G. E. W. Wolsterdaolme(eds.), Ciba Foundation Symposium 28 (New Series), The Structure and Function ofChromatin, p. 48 (1975). 7. Sun, T. T., Bollen, A., Kahan, L., and Traut, R. R.,Biochem. J. 13, 2339 (1974). 8. Olins, D. E. and Wright, E. B.,J. CellBiol. 59,304(1973). 9. Sun, T. T., Traut, R. R., and Kahan, L.,J. Mol. Biol. 87,509 (1974). 10. Olyin, Yu. V., Varshavsky, A. J., Mickelsaar, U. N., and Georgiev, G. P., Eur. J. Biochem. 22, 235 (1971). 11. Varshavsky, A. J., Ilyin, Yu. V., and Georgiev, G. P., Nature 250, 602 (1974). 12. Georgiev, G. P,, Ilyin, Yu. V., Tichonenko, A. S., and Stelmaschuk, V. I., Mol. Biol. (U.S.S.R.) 4, 246 (1970). 13. Bolund, L. A. and Johns, R. W.,Eur. J. Biochem. 35,546 (1973). 14. Panyim, S. and Chalkley, R., Biochemistry 8, 3972 (1969). 15. Studier, E. W.,J. Mol. Biol. 79,237 (1973). 16. Feldman, M. Ja.,Progr. Nucl. Acid. Res. andMol. Biol. 13, 1 (1973). 17. D'Anna, J. A., Jr. and Isenberg, I.,Biochemistry 12, 1035 (1973). 18. D'Anna, J. A., Jr. and Isenberg, I., Biochemistry 13, 2098 (1974). 19. Kelley, R. J., Biochem. Biophys. Res. Comm. 54, 1588 (1973). 20. Martinson, H. G. and McCarthy, B. I., Biochemistry, in press. 21. Elgin, S. C. R. and Weintraub, H., Ann. Rev. Biochem. 44 (1975)in press. 22. Georgiev, G. P., Ilyin, Yu. V., Tichonenko, A. S., Dobbert, N. N., and Ananieva, L. N., Mol. Biol. (U.S.S.R.) 1,815 (1967).
165
Yu. v. ILYIN and A. A. BAYEV,Jr. Institute of Molecular Biology, Academy of Sciences of the U.S.S.R., Moscow, U.S.S.R.
(Received 25 March, 1975) Abstract. Histone oligomers produced by formaldehyde treatment of chromatin were studied. It was shown that these histone oligomers could be converted into monomers by boiling in 0.1 N H2 SO4. Dimers H2B-H4 and H2B-H2A were identified by two-dimensional polyacrylamide gel electrophoresis. Abbreviations. Histones Nomenclature: H1 (formerly histone F 1); H2B (formerly histone F2b); H2A (formerly histone F2a2); H3 (formerly histone F3); H4 (formerly histone F2al). This nomenclature has been proposed at the recent Symposium on the Structure and Function of Chromatin. Ciba Foundation. London. April 1974. I. INTRODUCTION Bifunctional reagents are being widely used to reveal mutual arrangements of proteins in ribosomes and chromatin [1-10]. Diimidoesters [ 1-5], thioimidoesters and dithioimidoesters [6, 7] which cross-link adjacent amino groups in proteins were used for this purpose. Resulting oligomers can be converted into monomers by selective cleavage of the cross-links by ammonolysis or by 2-mercaptoethanol. We used recently bifunctional reagents to study neighbouring histones in chromatin [4-6] and discovered that all five histones take part in oligomer formation. However, the composition of histone dimers obtained was difficult to determine because a large number of different histone dimers was formed upon diimidoester treatment of the chromatin. Therefore, it was difficult to isolate individual dimers by means of polyacrylamide gel electrophoresis. On the other hand, treatment of chromatin with formaldehyde (HCHO), the simplest bifunctional reagent, produced only a few dimers [4] which could be completely resolved by acetic acid-urea gel electrophoresis. The only difficulty in this case was the determination dimer composition since HCHO is considered to be an irreversible cross-linking reagent. In the present work we have found that HCHO-induced histone dimers can be converted into initial monomers by boiling in 0.1 N H2 SO4. We report here the identification of two histone dimers (H2B-H2A and H2B-H4) in the formaldehyde-treated chromatin by means of the above-mentioned approach. 159 Molecular Biology Reports 2 (1975) 159-165. All Rights Reserved Copyright 9 1975 by D. Reidel Publishing Company, Dordrecht-Holland.
