/ . Biochem. 86, 789-794 (1979)
also Exist in Tetrahymena1 Koei HAMANA1 and Koichi IWAI Department of Protein Chemistry, Institute of Endocrinology, Gunma University, Maebashi, Gunma 371 Received for publication, April 5, 1979
High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HC1 from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HC1O4, 0.35 M NaCl, or 4 mM spermidine, together with HI histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.
In previous studies on histone species of the unicellular ciliated protozoan Tetrahymena pyriformis, we isolated and characterized a histonelike component in addition to 5 histone species (1, 2) and named it LG-histone (5) or H7 histone (4), because it was rich in lysine and glutamic acid, had a molecular weight of 12,500, and seemed to be a specific histone of Tetrahymena, by analogy with avian erythrocyte H5 histone and trout testis H6 histone (5). Later, Johmann and Gorovsky (6, 7) reported similar studies of Tetra1
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 1 Present address: College of Medical Care and Technology, Gunma University, Maebashi, Gunma 371. Abbreviations: HMG, high mobility group; SDS, sodium dodecyl sulfate; TX, Triton X-100; VI, Variety 1. Vol. 86, No. 3, 1979
789
hymena histone species, without referring to the existence of such a specific histone in nuclei isolated in a medium containing 0.1% (4 mM) spermidine hydrochloride. Meanwhile, Goodwin et al. (8, 9) isolated a new group of nonhistone chromosomal proteins, called the high mobility group (HMG) proteins, which can be extracted together with HI histone from chromatin with 0.35 M NaCl or 0.5 M HC1O4, and characterized by high mobilities on polyacrylamide gel electrophoresis and high contents of basic and acidic amino acids. These findings, as well as the above inconsistencies (6, 7), prompted us to re-examine the "Tetrahymena specific histone." Here we report that it can be extracted from chromatin with 0.35 M NaCl and removed from nuclei with 4 mM spermidine, and that this and other histone-like components, extractable with 0.25 M HC1 or 0.5 M HC1O4 from Tetrahymena chromatin, belong to the class of HMG proteins.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
High Mobility Group Nonhistone Chromosomal Proteins
790
K. HAMANA and K. 1WAI
Thus, the chromosomal H M G proteins have now been shown to exist in all 4 eukaryotic kingdoms: animals, plants, fungi (10), and protista.
Isolation of Nuclei and Chromatin—An amicronucleate strain, GL, and micronucleate strains, Variety 1 (VI) mating types 111 and IV, of Tetrahymena pyriformis were used. Exponentially growing cells were harvested and lysed with 0.2% Nonidet P40 in 0.25 M sucrose/10 mM Tris-HCl/ 3 mM CaClj/1 mM MgCl a , pH 7.5, as described previously (2). Macronuclei were isolated and purified by centrifugation in 1.9 M and 0 . 3 3 M sucrose/buffer (2). Chromatin was obtained by washing the nuclei with 0 . 1 5 M NaCI/lOmM Tris-HCl, pH 7.5 (2). Extraction of Chromosomal Proteins—Extraction of chromatin with 0.25 M HCl was performed as described previously (2); 0.5 M HC1O4 extraction was carried out directly from chromatin by Johns' method (11) and also from the 0.25 M HCl extract of chromatin according to Oliver et al. (12). Proteins were precipitated from these acid extracts, as well as from 0.15 to 0.60 M NaCl extracts with or without 4 mM spermidine trihydrochloride (Nakarai Chemicals), with 10 volumes of acetone. Gel Electrophorcsis and Gel Chromatography— Gel electrophoresis was carried out on 15% polyacrylamide gel (0.5x7.5 or 5.0 cm) in 6.25 M urea/0.9 M acetic acid, 6 mM Triton X-100 (TX)/ 0.9 M acetic acid, or 3 mM sodium dodecyl sulfate (SDS)/0.1 M glycine/NaOH, pH 10.0, as described previously (3, 4). Gel chromatography was performed on a Bio-Gel P-60 column (0.9x150 cm) in 10 mM HCl at 25°C (2). Amino Acid Analysis—Protein-dye (Amido black 10B) complex was extracted from the stained band slices of urea/acetic acid or TX/acetic acid gels with 90% formic acid at 2°C. The extract was dialyzed against 0.25 M HCl and then dried. These samples were hydrolyzed with 6 M HCl for 32 h at 110°C in evacuated tubes. Chromatographic fractions were hydrolyzed with 3 M mercaptoethanesulfonic acid (Pierce Chemicals) for 24 h at 110°C. The hydrolysates were analyzed with a Hitachi KLA-3B amino acid analyzer.
