F1 was, however, still a better template than chick erythrocyte chromatin depleted of both. F2C and F1. .... Template activity of chick-erythrocyte (e) and calf-.
Em. J. Riochem. 40,591-598 (1973)
Chick-ErythrocyteChromatin as a Template for RNA Synthesis in vitro Lars BOLUND and Ernest W. JOHNS Institute of Cancer Research: Royal Cancer Hospital, Chester Beatty Research Institute, Bulham Road, London (Received July 26/September 11, 1973)
The template activity of chick erythrocyte chromatin was measured with bacterial RNA polymerase and found to be low in comparison with that of calf thymus chromatin. The chromatins were precipitated in a standardized way from very dilute solutions prior to the assay in order t o minimize differences in solubility or physical state between the templates which might affect their ability t o support transcription. Selective and quantitative removal of the unique histone F2C from chick erythrocyte chromatin dramatically increased its template activity. Calf thymus chromatin devoid of histone F1 was, however, still a better template than chick erythrocyte chromatin depleted of both F2C and F1. Reconstitution of the depleted chromatins with the adequate amount of histone restored the original template activities whether erythrocyte F2C or calf thymus F1 was used. The results argue against a unique functional role of histone F2C as a super-repressor. Exchange of the majority of proteins between chick erythrocyte and calf thymus chromatin caused a n exchange of template properties. Non-histone proteins extracted from calf thymus chromatin with 0.35 M NaCl did not “activate” the chick erythrocyte chromatin although they were found to bind to it.
During erythropoiesis in the chick the erythroid cell is gradually inactivated [i].The cell nucleus loses its ability t o replicate its DNA and its RNA synthesis is switched off. Concomitant with these functional changes the erythroid chromatin is condensed. The dormant nucleus, which is retained even in the most mature erythrocyte, can however, be reactivated in somatic cell hybrids [2] a process which a t least superficially seems t o be the reverse of the inactivation during erythropoiesis [3]. The system is well suited as an experimental model and a study of the molecular mechanisms behind this extreme case of chromatin modification should give valuable information on chromatin structure and function in general. One approach towards the understanding of chick erythroid chromatin inactivation and reactivation is to study the template activity of mature erythrocyte chromatin in vitro. It has been found that chromatin isolated from mature erythrocytes is a significantly poorer template for RNA synthesis with bacterial RNA polymerase than is chromatin from more immature erythroid cells [4] or from other chick [5,6] or mammalian [7]tissues. It may also be possible t o activate chick erythrocyte chromatin in vitro [8,9]. The procedures for measuring the template activity of chromatin in vitro suffer from a series of Em. J. Biochem. 40 (1973)
serious limitations. It is not yet possible to use a true homologous RNA polymerase in the test since too little is known about the mammalian and avian polymerases and their cofactors. The template assay has to be performed a t ionic conditions under which chromatin is insoluble and it has been suggested [lo] that the physical state of the chromatin precipitate has overshadowing effects on transcription in vitro. I s is furthermore very difficult t o saturate the system with exogenous enzyme and thus to get a measure of the maximal template capacity of the chromatin. Since the changes in template activity inducible in chick erythrocyte chromatin can be expected t o be exceptionally large, we have in the present work attempted t o further analyse and activate this chromatin as a template in vitro. Methods of selective and quantitative reconstitution of chromatin with respect to specific histone and non-histone fractions were employed. We have used Escherichia coli RNA polymerase which a t presence seems to give as good a specificity of transcription as any other exogenous or “homologous” enzyme [ill. We have precipitated our chromatin preparations from very dilute solutions prior t o assay in order t o obtained a reproducible and finely divided precipitate, thus hopefully minimising the effects of the physical state of the chromatin on transcription. We have used enzyme concen-
592
trations which bring the template activity up t o a level where it is relatively insensitive to the enzyme/ DNA ratio although the chromatin still is not completely saturated with enzyme.