II. MATERIALS ANDMETHODS Chromatin simultaneously labelled in vivo with [aH]-thymidine (labelled in CH3) and with a hydrolysate of chlorella 14C-proteins was isolated from mouse Ehrlich ascites tumor cells as previously described [ 10, 12]. Chromatin isolation included the following steps: isolation of nuclei, extraction of the nuclei with 0.14 M NaCI, 0.01 M Na-EDTA; thereafter with 0.35 M NaC1 and additional purification of the chromatin by centrifugation through a layer of 1.7 M sucrose; 5 mM triethanolamine-HC1 buffer (TEA-HCI), pH 7.5 was present in all solutions. The specific radioactivity of DNA was about 2000 [3H] cpmgg -1 and that of histones approximately 500 [14C] cpm#g -1 histone contained about 50% of the total 14C-counts in the chromatin). The use of the labelled material facilitated calculations of yields after various procedures. The initial chromatin gel (from 300 to 500/ag of DNA per ml) was suspended in 5 mM TEA-HC1, pH 7.5, by 3 0 - 5 0 strokes with a tightly fitted Dounce homogenizer. Sheared chromatin (DNP) was obtained after passing through the water-cooled French press under a pressure drop of 1 kbar. The DNA in the sheared chromatin had a molecular weight of approximately 5• 10 s daltons [12]. Urea-treated DNP preparations were obtained by incubation of the DNP in 4 M deionized urea followed by dialysis against 5 mM TEA-HC1, pH 7.5 to remove urea [10, 12]. DNP preparations devoid of histone H1 were obtained according to Johns and Bolund [ 13]. Chromatin (2/ag of DNA per ml) was mixed with three volumes of 0.6 M NaC1; 0.05 M phosphate buffer, pH 7.0 and thereafter incubated with the resin AGW50• [13]. The soluble DNP devoid of histone H1 was separated from the resin by low-speed centrifugation and thereafter dialysed against 5 mM TEA-HCI, pH 7.5. HCHO treatment of the DNP was carried out as follows. The chromatin suspension or a DNP solution (200-300/ag of DNA per ml) in 5 mM TEA-HC1, pH 7.5 or in 0.12 M NaCI, 5 mM TEAHCI, pH 7.5 was mixed with a 5% HCHO solution (pH 7.5) to a final HCHO concentration of 0.2% or 1%. The mixture was incubated at 0 ~ After incubation the histones were extracted with 0.25 m HC1 [10], dialysed against 0.1 M CH3COOH and lyophilized. These samples were dissolved in 0.9 N CH3 COOH, 7 M urea, 0.1 M 2-mercaptoethanol and subjected to acetic acid-urea-polyacrylamide gel electrophoresis [14]. Twenty to fourty/ag of histone was applied to each gel. Two-dimensional gel electrophoresis was carried out in the apparatus of Studier [ 16], in the same buffer as was used in one-dimensional electrophoresis. The acrylamide concentration in the slab gel was 15%. III. RESULTS AND DISCUSSION An increase in the time of treatment of chromatin with HCHO results in a decrease of the percentage of total histone which can be extracted with 0.25 N HCI (Figure 1). With sufficiently long incubation with HCHO all histones become unextractable with 0.25 N HC1 (from the chromatin). The results of acetic acid-urea gel electrophoresis of histones isolated from chromatin treated with 1% HCHO in 0.12 M NaC1 are shown in Figure 2. One can see five dimer histone bands, the second and the third of which are the most prominent. Band 5 is the S - S dimer of histone H3 (dH3) [ 10, 14], because a similar dimer with the same electrophoretic mobility could be 160
100
90 80 70 13 ,,-I--,'
u cI
60
X
9
50
cO ffl
Z- 40 r
,_ 30 O_
20 10 I 10
I 20
I I 30 40 Time (min.)