Selective Extractability of LG Proteins—The macronuclear chromatins of an amicronucleate strain (GL) or a micronucleate strain (VI) of Tetrahymena pyriformis were found to contain 1 or 2 additional 0.25 M HCl-extractable components (LG-1 and LG-2) besides 5 histone species (HI, H2A, H2B, H3, and H4) when the extracts were examined by 3 kinds of gel electrophoresis, as shown in Fig. 1. These LG proteins as well as HI histone were also extracted selectively with 0.5 M HC1O4 from chromatin (Fig. 1) or its 0.25 M HCl extract precipitated with acetone (Figs. 4 and 5). Figure 2 shows that LG proteins were extracted more readily than HI histone with 0.35 to 0.60 M NaCl when the nuclei of GL strain (also VI strain; not shown) were treated with stepwise increasing concentrations of NaCl. LG proteins were preferentially released by 0.35 M NaCl, but were not released by 0.15 M NaCl. However,
Fig. 1. Polyacrylamide gel electrophoresis of 0.25 M HCl and 0.5 M HCIO, extracts from the chromatin of GL and VI strains on (A) 6.25 M urea/0.9 M acetic acid gel (0.5 x 7.5 cm) for 3 h at 2 milliamperes/gel, (B) 3 mM SDS/0.1 M glycine/NaOH gel (0.5x7.5 cm) for 6 h at 5 milliamperes/gel, and (C) 6 mM TX/0.9 M acetic acid gel (0.5 x 5.0 cm) for 2 h at 2 milliamperes/gel. Migration was from top to bottom. The two bands of LG-1 in ( Q are probably due to microheterogeneity or modification. Fig. 2. Polyacrylamide gel electrophoresis of proteins extracted stepwise with 0.15, 0.35, 0.45, 0.6 M NaCl, and then 0.25 M HCl from the nuclei of GL strain. Conditions were as in Fig. 1C. The mobility of HI histone was different in the presence and absence of the other 4 histones for an unknown reason. Fig. 3. Polyacrylamide gel electrophoresis of proteins extracted from the nuclei of GL strain with 0.15 M NaCl containing 0 or 4 mM spermidine. Conditions were as in Fig. 1C. The mobility of HI histone changed as described in the legend to Fig. 2. Fig. 4. Chromatography of the 0.5 M HCIO4-soluble fraction of the 0.25 M HCl extract from the chromatin of GL strain on a Bio-Gel P-60 column (0.9 x 150 cm) with 10 mM HCl at 25°C. Effluent fractions (1.2 ml each) were pooled and indicated, as subjected to analyses. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
EXPERIMENTAL PROCEDURES
RESULTS
HMG NONHISTONE PROTEINS IN Tetrahymena
791
I—HI H4—
v
' — LG-1
^ — LG-1
GL V I 0.25 M HC1
' — LG-1
GL VI 0.5 M HC10. 0.15 0.3b _. :- „ . . NaCl(M)
..25 HC1(M)
Fig. 2.