MATERIALS AND METHODS
Chromatin was isolated as previously described from chick erythrocyte ghosts [12] and calf thymus [13]. The final product was washed twice in absolute ethanol and stored under ethanol a t -10 "C until used [14]. Before each experiment the chromatin was washed in 0.075 M NaCl, 0.025 M EDTA p H 7.5 and dialysed overnight (against the salt solution to be employed in the experiment) t o remove some more protein and the ethanol. Chick erythrocyte chromatin was quantitatively depleted of FI histone by repeated extraction with 0.5 M NaCl and of erythrocyte-specific histone F2C by dissociation in 0.65 M NaC1, 0.05 M sodium phosphate buffer pH7.0 in the presence of ionexchange resin AG 50 x 2 as previously described [15]. F1 histone was quantitatively removed from calf thymus chromatin in the same way as F2C from chick erythrocyte chromatin. Fl and F2C histones were isolated from calf thymus and chick erythrocytes respectively as previously described [16,17]. Non-histone proteins were obtained from calf thymus chromatin by repeated extraction with 0.35 M NaCl p H 7 as described by Goodwin and Johns [18]. DNA was determined by absorption measurements a t 260 nm and by the methods of Dische [19] and Burton [20]. Protein concentration was measured by the method of Lowry et al. [21]. RNA polymerase was prepared from E . coli (RNAase-deficient strain MRE 600) by a modification of the method of Maryanka and Johnston [22]. All buffers contained 0.01 M MgCI,, 0.5 mM EDTA and 10 mM 2-mercaptoethanol. Enzyme was eluted from the protamine precipitate with 0.25M ammonium sulphate in 0.01 M imidazole-HC1p H 6.5. The protamine eluate was fractionated with saturated ammonium sulphate and the fraction containing the polymerase and less than 1 DNA-independent enzyme activity was used (30-45O/, or 45-55O/,). If none of these two fractions was free of DNA-independent enzyme, they were pooled and further purified either by repeated ammonium sulphate fractionation or on DEAE-cellulose according to Chamberlin and Berg [23]. The different enzyme preparations, which were stored saturated with sucrose at -70 "C, gave identical results in the type of assays performed in the present work. One unit of enzyme is here the amount capable of incorporating 1 nmole of [l*C]ATP into acid-insoluble product in 10 min at 37 "C (the normal assay conditions) in the presence of excess pure DNA.
RNA Synthesis by Erythrocyte Chromatin
The depleted chromatins were reconstituted with histones and/or non-histones by addition of the appropriate amounts of protein a t the ionic strength used for depletion, followed by dialysis against two changes of 0.14M NaCl p H 7.0. The precipitated, reconstituted chromatins were collected by centrifugation, dissolved in double-distilled water pH 7 and dialysed overnight, as were control and depleted chromatins. Histones were isolated from the control, depleted or reconstituted chromatins in H,O by the addition of 5 M HC1 under vigorous stirring to a final concentration of 0.25 M. The precipitated DNA was extracted once more with 0.25 M HC1. The protein was then precipitated from the pooled extracts with six volumes of acetone. Non-histone proteins were isolated from control and reconstituted erythrocyte chromatin in the same way as described above for calf thymus chromatin [IS]. The isolated proteins were analysed polyacrylby electrophoretic separation on 20 amide gels as previously described [24]. The chromatin preparations to be tested in the template assay were diluted with varying amounts of double-distilled water and precipitated by rapid mixing with half the volume of either 0.42 M NaC1, or 3-times-concentrated assay buffer, or 0.15 M MgC1, with the aid of a Vortex mixer. When the mixtures turned slightly opalescent the finely divided precipitate could be recovered by centrifugation (2500 rev./min, Mistral 6L, 30 min). The supernatants were checked for remaining chromatin and it was found that chromatins containing a full complement of histone were quantitatively precipitated in all the three salt solutions whereas the preparations depleted of F1 and/or F2C could only be quantitatively recovered from 0.05 M MgC1,. The chromatin precipitates were resuspended in 0.5 ml assay medium, i.e. (0.05M Tris-HC1 buffer pH 7.8 containing 8 mM MgCl,, 2 mM MnCI,, 10 mM 2-mercaptoethanol, 0.4mM ATP, CTP, GTP and UTP and 0.5 pM [14C]ATP(specific activity 196 mCi/ mmol), and kept on ice until the addition of enzyme. The assay for RNA synthesis was carried out for 10 min a t 37 "C as described previously [25]. Controls were chromatins incubated with enzyme and the complete assay medium for zero time and enzyme incubated without template. The highest value of these controls was subtracted from the incorporation data of the experiment. As can be seen from Fig. 1 chromatin precipitated from a more dilute solution is a substantially better template than the same chromatin recovered from a solution of higher concentration. This confirms the results of Hoare and Johns [7]. Extreme diIution created problems in quantitatively precipitating calf thymus chromatin even with MgCI, and could thus not be used. Eur. J. Biochem. 40 (1973)
