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Equimolar proportions of somatic-type, core histones in chromatin of spermatogonia
Biochimica et Biophysica Actao 740 (1983) 342-345 Elsevier
342
BBA Report
BBA 90023 EQUIMOLAR P R O P O R T I O N S
OF SOMATIC-TYPE, CORE H I S T O N E S IN C H R O M A T I N OF
SPERMATOGONIA
MARIE L. CHIU and J. LOGAN 1RVIN Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) (Received March 24th, 1983)
Key words: Histone," Leucine incorporation; Spermatogenesis; (Rat testis)
[3H]Leucine incorporation into histones of seminiferous epithelial cells of hypophysectomized rats was used to calculate the molar proportions of the core histones of spermatogonia. The molar proportions H 3 : H 2 B : ( H 2 A + protein A24):H4 are I : I : I : I , viz. identical with those reported by others for somatic cells. Similar results were obtained when molar proportions of histones of seminiferous epithelial cells from immature rat testis (predominantly populated with spermatogonia) were determined by the. dye-binding method. These data are relevant to mechanisms for the replacement of some of the core histones by variants during the primary spermatocyte stages.
During spermatogenesis new histone variants appear at the primary spermatocyte stages [1-8], viz. TH2B-x (a variant of H2B), X 2 (a variant of H2A) and T H I - x B (a variant of the H1 group of histones). We have reported [3,9] that in seminiferous epithelial cell nuclei the molar proportions of the major classes of histones (summation of sub-species of each class) relative to histone H4 are similar to those found in somatic cell nuclei and chromatin. This observation suggests that during the primary spermatocyte stages TH2B-x replaces an equivalent molar amount of H2B, X 2 an equivalent amount of H2A and T H I - x B an equivalent amount of one or more of the usual H1 species. Alternatively, the nucleosomes and chromatin produced during the spermatogonial stages might be deficient in the H2B, H2A, and HI species, and the deficits might be eliminated at the primary spermatocyte stages by insertion of the corresponding variants. To aid in determining which of these alternatives is the correct one it is important to know the molar proportions of the somatic species of histones, relative to H4, at the spermatogonial 0167-4781/83/$03.00 © 1983 ElsevierSciencePublishers B.V.
stages. Although it is not possible to isolate spermatogonia in sufficient purity and quantity to determine the molar proportions of the histones directly, these proportions can be determined indirectly. The procedure used here was similar to the [3H]leucine incorporation method previously applied [9]. Based upon the kinetic data for spermatogenesis in the hypophysectomized rat reported by Clermont and Morgentaler [10] and Clermont and Harvey [11], 11-13 days after injection of [3H]leucine into rats which were hypophysectomized 10 days earlier, some of the label incorporated into histones (principally the variants TH2Bx and THI-x) of pachytene cells will have progressed to the spermatid stages, but approx. 75% of the pachytene cells will have degenerated as a result of the hypophysectomy [10]. The label initially incorporated into histones of spermatogonia will have progressed to the leptotene stage, but further progress to the pachytene stage is greatly impeded due to degeneration of cells at those stages as a result of hypophysectomy [10]. Also, the 'excess' histones initially synthesized [9] will
343
have been degraded or removed from the chromatin 11-13 days after injection of [3H]leucine [9]. Consequently, at that time the molar proportions of the electrophoretically separated [3H]leucinelabeled histones relative to H4 can be calculated as described previously [9], and these molar proportions will correspond closely to those of the nuclei of spermatogonia at the time of initial labeling. These conclusions were also confirmed by determining the molar proportions of histones, extracted from immature rat testis, by the dye-binding method [3]. Hypophysectomized rats, immature rats, and L-[4,5-3H]leucine were obtained from the sources stated previously [2,9]. The techniques of intratesticular injection, isolation of nuclei, extraction and polyacrylamide gel electrophoresis of histones, liquid scintillation counting of histones in the gel slices, and calculation of molar proportions relative to histone H4 were as described previously [9]. The data of Table I show that in rats injected with [3H]leucine 10 days after hypophysectomy the molar proportions of the somatic types of core histones relative to H4, viz. H3 : H2B : (H2A + protein A24) : H4, are 1 : 1 : 1 : 1 as calculated from the data obtained 11 and 13 days after the [3H]leucine injections. As stated in the introduction, we interpret these data as representing the