>— HI
HI —
— LG-2
— H2A — H3 — H2B — H4
— LG-1
— HI
-LG-2
H3
G
— LG-1 GL V I 0.25 M HC1
GL 0.5 M
vi HC10-
0.15 M 0.25 M 0.15 M 0.25 M NaCl HC1 NaCl HC1 + 4 mM spermidine
H2A H3 H2B H4 HI
Fig. 3.
= LG-1
-LG-2 -HI
0.15
'LG-1
o.io -
0.05 -
GL VI 0.25 M HC1
GL V I 0 . 5 M HC10. Fig. 1.
Vol. 86, No. 3, 1979
20
40
FRACTION NUMBER
Fig. 4.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
— LG-2
-LG-2
792
K. HAMANA and K. IWAI
HI —
LG-1
I Peak Fig. 5. Polyacrylamide gel electrophoresis of the peaks from gel chromatography (Fig. 4), performed as described in the legend to Fig. 1C.
TABLE I. Amino acid compositions of LG proteins and HI histone of GL and VI strains. Values are given as mol/100 mol of amino acids found. Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp
GL
strain
VI strain b
HI*
LG-1»
Hlb
LG-l
33.4
26.9
30.6
2.1 2.9 7.6
2.2 1.3
1.3 2.3 7.9
10.5
3.7 5.2
26.1 1.4 10 7.8 3.6 5.8 14.6 8.2 5.3 7.2 0.0 3.5 1.7 Z9 3.6 3.9 . 3.0 0.0
7.8 4.8 6.1 2.2
12.2 0.0 6.9 0.5 2.3 1,1 0.2 0.0 —
7. 1
14.5 6.8 4.7 7.5 0.0 4.8 1.7
3. 1 3.9 4.0
Z9 —
11. 1 9.0 5.3 7.9 2.6
12.4 0.0 5.3 0.6 2.0 1.2 0.3
a2 0.0
Hie 32.3 1.6 2.0 7. 1 9.0 8.2 5.8 4.9 5.5 14.6 0.0 4.6 0.5 2.5 1. 1 0.2 0.2 —
LG-1
also Exist in Tetrahymena1 Koei HAMANA1 and Koichi IWAI Department of Protein Chemistry, Institute of Endocrinology, Gunma University, Maebashi, Gunma 371 Received for publication, April 5, 1979
High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HC1 from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HC1O4, 0.35 M NaCl, or 4 mM spermidine, together with HI histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.
In previous studies on histone species of the unicellular ciliated protozoan Tetrahymena pyriformis, we isolated and characterized a histonelike component in addition to 5 histone species (1, 2) and named it LG-histone (5) or H7 histone (4), because it was rich in lysine and glutamic acid, had a molecular weight of 12,500, and seemed to be a specific histone of Tetrahymena, by analogy with avian erythrocyte H5 histone and trout testis H6 histone (5). Later, Johmann and Gorovsky (6, 7) reported similar studies of Tetra1
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 1 Present address: College of Medical Care and Technology, Gunma University, Maebashi, Gunma 371. Abbreviations: HMG, high mobility group; SDS, sodium dodecyl sulfate; TX, Triton X-100; VI, Variety 1. Vol. 86, No. 3, 1979
789
hymena histone species, without referring to the existence of such a specific histone in nuclei isolated in a medium containing 0.1% (4 mM) spermidine hydrochloride. Meanwhile, Goodwin et al. (8, 9) isolated a new group of nonhistone chromosomal proteins, called the high mobility group (HMG) proteins, which can be extracted together with HI histone from chromatin with 0.35 M NaCl or 0.5 M HC1O4, and characterized by high mobilities on polyacrylamide gel electrophoresis and high contents of basic and acidic amino acids. These findings, as well as the above inconsistencies (6, 7), prompted us to re-examine the "Tetrahymena specific histone." Here we report that it can be extracted from chromatin with 0.35 M NaCl and removed from nuclei with 4 mM spermidine, and that this and other histone-like components, extractable with 0.25 M HC1 or 0.5 M HC1O4 from Tetrahymena chromatin, belong to the class of HMG proteins.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
High Mobility Group Nonhistone Chromosomal Proteins
790
K. HAMANA and K. 1WAI
Thus, the chromosomal H M G proteins have now been shown to exist in all 4 eukaryotic kingdoms: animals, plants, fungi (10), and protista.