L. Bolund and E. W. Johns
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-3 4
Z 0
cn
A
I
0
\
. 0 LD
\ \ \
0
-E 2 a -
\
0
\ \
2 4
0
2
I
L
0 0 c .- 1
E
? 8 50 25 Chromatin DNA ( k g / r n l )
100
10
Fig. 1. Template activity of chick-erythrocyte (e) and calfthymus (A) chromatin as a function of the concentration of the solutions from which the same quantities of chromatins were precipitated with 0.14 M NaCl
0 Control
Dissociated
Separated
Fig. 3. Template activity of chick-erythrocyte (0, e) and calfthymus ( A , A) chromatin precipitated as in the experiment of Pig.2 (control) or by 14-fold dilution with distilled water of preparations dissociated in 2-M NaCl p H 8.0 (“dissociated”). The filled symbols a t position “separated” show the template activities of the DNAs separated from the bulk of the chromatin proteins by centrifugation (2000OOxg, 8 h) of the dissociated solution in 2 M NaCl p H 8.0 and then remixed with the supernatant and precipitated by 14-fold dilution with distilled water. The open symbols at position “separated” show the template activities of erythrocyte (0) and calf thymus ( A ) DNA prepared as above but remixed with the protein supernatant from the same amount of chromatin from the other species, treated identically
/
i
I /
CI
/
chromatin/ml and tested for template activity with enzyme/DNA ratios of 0.04 to 0.12. RESULTS
0.0160.032 0.040 0.080 Enzyme / D N A ( u n i t s l p g )
0.120
Fig. 2. Template activity of chick-erythrocyte (e) and calfthymus (A) chromatin, precipitated with 0.14-M NaCl from solution with 25 pg DNAIm1, as a function of the amount of RNA polymerase per unit D N A in the assay. Enzyme units are arbitrary
Fig.2 shows the effect of increasing the ratio of RNA polymerase to DNA in chromatin under the assay conditions used. The chromatin was never completely saturated with enzyme but a level was reached where the template activity is relatively insensitive to the enzyme/DNA ratio. I n the rest of the work the chromatins were precipitated from solutions with 10-25 pg DNA in Eur. J. Biochem. 40 (1973)
Difference in Template Activity between ChickErythrocyte and Calf-Thymus Chromatin Chick-erythrocyte chromatin is a much poorer template for RNA synthesis in vitro than is calf thymus chromatin irrespective of degree of subdivision of the precipitated chromatin and enzyme/ DNA ratio (Fig.1 and 2). Even if the chromatins are dissolved in 2 M NaCl pH 8.0, which dissociates most of the DNA-bound proteins [26], and reconstituted and precipitated by rapid addition of 13 volumes of double-distilled water (final NaCl concn = 0.14 M) the difference in ternplate activity remains (Fig.3). Chick erythrocyte and calf thymus chromatins were dissociated in 2 M NaCl p H 8.0 and the DNA was recovered as a pellet by centrifugation (MSE 65, 200000 x y for 8 h). The chick erythrocyte DNA was
RNA Synthesis by Erythrocyte Chromatin
594
c
0
c
e
.-ol
E
F1 -F2C -F3 +F 2 B -F2A2 -F2A1
F1, F2CF3+F2B F2A2 F2A1
.+-
0
c .P c
.-F
n
B
A
C
D
0 c .c
e
m .-
E
0
c 0 .c
F 1 ~ F3 F2BF2A2F2A1-
-F2C -F3tF2B -F2 A2 -F2 A1
?! .-
0
t 0 E
F
G
H
Fig. 4. Polyacrylamide-gel electrophoretic pattern of histones extracted with 0.25 M H C l from ( A ) chick-erythrocyte chromatin, ( B ) chick-erythrocyte chromatin depleted of F2C, ( C ) preparation B reconstituted with calf thymus F l , ( D )
preparation B reconstituted with chick erythrocyte F2C, ( E ) calf-thymus chromatin, ( F ) calf-thymus chromatin depleted of F1, ( G ) preparation F reconstituted with calf-thymus F1, ( H ) preparation F reconstituted with chick erythrocyte F2C
then redissolved in either the protein-containing supernatant of the same centrifuge tube or the supernatant of a tube in which the same amount of dissociated calf thymus chromatin had been centrifuged. The reverse was done with the calf thymus DNA. The chromatins reconstituted and precipitated from these solutions by rapid dilution with 13 volumes of double-distilled water were tested for template activity. The chromatins reconstituted with their own proteins had similar template activities to the original materials whereas chick erythrocyte chromatin proteins made calf thymus DNA a much poorer template than chick erythrocyte DNA with calf thymus chromatin proteins (Fig.3). Although the experiment is crude, partly because the DNAs are difficult t o redissolve properly in the protein super-
natants, the result indicates that the low template activity of chick erythrocyte chromatin in vitro (whether or not related to the situation in viwo) is due to some coarse repressing function of its chromatin proteins or else to a lack of activating proteins.