I 50
I 60
I 70
Fig. 1. Time dependence of histone extraction after treatment of chromatin with 1% HCHO in 0.12 M NaC1, 5 mM TEA-HC1, pH 7.5.
161
Fig. 2. Acetic acid-urea gel electrophoresis of histones from chromatin treated with 1% HCHO in 0.12 M NaCI, 5 mM TEA-HCI, pH 7.5. Histones from untreated chromatin (A) and from chromatin treated with HCHO for 5 min (B), 15 min (C), 30 min (D) and 60 min (E).
observed in the chromatin not treated with HCHO (Figure 2A; Figure 3, O) and since dithiotreitol treatment of histone samples before electrophoresis completely eliminates this dimer. The dimer band 1 is the weakest one, it can be seen only after prolonged chromatin treatment with HCHO (Figure 2E). Figure 3 represents the acetic acid-urea gel electrophoresis pattern of histones which were extracted from the chromatin and from various DNP preparations previously subjected to HCHO treatment at an ionic strength of 0.005 and 0.12, respectively. The removal of histone H 1 does not influence the formation of histone dimers upon HCHO treatment of chromatin [4, 6]. On the HCHO-treated DNP lacking histone H 1 the same band of histone dimers were observed as in the initial chromatin. The yield of dimers was less at low ionic strength than at high ionic strength (Figure 3). This yield was almost negligible upon the HCHO-treatment of the urea-treated chromatin (Figure 3, III A). The absence of dimers in this case can be explained in two ways: Firstly, the observed dimers may reflect side-by-side interactions between neughbouring DNP fibrils. In the urea-treated chromatin which is a true DNP solution [ 12] these interactions can be negligible. Secondly, if the formation of histone dimers is due to histone-histone interaction within DNP fibrils one may suggest that urea treatment of the chromatin results in a strong 162
weakening of such interactions. Furthermore, it should be noted that the virtual absence of histone dimers in the chromatin which was treated with HCHO at low ionic strength also can be explained by the same line of reasoning.
Fig. 3. Acetic acid-ureagel electrophoresisof histones from chromatin and DNP treated with 0.2%HCHO for 2 hr in 5 mM TEA-HCI,pH 7.5 (A), or in 0.12 M NaCI,5 mM TEA-HC1,pH 7.5 (B). O -Histones from untreated chromatin. I - chromatin. II - DNP (sheared chromatin). III - DNP obtained by urea treatment of sheared chromatin. IV - DNP lackinghistones H1.
The selective hydrolysis of methylene bridges [ 16] in histone dimers was carried out by boiling in 0.1 N H2 SO4. If histones isolated from the HCHO-treated chromatin were boiled for 5-10 min in 0.1 N H2 SO4, all dimers were converted into monomers as could be seen by gel electrophoresis. Identification of dimers was performed by two-dimensional gel electrophoresis. After electrophoresis in the first direction [ 15] the gel was removed from the tube and put into 0.1 N H2 SO4 at 100 ~ for 5-7 min. Thereafter H2 SO4 was removed from the gel by soaking it in a large volume of 0.9 N CH3COOH, 2.5 M urea and 1% 2-mercaptoethanol. The gel was cut longitudinally using a thin wire and thereafter one of the halves was put onto a slab-gel. As can be seen in Figure 4 the bands 1,2, 3 correspond to the dimers which were formed by the same pair of histones H2B-H4. The presence of three conformers of the same dimer H2B-H4 can be explained by the close proximity of histone H2B and H4 in chromatin. Therefore HCHO can probably form bonds between different sites of the two histones. Band 4 is a histone dimer H2B-H2A. Our data on the pattern of histone dimers produced by the formaldehyde treatment of the chromatin can be compared with those obtained by Olins and Wright [8]. In the latter work glutaraldehyde treatment of erythrocyte nuclei has been studied. It was shown that besides histone dimers a significant proportion of higher histone oligomers have been formed upon glutaraldehyde treatment. The authors did not identify obtained oligomers but showed that many of them included histones H1 and H5. Pairs similar to those discovered by us in chromatin have been obtained also upon mixing of solutions of different histone fractions [ 17, 18, 19]. The 163
pair H2B-H4 was also observed in chromatin after tetranitromethane treatment [20]. When this manuscript was in preparation we became alware of the investigation of Weintraub et al. who obtained analogous histone dimers after treating chromatin with HCHO [21 ]. The method of selective cleavage of methylene cross-links in HCHO-produced histone oligomers which was developed in the present work, probably, can be used in many other cases, for example, to reveal the protein topography of the ribosome. It is not yet clear whether the formation of histone dimers is due to histone-histone interaction along a single deoxyribonucleoprotein fibril or due to side-by-side interactions between different DNP fibrils in the chromatin. The first possibility appears to be more probable since the same histone dimers are formed upon HCHO treatment either in the original insoluble chromatin gel or in the soluble histone HI-depleted chromatin [22]. This question requires further study.