molar proportions of the histones synthesized by spermatogonia at the time of the [3H]leucine injection. These molar proportions are identical with those reported for somatic type cells [12,13]. The data from the experiments involving injection of [ 3 H]leucine 5 days after hypophysectomy also show molar proportions of the somatic species of histones which approach 1 : 1 relative to H4, but they are less satisfactory than the data from the 10-day hypophysectomized rats, probably because the developmental progression from leptotene to pachytene stages is less severely affected 5 days after hypophysectomy than at 10 days. If these stages are not inhibited, then some of the labeled histones synthesized in spermatogonia will be replaced by non-labeled variant histones when the spermatogonia progress to the pachytene stage. In both sets of experiments some radioactivity was found in the variants TH 1-x and TH2B-x since the spermatid nuclei were not removed by centrifugation. These histone variants would have been labeled by cells, already at the pachytene stage at the time of [3H]leucine injection, which progressed to the spermatid stages in the ensuing 11-13 days as discussed previously. The equimolar deposition of somatic core histones in spermatogonial stages was further confirmed by determining with the dye-binding
TABLE I M O L A R P R O P O R T I O N S O F HISTONES OF S E M I N I F E R O U S EPITHELIAL CELLS F R O M TESTES OF HYPOPHYSECTOM I Z E D RATS D E T E R M I N E D BY T H E L E U C I N E I N C O R P O R A T I O N M E T H O D . [3H]LEUCINE WAS INJECTED 5 DAYS A N D l0 DAYS A F T E R H Y P O P H Y S E C T O M Y , A N D M O L A R RATIOS W E R E D E T E R M I N E D AT V A R I O U S T I M E INTERVALS A F T E R T H E INJECTION. (AVERAGES OF 3 - 6 D E T E R M I N A T I O N S A T EACH T I M E INTERVAL) Histone fraction
Molar ratios of histones relative to H4 Injection of [3H]leucine at 5 d after hypophysectomy (days after the injection)
Injection of [ 3 H]leucine at 10 d after hypophysectomy (days after the injection)
3
7
11
13
17
11
13
H1 H3 H2B H2A A24 (H2A + A24) H4
0.16 1.29 0.74 1.14 0.06 1.2 1.0
0.25 1.15 0.74 0.96 0.05 1.01 1.0
0.25 1.0 0.74 0.91 0.06 0.97 1.0
0.26 1.0 0.80 0.85 0.07 0.92 1.0
0.30 0.98 0.81 0.75 0.08 0.83 1.0
0.34 1.02 0.98 0.89 0.09 0.98 1.0
0.32 1.05 0.98 0.89 0.08 0.97 1.0
THl-x TH2B-x
0.26 1.14
0.29 I. 14
0.21 I. 12
0.18 0.92
0.14 0.70
0.16 0.71
0.1 l 0.66
344
method [3] the molar proportions of histones extracted from nuclei of seminiferous epithelial cells from testes of immature rats. Typical electrophoretic patterns of histones of seminiferous epithelial cells from testes of rats at 14 and 17 days after birth are shown in Fig. 1 for comparison with a pattern for histones of seminiferous epithelial cells of testes of adult rats. It can be observed that TH2B-x is well resolved from both H2B and H3 during gel electrophoresis in 6.25 M urea in comparison with the results reported previously [2] for electrophoresis in 2.5 M urea. Electrophoresis was
1
A24
2
1 [1
also performed in 0.5 M urea for use in calculation of molar proportions of the histones by dye-binding [3], but the patterns are not shown here. Faint bands for TH2B-x and X 2 are observed (Fig. 1) at 14 days when primary spermatocytes are just beginning to appear, but these variants were not found at 7 days when primary spermatocytes were absent [2]. As observed in Table II, the molar proportions of core histones in immature rats at t4 and 17 days after birth are similar to those reported in Table I and are in agreement with the proportions of the core histones reported earlier [ 12,13] for somatic-type cells of other tissues. It was reported [14] that in the seminiferous epithelial cells of rats at 14 days after birth spermatogonia predominate (67.9 _+ 3.9% of total seminiferous epithelial cells), but also leptotene + zygotene spermatocytes (14.2 _+ 4.0%) and a few pachytene and diplotene spermatocytes (2.2 _+ 0.8%) are present. Seminiferous epithelial cells of new born rats contain gonocytes, but those cells degenerate and disappear from seminiferous epithelial cells by 9 days (15). Seminiferous epithelial cells of 13-day-old rats also contain some supporting cells [15]. Therefore, the molar proportions of histones reported in Table II from the dye-binding measurements are averages for the various types of cells present. Nevertheless, spermatogonia pre-