T h e Role of Chick-Erythrocyte-Specific Histones Chick erythrocyte chromatin has relatively little very lysine-rich histone F1 but contains a unique serine, lysine and arginine-rich histone F2C which has been thought t o be responsible for the final shutoff of RNA synthesis during erythropoiesis. We thus attempted to study the importance of histones F2C and F1 for the template activity by selective depletion of the chromatins [15]. Eur. J. Biochem. 40 (1973)
L. Bolund and E. W. Johns
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-
20
Q Z
n Is)
8 . -
10
0
0
s
- e Q
z Q
0 f
6
I
L 0
0 c ._ 5
4
0
Q L
0
c
“
2
C
Control Depleted Reconstituted with F1 F2C
Fig.5. Template activity of chick erythrocyte (0, e, 8) and calf thymus ( A , A, A) chromatin precipitated with 0.14 M NaCl (a, A), assay buffer (0, A ) or 0.05 M MgC1, (@, A) Control = chromatins A and E, depleted = chromatins B and F , reconstituted with F1 = chromatins C and G and reconstituted with F2C = chromatins D and H of Fig. 4
Chick erythrocyte chromatin selectively depleted of histone F2C (Fig.4) is a substantially better template for RNA synthesis in vitro than is control erythrocyte chromatin (Fig.5 ) . The original template activity is restored upon reconstitution with the physiological amount of F2C by dialysis against 0.14 M NaCl (Fig.4 and 5 ) . However, removal of the very lysine-rich histone F1 from calf thymus chromatin (Fig.4) increases also its originally higher template activity (Fig.5 ) . Furthermore, the original template activity is restored for both F2C-depleted chick erythrocyte chromatin or Fl-depleted calf thymus chromatin whether reconstituted with F1 or F2C (Fig.4 and 5 ) . F2C may depress the template activity of FI-depleted calf thymus chromatin slightly more than F1 (Fig.5 and 7) but i t is not clear whether this difference is significant. The results do not support the idea of a unique role of F2C as a final repressor. Chick erythrocyte chromatin also contains some specific histone F1. This histone fraction is easily extracted with 0 . 5 M NaCl p H 7 . Removal of F1 (Fig.6) has no or very little effect on the template activity of chick erythrocyte chromatin (Fig.7). Additional depletion of F2C increases the template activity to a similar extent as removal of F2C alone Eur. J. Biochem. 40 (1973)
595
(Fig.5 and 7) and, although F1 and F2C-depleted chick erythrocyte chromatin have a histone composition similar to Fi-depleted calf thymus chromatin (Fig.6), it is still a poorer template in the present assay (Fig.7). Furthermore, preparations depleted of both F1 and F2C return to their original template activities when reconstituted either with F2C or calf thymus F1 (Fig.6 and 7). Thus, neither chick erythrocyte histone F2C nor Fi can be ascribed a unique functional role in this system. It may be of interest to note that the chromatin preparations completely devoid of F2C or F1 could not be quantitatively precipitated in 0.14 M NaCl or assay buffer but only in 0 . 5 M MgC12. The partial precipitates of depleted chromatins recovered from the first two salt solutions had a significantly lower template activity than the total depleted preparation precipitated by 0.05 M MgC1, whereas no difference was found between the quantitative precipitates of chromatins with a full histone complement obtained from the three salt solutions (Fig.5). This indicates a functional as well as a structural heterogeneity in both calf thymus and chick erythrocyte chromatin. Chromatins depleted of additional histone species such as F2A2 and F2B [15] could not be precipitated even in MgC1, and their template activity could thus not be assayed by the present method.
