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A very rapidly migrating f1 histone associated with gene-sized pieces of DNA in the macronucleus of Oxytricha sp.
109
Biochimica et Biophysica Acta, 407 (1975) 109--113 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 91416
A VERY RAPIDLY MIGRATING f l HISTONE ASSOCIATED WITH GENE-SIZED PIECES OF DNA IN THE MACRONUCLEUS OF OXYTRICHA SP.
ELIZABETH
B. C A P L A N
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo., 80302 (U.S.A.) (Received May 22nd, 1975)
Summary Histones bound to gene-sized pieces of DNA in the macronucleus of
Oxytricha sp. consist of five main fractions: fl, f2al, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxy tricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f l fraction differs. Oxytricha f l is unique among eukaryotes in that it migrates faster than f2al and is located nearest the cathode in polyacrylamideurea gels.
Histones found in the macronucleus of the hypotrich ciliate, Oxytricha sp., are associated with DNA that has an average molecular weight of 2.08 • 106 [1] and a mean length of 1.1/~m (Wesley, R.D., personal communication). These short DNA segments are not artifacts caused by mechanical shearing or enzymatic digestion, but are small DNA duplexes that occur naturally in hypotrichs and provide all genetic instructions for vegetative growth [2--4]. The number of nucleotide bases of hypotrich macronuclear DNA segments has been estimated, and is the equivalent of one to a few structural genes of average size [3,4]. With the exception of fraction fl, the histones bound to gene-sized pieces of DNA in Oxytricha do not differ significantly from vertebrate histones in electrophoretic mobility or molecular weight. It has been established that histones f2al, f2a2, f2b and f3 have been conserved in structure during the evolution of eukaryotes, but that the lysine-rich f l fraction has become diversified. The heterogeneity of the f l fraction has been demonstrated by varied f l electrophoretic mobility patterns among vertebrates and invertebrates [5,6], by unlike chromatographic patterns of f l extracted from tissues of the same organism [7], and by microheterogeneity in partial
110
primary sequences of f l polypeptides [8]. In Oxytricha macronuclei, the f l fraction is similar to vertebrate f l in that it is soluble in 5% HC104 [9] and rich in lysine residues. Unlike vertebrate f l , however, Oxytricha macronuclear f l migrates ahead of all other histone fractions toward the cathode in polyacrylamide-urea gels. Histones were isolated from Oxytricha that were grown in sterile cultures of 0.2% cerophyl and maintained on the food organism, Tetrahymena pyriformis. Tetrahymena were grown axenically in 2% proteose peptone. All isolation procedures were carried o u t at 4°C. Cells were collected by sedimentation and lyzed in 0.05% Triton X-100, 0.02% spermidine-HC1, 3% sucrose, pH 6.8 (approximately 0.5 ml packed cells/10 ml Triton solution). Macronuclei were separated as a pellet from cytoplasmic elements and from micronuclei by centrifuging the lysate 10 min at 90 × g through 40 ml of 0.05% Triton, 0.02% spermidine-HC1, 0.05% CaC12, 10% sucrose (pH 6.8). Macronuclei were further purified by centrifugation for 10 min at 130 X g through 40 ml of 0.05% Triton, 0.02% spermidine-HC1, 0.05% CaCl~, 30% sucrose (pH 6.8). The last step was repeated 3 times. Chromatin was prepared in the following manner by a modification of the procedure of Yabuki and Iwai [10]. The nuclear pellet was suspended in 3 ml of 0.14 M NaC1 / 0.05 M NaHSO3 and the suspension was stirred gently for 15 min. A chromatin pellet was collected after centrifugation of the suspension 10 min at 3300 × g. The removal of R N A and soluble non-histone proteins from chromatin was monitored by measuring the absorbance of the supernatant fraction at 260 and 280 nm, and the saline washes were repeated a total of 4 times. Histones were extracted from the chromatin pellet with 1.5 ml of 0.4 M HC1 by gentle stirring for 20 min. After the suspension was