Isolation of Nuclei and Chromatin—An amicronucleate strain, GL, and micronucleate strains, Variety 1 (VI) mating types 111 and IV, of Tetrahymena pyriformis were used. Exponentially growing cells were harvested and lysed with 0.2% Nonidet P40 in 0.25 M sucrose/10 mM Tris-HCl/ 3 mM CaClj/1 mM MgCl a , pH 7.5, as described previously (2). Macronuclei were isolated and purified by centrifugation in 1.9 M and 0 . 3 3 M sucrose/buffer (2). Chromatin was obtained by washing the nuclei with 0 . 1 5 M NaCI/lOmM Tris-HCl, pH 7.5 (2). Extraction of Chromosomal Proteins—Extraction of chromatin with 0.25 M HCl was performed as described previously (2); 0.5 M HC1O4 extraction was carried out directly from chromatin by Johns' method (11) and also from the 0.25 M HCl extract of chromatin according to Oliver et al. (12). Proteins were precipitated from these acid extracts, as well as from 0.15 to 0.60 M NaCl extracts with or without 4 mM spermidine trihydrochloride (Nakarai Chemicals), with 10 volumes of acetone. Gel Electrophorcsis and Gel Chromatography— Gel electrophoresis was carried out on 15% polyacrylamide gel (0.5x7.5 or 5.0 cm) in 6.25 M urea/0.9 M acetic acid, 6 mM Triton X-100 (TX)/ 0.9 M acetic acid, or 3 mM sodium dodecyl sulfate (SDS)/0.1 M glycine/NaOH, pH 10.0, as described previously (3, 4). Gel chromatography was performed on a Bio-Gel P-60 column (0.9x150 cm) in 10 mM HCl at 25°C (2). Amino Acid Analysis—Protein-dye (Amido black 10B) complex was extracted from the stained band slices of urea/acetic acid or TX/acetic acid gels with 90% formic acid at 2°C. The extract was dialyzed against 0.25 M HCl and then dried. These samples were hydrolyzed with 6 M HCl for 32 h at 110°C in evacuated tubes. Chromatographic fractions were hydrolyzed with 3 M mercaptoethanesulfonic acid (Pierce Chemicals) for 24 h at 110°C. The hydrolysates were analyzed with a Hitachi KLA-3B amino acid analyzer.
Selective Extractability of LG Proteins—The macronuclear chromatins of an amicronucleate strain (GL) or a micronucleate strain (VI) of Tetrahymena pyriformis were found to contain 1 or 2 additional 0.25 M HCl-extractable components (LG-1 and LG-2) besides 5 histone species (HI, H2A, H2B, H3, and H4) when the extracts were examined by 3 kinds of gel electrophoresis, as shown in Fig. 1. These LG proteins as well as HI histone were also extracted selectively with 0.5 M HC1O4 from chromatin (Fig. 1) or its 0.25 M HCl extract precipitated with acetone (Figs. 4 and 5). Figure 2 shows that LG proteins were extracted more readily than HI histone with 0.35 to 0.60 M NaCl when the nuclei of GL strain (also VI strain; not shown) were treated with stepwise increasing concentrations of NaCl. LG proteins were preferentially released by 0.35 M NaCl, but were not released by 0.15 M NaCl. However,