Lack of “Activation” with Non-histone Proteins Extracted with 0.35 M NaCl from Calf-Thymus Chromatin The low template activity of chick erythrocyte chromatin might also be due to a lack of activating factors. Chick erythrocyte chromatin contains less and, a t least in part, different non-histone proteins than chromatins from other sources [i]. Since non-histone proteins have been suggested as gene activators in systems in vitro [27-291 and chick erythrocyte nuclei accumulate substantial amounts of non-histone proteins during activation in cell hybrids [3], we tried to “activate” chick erythrocyte ghosts by incubation with non-histone proteins extracted by 0.35 M NaCl from calf thymus chromatin. Chromatin isolated from these ghosts had the same low template activity as control chick erythrocyte chromatin. The experiment was repeated with fresh chick erythrocyte chromatin which was mixed, in 0 . 3 5 M NaC1, with the proteins extracted from calf thymus chromatin and was then dialysed against distilled water until completely precipitated. The precipitate was washed 3 times in 0.075M NaCl, 0.025 M EDTA pH 7, dissolved in distilled water and after dialysis overnight analysed for template activity and content of proteins extractable with 0.35 M NaC1. No change in template activity was found although the chick erythrocyte chromatin had bound
RNA Synthesis by Erythrocyte Chromatin
596
-F2C -F3+ F2B -F2A2
F2A2U
Y
F2A1-
-F2A1
b-
B
A
D
C
E
c
..e
.-0
.-ul E
L
0
C 0 .c
u
Y
F1F3+ F2BF2A2F2 A1-
-F2 C -F3+F28 -F2 A2 -F2 A1
a 7 F
G
H
I
Fig. 6. Polyacrylamide-gel electrophoresis patterns of histones extracted with 0.25 M HCl from ( A ) chick-erythrocyte chromatin ( B ) chick-erythrocyte chromatin depleted of PI, (C) prepuration B depleted of F2C, (0) preparation C reconstituted with calf-thymus F I and calf-thymus non-histone proteins, ( E ) preparation C reconstituted with chick-erythrocyte F2 C and calf-
thymus non-histone proteins, ( P ) calf-thymus chromatin, ( G ) calf-thymus chromatin depleted of Fl, ( H ) preparation G reconstituted with calf-thymus PI and calf-thymus non-histone proteins, ( I ) preparation G reconstituted with chick erythrocyte F2C and calf-thymus non-histone proteins
most if not all of the calf t'hymus non-histone species present in the 0.35 M NaCl extract (Fig.8). The same negative result was obtained when the chick erythrocyte chromatin was repeatedly extracted with 0.35 M NaCl prior to mixing with the calf thymus non-histone proteins. It could be argued that these non-histone proteins can only perform their function in the presence of the right histone complement. Chick erythrocyte chromatin depleted of histones F1 a,nd F2C was
therefore reconstituted with calf thymus F1 and nonhistone proteins. This reconstituted chromatin had the same low template activity as control chick erythrocyte chromatin and depleted chromatins reconstituted with only F1, only F2C or F2C plus calf thymus non-histone proteins (Fig. 7). Calf thymus chromatin depleted of histone F1 was also reconstituted with F1 or F2C in the presence and absence of the proteins extracted with 0.35 M NaCl from another batch of calf thymus chromatin. I n Em. J. Biochem. 40 (1973)
L. Bolund and E. W. Johns
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this case a slightly higher template activity was found for the preparations reconstituted in the presence of the non-histone proteins (Fig. 7).