TABLE II MOLAR PROPORTIONS OF HISTONES (RELATIVE TO H4) OF S E M I N I F E R O U S EPITHELIAL CELLS FROM TESTES OF I M M A T U R E RATS, D E T E R M I N E D BY THE D Y E - B I N D I N G M E T H O D (AVERAGES OF 3-6 DETERMINATIONS). Histone
Molar ratios of histone relative to H4
fractions
14-day
17-day
Adult
H1 H3 H2B H2A A24 (H2A + A24) H4
0.40 0.98 0.94 0.88 0.08 0.96 1.0
0.40 1.10 0.92 0.88 0.06 0.94 1.0
0.15 0.97 0.48 0.74 0.03 0.77 1.0
THI-x TH2B-x X2
0.11 0.23 0.08
0.16 0.30 0.12
0.23 0.64 0.15
rats
Fig. 1. Polyacrylamide gel electrophoretic patterns of histones of the seminiferous epithelial cells from the testis of rats at various time intervals (days) after birth. Electrophoresis for 24 h at 200 V in downward direction with 6.25 M urea concentration. Gel 1, 14 days after birth; Gel 2, 17 days after birth; Gel 3, adult.
345 d o m i n a t e , a n d c o n s e q u e n t l y if m o l a r p r o p o r t i o n s of core histones differed significantly f r o m a 1:1:1:1 r a t i o for ( H 2 A + A 2 4 ) : H 2 B : H 3 : H 4 , t h e n such d e v i a t i o n w o u l d have been reflected in the d a t a of T a b l e II. Both a n a l y t i c a l m e t h o d s r e p o r t e d here l e a d to the conclusion that the core histories are p r e s e n t in s p e r m a t o g o n i a in e q u i m o l a r p r o p o r t i o n s relative to histone H4. Consequently, the histone variants T H 2 B - x a n d X 2 which a p p e a r at the p r i m a r y s p e r m a t o c y t e stages m u s t r e p l a c e e q u i v a l e n t a m o u n t s of H2B a n d H 2 A , respectively, i n s t e a d of c o m p l e t i n g preexisting deficiencies in the chromatin. This research was a i d e d b y a g r a n t f r o m the N a t i o n a l I n s t i t u t e of C h i l d H e a l t h a n d H u m a n Development (NIH) HDO5277.
References 1 Branson, R.E., Grimes, S.R., Jr., Yonuschot, G. and Irvin, J.L. (1975) Arch. Biochem. Biophys. 168, 403-412
2 Grimes, S.R., Jr., Chae, C.-B. and Irvin, J.L. (1975) Biochem. Biophys. Res. Commun. 64, 911-917 3 Cb_iu, M.L. and Irvin, J.L. (1980) Anal. Biochem. 109, 102-108 4 Chiu, M.L. and Irvin, J.L. (1978) Biol. Reprod. 19, 984-993 5 Shires, A., Carpenter, M.P. and Chalkley, R. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2714-2718 6 Shires, A., Carpenter, M.P. and Chalkley, R. (1976) J. Biol. Chem. 251, 4155-4158 7 Mills, N.C., Van, N.T. and Means, A.R. (1977) Biol. Reprod. 17, 760-768 8 Kumaroo, K.K. and Irvin, J.L. (1980) Biochem. Biophys. Res. Commun. 94, 49-54 9 Chiu, M.L. (1982) Biochim. Biophys. Acta 699, 110-120 10 Clermont, Y. and Morgentaler, H. (1955) Endocrinology 57, 369-382 11 Clermont, Y. and Harvey, S.C. (1965) Endocrinology 76, 80-89 12 Kornberg, R.D. (1974) Science 184, 868-871 13 Olins, A.L., Carlson, R.D., Wright, E.G. and Olins, D.E. (1976) Nucleic Acids Res. 3, 3271-3291 14 Vernon, R.G., Go, V.LW. and Fritz, I.B. (1971) Can. J. Biochem. 49, 761-767 15 Clermont, Y. and Perey, B. (1957) Am. J. Anat. 100, 241-267
342
BBA Report
BBA 90023 EQUIMOLAR P R O P O R T I O N S
OF SOMATIC-TYPE, CORE H I S T O N E S IN C H R O M A T I N OF
SPERMATOGONIA
MARIE L. CHIU and J. LOGAN 1RVIN Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) (Received March 24th, 1983)
Key words: Histone," Leucine incorporation; Spermatogenesis; (Rat testis)
[3H]Leucine incorporation into histones of seminiferous epithelial cells of hypophysectomized rats was used to calculate the molar proportions of the core histones of spermatogonia. The molar proportions H 3 : H 2 B : ( H 2 A + protein A24):H4 are I : I : I : I , viz. identical with those reported by others for somatic cells. Similar results were obtained when molar proportions of histones of seminiferous epithelial cells from immature rat testis (predominantly populated with spermatogonia) were determined by the. dye-binding method. These data are relevant to mechanisms for the replacement of some of the core histones by variants during the primary spermatocyte stages.