Fig. 4. Two-dimensionalacetic acid-urea gel electrophoresis of histone dirners treated with 0.1 N H2SO4. (A) original gel. (B) schematic drawing.
ACKNOWLEDGEMENTS We would like to thank Prof. G. P. Georgiev for helpful discussions and Dr. A. J. Varshavsky for critical reading of the manuscript. REFERENCES 1. Slobin, L. J.,J. Mol. Biol. 64, 297 (1972). 2. Lutter, L. S., Zeichhardt, H., Kurland, C. G., and St6ffler, G.,Mol. Gen. Genet. 119,357 (1972)o 164
3. Bickle, T. A., Hershey, J. W. B., and Traut, R. R.,Proc. Nat. Acad. ScL U.S. 69, 1327 (1972). 4. Ilyin, Yu. V., Bayev, A. A., Jr., Zhuze, A. L., and Varshavsky, A. J., Mol. Biol. Reports 1, 343 (1974). 5. Kornberg, R. D. and Thomas, J. O.,Science 184, 865 (1974). 6. Georgiev, G. P., in D. W. Fitzsimons and G. E. W. Wolsterdaolme(eds.), Ciba Foundation Symposium 28 (New Series), The Structure and Function ofChromatin, p. 48 (1975). 7. Sun, T. T., Bollen, A., Kahan, L., and Traut, R. R.,Biochem. J. 13, 2339 (1974). 8. Olins, D. E. and Wright, E. B.,J. CellBiol. 59,304(1973). 9. Sun, T. T., Traut, R. R., and Kahan, L.,J. Mol. Biol. 87,509 (1974). 10. Olyin, Yu. V., Varshavsky, A. J., Mickelsaar, U. N., and Georgiev, G. P., Eur. J. Biochem. 22, 235 (1971). 11. Varshavsky, A. J., Ilyin, Yu. V., and Georgiev, G. P., Nature 250, 602 (1974). 12. Georgiev, G. P,, Ilyin, Yu. V., Tichonenko, A. S., and Stelmaschuk, V. I., Mol. Biol. (U.S.S.R.) 4, 246 (1970). 13. Bolund, L. A. and Johns, R. W.,Eur. J. Biochem. 35,546 (1973). 14. Panyim, S. and Chalkley, R., Biochemistry 8, 3972 (1969). 15. Studier, E. W.,J. Mol. Biol. 79,237 (1973). 16. Feldman, M. Ja.,Progr. Nucl. Acid. Res. andMol. Biol. 13, 1 (1973). 17. D'Anna, J. A., Jr. and Isenberg, I.,Biochemistry 12, 1035 (1973). 18. D'Anna, J. A., Jr. and Isenberg, I., Biochemistry 13, 2098 (1974). 19. Kelley, R. J., Biochem. Biophys. Res. Comm. 54, 1588 (1973). 20. Martinson, H. G. and McCarthy, B. I., Biochemistry, in press. 21. Elgin, S. C. R. and Weintraub, H., Ann. Rev. Biochem. 44 (1975)in press. 22. Georgiev, G. P., Ilyin, Yu. V., Tichonenko, A. S., Dobbert, N. N., and Ananieva, L. N., Mol. Biol. (U.S.S.R.) 1,815 (1967).
165