DISCUSSION
0 Control
- F1 - F l - F P C
1 Reconstituted w i t h F1 F l + N H F2CF2C+NH
Fig. 7. Template activity of chick-erythrocyte (@) and calfthymus ( A ) chromatin precipitated with 0.05-M MgCl,. Control = chromatins A and F; -F1 = chromatins B and G ; -F1 -F2C = chromatin C; reconstituted with Fl nonhistone proteins = chromatins D and H; reconstituted with non-histone proteins = chromatins E and I of Fig.6. F2C Reconstituted with F1 and F2C denotes preparations like (D and H) and (E and I) respectively where non-histone proteins have not been present during reconstitution
+
+
0 C .-c I
.-m
E
* 0
0 A B C Fig. 8. Polyacrylamide-gel-electrophoresispatterns of proteins extracted with 0.35-11f N a C l p H 7.0 from ( A ) calf-thymus chromatin, ( B ) chick-erythrocyte chromatin and ( C ) chickerythrocyte chromatin reconstituted with the proteins from A Eur. J. Biochem. 40 (1973)
Chick erythrocyte chromatin was found to be a much poorer template for RNA synthesis in vitro than was calf thymus chromatin. This agrees with previous findings [4-71. The chromatin preparations were all analysed in the form of finely divided precipitates which had been obtained in a standardised way which argues against precipitation artefacts being the cause for the different template activities although differences in the precipitation unrelated to the biological control of transcription of course can not be definitely excluded. Removal of the unique chick erythrocyte histone F2C dramatically increases the template activity of chick erythrocyte chromatin whereas depletion of Fi has hardly any effect. This agrees with previous results of Seligy and Neelin [30]. F2C does not, however, play a unique functional role in this system since exchange of F2C and F1 between chick erythrocyte and calf thymus chromatin had no significant effect on template activities. This argues against F2C alone being some kind of super-repressor. There are many reasons to expect that nonhistone proteins should be capable of activating chick erythrocyte chromatin. During activation in somatic cell hybrids erythrocyte nuclei accumulate large amounts of non-histone proteins [3] and many groups have reported that non-histone chromatin proteins make DNA regions in chromatin available for transcription in vitro [27 -291. However, all attempts to activate chick erythrocyte chromatin with non-histone proteins isolated by extraction of calf thymus chromatin with 0.35M NaCl were unsuccessful in the present work. One explanation for this negative result may be that this particular protein fraction does not contain the activating molecules. Many of the the nonhistone proteins isolated from chromatin may be contaminants from the cytoplasm [31] or the membranes [32]. I n most of the experiments where a pronounced effect on transcription has been found a larger spectrum of non-histone proteins have been used [27,28]. It may also be noted that Wang et al. [29,33], using a similar fraction of non-histone proteins as we have done, found significant effects mainly on chromatin from the same source. This was also the case in the present experiments. The activation of chick erythrocyte chromatin in somatic cell hybrids, however, shows no organ or species specificities. Another possibility is that more complex protein exchanges have to take place in order that activation
598
L. Bolund and E. W. Johns: RNA Synthesis by Erythrocyte Chromatin
of chick erythrocyte chromatin can occur. The physiological activation might require some subtle quantitative balance or co-operation between many different proteins which can not be properly mimicked in a biochemical experiment. However, the exchange of the majority of DNAbound proteins between chick erythrocyte and calf thymus chromatin caused a reversal of template properties. This result indicates a t least that the DNA bound macromolecules are responsible €or the differences in template activity in this system in vitro. We can not yet rule out the possibility that our model system is too artificial to give relevant information on the mechanism of Chromatin activation in vivo. An analysis of the RNA synthesised, with respect to the presence of specific gene products, will probably be necessary to decide if a true picture of the template properties of the chick erythrocyte genome can be obtained with bacterial RNA polymerase and precipitated chromatin. This investigation has been supported by grants from the Swedish Cancer Society, the Medical Research Council and the Cancer Research Campaign. One of us (L.B.)acknowledges the receipt of a Fellowship in the European Sciences Exchange Program awarded jointly by the Royal Academy of Sciences in Stockholm, Sweden, and the British Royal Society.