During spermatogenesis new histone variants appear at the primary spermatocyte stages [1-8], viz. TH2B-x (a variant of H2B), X 2 (a variant of H2A) and T H I - x B (a variant of the H1 group of histones). We have reported [3,9] that in seminiferous epithelial cell nuclei the molar proportions of the major classes of histones (summation of sub-species of each class) relative to histone H4 are similar to those found in somatic cell nuclei and chromatin. This observation suggests that during the primary spermatocyte stages TH2B-x replaces an equivalent molar amount of H2B, X 2 an equivalent amount of H2A and T H I - x B an equivalent amount of one or more of the usual H1 species. Alternatively, the nucleosomes and chromatin produced during the spermatogonial stages might be deficient in the H2B, H2A, and HI species, and the deficits might be eliminated at the primary spermatocyte stages by insertion of the corresponding variants. To aid in determining which of these alternatives is the correct one it is important to know the molar proportions of the somatic species of histones, relative to H4, at the spermatogonial 0167-4781/83/$03.00 © 1983 ElsevierSciencePublishers B.V.
stages. Although it is not possible to isolate spermatogonia in sufficient purity and quantity to determine the molar proportions of the histones directly, these proportions can be determined indirectly. The procedure used here was similar to the [3H]leucine incorporation method previously applied [9]. Based upon the kinetic data for spermatogenesis in the hypophysectomized rat reported by Clermont and Morgentaler [10] and Clermont and Harvey [11], 11-13 days after injection of [3H]leucine into rats which were hypophysectomized 10 days earlier, some of the label incorporated into histones (principally the variants TH2Bx and THI-x) of pachytene cells will have progressed to the spermatid stages, but approx. 75% of the pachytene cells will have degenerated as a result of the hypophysectomy [10]. The label initially incorporated into histones of spermatogonia will have progressed to the leptotene stage, but further progress to the pachytene stage is greatly impeded due to degeneration of cells at those stages as a result of hypophysectomy [10]. Also, the 'excess' histones initially synthesized [9] will
343
have been degraded or removed from the chromatin 11-13 days after injection of [3H]leucine [9]. Consequently, at that time the molar proportions of the electrophoretically separated [3H]leucinelabeled histones relative to H4 can be calculated as described previously [9], and these molar proportions will correspond closely to those of the nuclei of spermatogonia at the time of initial labeling. These conclusions were also confirmed by determining the molar proportions of histones, extracted from immature rat testis, by the dye-binding method [3]. Hypophysectomized rats, immature rats, and L-[4,5-3H]leucine were obtained from the sources stated previously [2,9]. The techniques of intratesticular injection, isolation of nuclei, extraction and polyacrylamide gel electrophoresis of histones, liquid scintillation counting of histones in the gel slices, and calculation of molar proportions relative to histone H4 were as described previously [9]. The data of Table I show that in rats injected with [3H]leucine 10 days after hypophysectomy the molar proportions of the somatic types of core histones relative to H4, viz. H3 : H2B : (H2A + protein A24) : H4, are 1 : 1 : 1 : 1 as calculated from the data obtained 11 and 13 days after the [3H]leucine injections. As stated in the introduction, we interpret these data as representing the