Fig. 1. Polyacrylamide gel electrophoresis of 0.25 M HCl and 0.5 M HCIO, extracts from the chromatin of GL and VI strains on (A) 6.25 M urea/0.9 M acetic acid gel (0.5 x 7.5 cm) for 3 h at 2 milliamperes/gel, (B) 3 mM SDS/0.1 M glycine/NaOH gel (0.5x7.5 cm) for 6 h at 5 milliamperes/gel, and (C) 6 mM TX/0.9 M acetic acid gel (0.5 x 5.0 cm) for 2 h at 2 milliamperes/gel. Migration was from top to bottom. The two bands of LG-1 in ( Q are probably due to microheterogeneity or modification. Fig. 2. Polyacrylamide gel electrophoresis of proteins extracted stepwise with 0.15, 0.35, 0.45, 0.6 M NaCl, and then 0.25 M HCl from the nuclei of GL strain. Conditions were as in Fig. 1C. The mobility of HI histone was different in the presence and absence of the other 4 histones for an unknown reason. Fig. 3. Polyacrylamide gel electrophoresis of proteins extracted from the nuclei of GL strain with 0.15 M NaCl containing 0 or 4 mM spermidine. Conditions were as in Fig. 1C. The mobility of HI histone changed as described in the legend to Fig. 2. Fig. 4. Chromatography of the 0.5 M HCIO4-soluble fraction of the 0.25 M HCl extract from the chromatin of GL strain on a Bio-Gel P-60 column (0.9 x 150 cm) with 10 mM HCl at 25°C. Effluent fractions (1.2 ml each) were pooled and indicated, as subjected to analyses. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
EXPERIMENTAL PROCEDURES
RESULTS
HMG NONHISTONE PROTEINS IN Tetrahymena
791
I—HI H4—
v
' — LG-1
^ — LG-1
GL V I 0.25 M HC1
' — LG-1
GL VI 0.5 M HC10. 0.15 0.3b _. :- „ . . NaCl(M)
..25 HC1(M)
Fig. 2.
>— HI
HI —
— LG-2
— H2A — H3 — H2B — H4
— LG-1
— HI
-LG-2
H3
G
— LG-1 GL V I 0.25 M HC1
GL 0.5 M
vi HC10-
0.15 M 0.25 M 0.15 M 0.25 M NaCl HC1 NaCl HC1 + 4 mM spermidine
H2A H3 H2B H4 HI
Fig. 3.
= LG-1
-LG-2 -HI
0.15
'LG-1
o.io -
0.05 -
GL VI 0.25 M HC1
GL V I 0 . 5 M HC10. Fig. 1.
Vol. 86, No. 3, 1979
20
40
FRACTION NUMBER
Fig. 4.
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/789/2186323 by Stockholm University Library user on 19 January 2019
— LG-2
-LG-2
792
K. HAMANA and K. IWAI
HI —
LG-1
I Peak Fig. 5. Polyacrylamide gel electrophoresis of the peaks from gel chromatography (Fig. 4), performed as described in the legend to Fig. 1C.
TABLE I. Amino acid compositions of LG proteins and HI histone of GL and VI strains. Values are given as mol/100 mol of amino acids found. Amino acid Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp
GL
strain
VI strain b
HI*
LG-1»
Hlb
LG-l
33.4
26.9
30.6
2.1 2.9 7.6
2.2 1.3
1.3 2.3 7.9
10.5
3.7 5.2
26.1 1.4 10 7.8 3.6 5.8 14.6 8.2 5.3 7.2 0.0 3.5 1.7 Z9 3.6 3.9 . 3.0 0.0
7.8 4.8 6.1 2.2
12.2 0.0 6.9 0.5 2.3 1,1 0.2 0.0 —
7. 1
14.5 6.8 4.7 7.5 0.0 4.8 1.7
3. 1 3.9 4.0
Z9 —
11. 1 9.0 5.3 7.9 2.6
12.4 0.0 5.3 0.6 2.0 1.2 0.3
a2 0.0
Hie 32.3 1.6 2.0 7. 1 9.0 8.2 5.8 4.9 5.5 14.6 0.0 4.6 0.5 2.5 1. 1 0.2 0.2 —
LG-1