REFERENCES 1. Ringertz, N. R. & Bolund, L. (1974) T h e Cell Nucleus (Busch, H. ed.) Academic Press, New York, in press. 2. Harris, H. (1967) J . Cell Sci. 2, 23-32. 3. Ringertz, N. R. & Bolund, L. (1974) I n t . Rev. E x p . Pathol. 13, 83-115. 4. Appels, R., Harlow, R., Tolstoshev, P. & Wells, J. R. E. (1973) Biochemistry of Gene Expression in Higher Organisms, (Pollack, J. K. & Wilson Lee, J., eds) pp. 191-205, Australia and New Zealand Book Co. Ltd. 5. Dingman, C. W. and Sporn, S. B. (1964) J . Biol. Chem. 239, 3483-3492. 6. Seligy, V. & Miyagi, M. (1969) Exp. Cell Res. 58, 27-34.
7. Hoare, T. A. & Johns, E. W. (1970) Biochem. J . 119, 931-932. 8. Thompson, L. R. & McCarthy, B. J. (1968) Biochem. Biophys. Res. Commun. 30, 166-172. 9. Leake, R. E., Trench, M. E. & Barry, J. M. (1972) Exp. Cell Res. 71, 17-26. 10. Hoare, T. A. & Johns, E. W. (1971) Biochim. Biophys. Acta, 247, 408-411. 11. Smith, K. D., Church, R. B. & McCarthy, B. J. (1969) Bioehemistru. 8. 4271 -4277. 12. Murray, K., fidali, G. & Neelin, J. M. (1968) Biochem. J . 107. 207-215. 13. Davison, P. F., James, S. W. F., Shooter, K. V. & Butler, J. A. V. (1954) Biochim. Biophys. Acta, 15, 415424. 14. Johns, E. W. (1971) in Histones and Nucleohistones (Phillips, D. M. P. ed.) pp. 1-45, Plenum Press, London. 15. Bolund. L. & Johns. E. VIr. (1973) Eur. J . Biochem. 35. 546-553. 16. Johns, E. W. (1964) Bioehem. J . 92, 55-59. 17. Johns, E. W. & Diggle, J. H. (1969) Eur. J . Biochem. 11, 495-498. 18. Goodwin, G. W. & Johns, E. W. (1972) F E B S Lett. 21, 103-104. 19. Dische, Z. (1930) Mikrochemie, 8, 4-13. 20. Burton, K. (1956) Biochem. J . 62, 315-323. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. S. (1951) J. Biol. Chem. 193, 265-275. 22. Maryanka, D. & Johnston, J. R. (1970) FEBS Lett. 7 , 125-128. 23. Chamberlin, M. & Berg, P. (1962) Proc. Natl. Acad. Sci. U . S. A. 48, 81-94. 24. Johns, E. W. (1967) Biochem. J . 104, 78-82. 25. Johns, E. W. & Hoare, T. A. (1970) Nature (Lond.) 226, 650-651. 26. Spelsberg, T. C. & Hnilica, L. S. (1971) Biochim. Biophys. A&, 228, 202-211. 27. Paul, J. & Gilmour, R. S. (1968) J . Mol. Biol. 34, 305316. 28. Spelsberg, T. C., Hnilica, L. S. & Ansevin, A.T. (1971) Biochim. Biovhus. Acta. 228. 550-562. 29. Kamiyama, M.; &YWang,T. Y.' (1971) Biochim. Biophys. Acta, 228, 563-576. 30. Seligy, V. L. & Neelin, J. M. (1970) Biochim. Biophys. Acta, 213, 380-390. 31. Johns, E. W. & Forrester, S. (1969) Eur. J . Biochem. 8, 547-551. 32. Harlow, R., Tolstoshev, P. & Wells, J. R. E. (1972) Cell Differentiation, 1, 341-349. 33. Wang, T. Y. (1971) E x p . Cell Res. 69, 217-219. \
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L. Bolund's present address: Institutionen for Medicinsk Cellforskning och Genetik, Medicinska Nobelinstitutet, Karolinska Institutet, Solnavagen 1, S-10401 Stockholm 60, Sweden
E. W. Johns, Chester Beatty Research Institute, Institute of Cancer Research: Royal Cancer Hospital, Fulham Road, London, Great Britain, SW3 6JB
Eur. J. Biochem. 40 (1973)