molar proportions of the histones synthesized by spermatogonia at the time of the [3H]leucine injection. These molar proportions are identical with those reported for somatic type cells [12,13]. The data from the experiments involving injection of [ 3 H]leucine 5 days after hypophysectomy also show molar proportions of the somatic species of histones which approach 1 : 1 relative to H4, but they are less satisfactory than the data from the 10-day hypophysectomized rats, probably because the developmental progression from leptotene to pachytene stages is less severely affected 5 days after hypophysectomy than at 10 days. If these stages are not inhibited, then some of the labeled histones synthesized in spermatogonia will be replaced by non-labeled variant histones when the spermatogonia progress to the pachytene stage. In both sets of experiments some radioactivity was found in the variants TH 1-x and TH2B-x since the spermatid nuclei were not removed by centrifugation. These histone variants would have been labeled by cells, already at the pachytene stage at the time of [3H]leucine injection, which progressed to the spermatid stages in the ensuing 11-13 days as discussed previously. The equimolar deposition of somatic core histones in spermatogonial stages was further confirmed by determining with the dye-binding
TABLE I M O L A R P R O P O R T I O N S O F HISTONES OF S E M I N I F E R O U S EPITHELIAL CELLS F R O M TESTES OF HYPOPHYSECTOM I Z E D RATS D E T E R M I N E D BY T H E L E U C I N E I N C O R P O R A T I O N M E T H O D . [3H]LEUCINE WAS INJECTED 5 DAYS A N D l0 DAYS A F T E R H Y P O P H Y S E C T O M Y , A N D M O L A R RATIOS W E R E D E T E R M I N E D AT V A R I O U S T I M E INTERVALS A F T E R T H E INJECTION. (AVERAGES OF 3 - 6 D E T E R M I N A T I O N S A T EACH T I M E INTERVAL) Histone fraction
Molar ratios of histones relative to H4 Injection of [3H]leucine at 5 d after hypophysectomy (days after the injection)
Injection of [ 3 H]leucine at 10 d after hypophysectomy (days after the injection)
3
7
11
13
17
11
13
H1 H3 H2B H2A A24 (H2A + A24) H4
0.16 1.29 0.74 1.14 0.06 1.2 1.0
0.25 1.15 0.74 0.96 0.05 1.01 1.0
0.25 1.0 0.74 0.91 0.06 0.97 1.0
0.26 1.0 0.80 0.85 0.07 0.92 1.0
0.30 0.98 0.81 0.75 0.08 0.83 1.0
0.34 1.02 0.98 0.89 0.09 0.98 1.0
0.32 1.05 0.98 0.89 0.08 0.97 1.0
THl-x TH2B-x
0.26 1.14
0.29 I. 14
0.21 I. 12
0.18 0.92
0.14 0.70
0.16 0.71
0.1 l 0.66
344
method [3] the molar proportions of histones extracted from nuclei of seminiferous epithelial cells from testes of immature rats. Typical electrophoretic patterns of histones of seminiferous epithelial cells from testes of rats at 14 and 17 days after birth are shown in Fig. 1 for comparison with a pattern for histones of seminiferous epithelial cells of testes of adult rats. It can be observed that TH2B-x is well resolved from both H2B and H3 during gel electrophoresis in 6.25 M urea in comparison with the results reported previously [2] for electrophoresis in 2.5 M urea. Electrophoresis was
1
A24
2
1 [1
also performed in 0.5 M urea for use in calculation of molar proportions of the histones by dye-binding [3], but the patterns are not shown here. Faint bands for TH2B-x and X 2 are observed (Fig. 1) at 14 days when primary spermatocytes are just beginning to appear, but these variants were not found at 7 days when primary spermatocytes were absent [2]. As observed in Table II, the molar proportions of core histones in immature rats at t4 and 17 days after birth are similar to those reported in Table I and are in agreement with the proportions of the core histones reported earlier [ 12,13] for somatic-type cells of other tissues. It was reported [14] that in the seminiferous epithelial cells of rats at 14 days after birth spermatogonia predominate (67.9 _+ 3.9% of total seminiferous epithelial cells), but also leptotene + zygotene spermatocytes (14.2 _+ 4.0%) and a few pachytene and diplotene spermatocytes (2.2 _+ 0.8%) are present. Seminiferous epithelial cells of new born rats contain gonocytes, but those cells degenerate and disappear from seminiferous epithelial cells by 9 days (15). Seminiferous epithelial cells of 13-day-old rats also contain some supporting cells [15]. Therefore, the molar proportions of histones reported in Table II from the dye-binding measurements are averages for the various types of cells present. Nevertheless, spermatogonia pre-
TABLE II MOLAR PROPORTIONS OF HISTONES (RELATIVE TO H4) OF S E M I N I F E R O U S EPITHELIAL CELLS FROM TESTES OF I M M A T U R E RATS, D E T E R M I N E D BY THE D Y E - B I N D I N G M E T H O D (AVERAGES OF 3-6 DETERMINATIONS). Histone
Molar ratios of histone relative to H4
fractions
14-day
17-day
Adult
H1 H3 H2B H2A A24 (H2A + A24) H4
0.40 0.98 0.94 0.88 0.08 0.96 1.0
0.40 1.10 0.92 0.88 0.06 0.94 1.0
0.15 0.97 0.48 0.74 0.03 0.77 1.0
THI-x TH2B-x X2
0.11 0.23 0.08
0.16 0.30 0.12
0.23 0.64 0.15
rats
Fig. 1. Polyacrylamide gel electrophoretic patterns of histones of the seminiferous epithelial cells from the testis of rats at various time intervals (days) after birth. Electrophoresis for 24 h at 200 V in downward direction with 6.25 M urea concentration. Gel 1, 14 days after birth; Gel 2, 17 days after birth; Gel 3, adult.
345 d o m i n a t e , a n d c o n s e q u e n t l y if m o l a r p r o p o r t i o n s of core histones differed significantly f r o m a 1:1:1:1 r a t i o for ( H 2 A + A 2 4 ) : H 2 B : H 3 : H 4 , t h e n such d e v i a t i o n w o u l d have been reflected in the d a t a of T a b l e II. Both a n a l y t i c a l m e t h o d s r e p o r t e d here l e a d to the conclusion that the core histories are p r e s e n t in s p e r m a t o g o n i a in e q u i m o l a r p r o p o r t i o n s relative to histone H4. Consequently, the histone variants T H 2 B - x a n d X 2 which a p p e a r at the p r i m a r y s p e r m a t o c y t e stages m u s t r e p l a c e e q u i v a l e n t a m o u n t s of H2B a n d H 2 A , respectively, i n s t e a d of c o m p l e t i n g preexisting deficiencies in the chromatin. This research was a i d e d b y a g r a n t f r o m the N a t i o n a l I n s t i t u t e of C h i l d H e a l t h a n d H u m a n Development (NIH) HDO5277.
References 1 Branson, R.E., Grimes, S.R., Jr., Yonuschot, G. and Irvin, J.L. (1975) Arch. Biochem. Biophys. 168, 403-412
2 Grimes, S.R., Jr., Chae, C.-B. and Irvin, J.L. (1975) Biochem. Biophys. Res. Commun. 64, 911-917 3 Cb_iu, M.L. and Irvin, J.L. (1980) Anal. Biochem. 109, 102-108 4 Chiu, M.L. and Irvin, J.L. (1978) Biol. Reprod. 19, 984-993 5 Shires, A., Carpenter, M.P. and Chalkley, R. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2714-2718 6 Shires, A., Carpenter, M.P. and Chalkley, R. (1976) J. Biol. Chem. 251, 4155-4158 7 Mills, N.C., Van, N.T. and Means, A.R. (1977) Biol. Reprod. 17, 760-768 8 Kumaroo, K.K. and Irvin, J.L. (1980) Biochem. Biophys. Res. Commun. 94, 49-54 9 Chiu, M.L. (1982) Biochim. Biophys. Acta 699, 110-120 10 Clermont, Y. and Morgentaler, H. (1955) Endocrinology 57, 369-382 11 Clermont, Y. and Harvey, S.C. (1965) Endocrinology 76, 80-89 12 Kornberg, R.D. (1974) Science 184, 868-871 13 Olins, A.L., Carlson, R.D., Wright, E.G. and Olins, D.E. (1976) Nucleic Acids Res. 3, 3271-3291 14 Vernon, R.G., Go, V.LW. and Fritz, I.B. (1971) Can. J. Biochem. 49, 761-767 15 Clermont, Y. and Perey, B. (1957) Am. J. Anat. 100, 241-267