centrifuged 20 min at 3300 × g, supernatant histone chloride was decanted. The extraction was repeated twice more with 0.75 ml of 0.4 M HC1. Cold acetone (10 vol./volume) was added to the pooled supernatant fractions and histones were allowed to precipitate overnight at -20°C. Histones were fractioned by the methods of Johns [9], and f l , f 2 a l , f2a2, f2b and f3 were identified. The molecular weights of Oxytricha histones were determined b y the m e t h o d Of Panyim and Chalkley [11]. Amino acid analysis was carried o u t according to the methods outlined by Blackburn [12]. Short (7.5 cm) and long (25 cm) polyacrylamide-urea gels were made according to the methods of Panyim and Chalkley [13] with a final pH of 2.7 and a concentration of 15% acrylamide and 2.5 M urea. 11 cm gels containing sodium dodecyl sulfate were made according to the m e t h o d of Panyim and Chalkley [11] with a final pH of 10 and a concentration of 15% acrylamide and 0.5% sodium dodecyl sulfate. Histones were reduced with fresh 0.5 M g-mercaptoethanol before electrophoresis. Pre-electrophoresis was carried o u t for 3 h (7.5-cm gels) or 17 h (25-cm gels), and electrophoresis was performed at 2 mA/gel for 2 h on 7.5-cm gels and at 200 V for 16 h on 25-cm gels. Electrophoresis was performed at 1 mA/gel for 5.5 h on l l - c m (sodium dodecyl sulfate) gels. Gels were stained with 1% Amido Schwartz, 7% acetic acid, 40% ethanol, and destained in the acid-ethanol solvent. Oxytricha macronuclear histones separated in 25-cm polyacrylamideurea gels are shown in Fig. 1. Fractions f3, f2b, f2a2, f 2 a l , f l a and f l b are
111
(,)
(-)
!liiI~!i~llI!~ ~ ! ~III1~IllIil Jl~lltllii ~1111 IIIHIHIIllll:l~lll filllll~]ll]lllll IIIilHii lll!lll~l lil!]ll*I !lllilil!~l~lll!II !111]11!I !liii!ll~ ~II~II
"":' ~ ~.~S.,.!,"!.,.,.: ~.. ~] ..~*~,..~.,'__~' 'I° ' z ~
, ~Is , ,i,~ ~ ~
, ~ ,:,IB,:~ , ~
~ ,I~ ' . . ~
Fig. I, High resolution eleetaophoresis of Oxytricha macronueleax histones on polyaerylamlde-urea gels ( I ) f3 s u b f r a e t i o n s , (2) f2b f r a c t i o n , (3) f2a2 s u b f r a c t i o n s , (4) f 2 a l s u b f r a c t i o n s , (5) f l a subf r a c t i o n (5') f l b s u b f r a c t i o n . Oxytricha m a c r o n u c l e a r h i s t o n e s w e r e p r e p a r e d as d e s c r i b e d in the t e x t . E l e c t r o p h o r e s i s was c a r r i e d o u t at 2 0 0 V for 16 h o n 25 c m gels m a d e o f 15% a c r y l a m i d e , 2.5 M u r e a (pH 2.7). M i g r a t i o n is f r o m l e f t (+) t o right (--).
designated 1, 2, 3, 4, 5 and 5', respectively. Fig. 2 shows the position of Oxytricha f l compared to the position of calf thymus f l in 7.5 cm polyacrylamide-urea gels. In vertebrates, f l histone migrates slowest in polyacrylamide-urea gels, and is found nearer the anode than other histone fractions. In Oxytricha, however, f l migrates ahead of f2al and is found nearer the cathode than other histone fraction3. The position of a histone in polyacrylamide-urea gels is a function of the size and the net positive charge of the molecule. For the following reasons, it is not likely that the position of Oxytricha f l in polyacrylamide-urea gels is a result of the degradation of f l into smaller molecules that run more rapidly than an undegraded species. First, precautions were taken to avoid proteolysis by using 0.05 M NaHSO3 during chromatin isolation. 0.05 M NaHSO3 inhibits the activity of a protease that attacks nucleohistone and histone in both calf thymus and rat liver [14,15]. According to Bartley and Chalkley [14], the earliest sign of proteolysis is a degradation of f l with the resultant production of small components that appear as faint bands migrating faster than f l histone in electrophoresis. Oxytricha fl, extracted with 5% HC104 or with whole histone in 0.4 M HC1, exhibits identical patterns in electrophoresis that do not include minor components migrating faster than the major f l subfractions. Secondly, the molecular weight of Oxytricha f l has been estimated as 21 000 by the method of Panyim and Chalkley [11] and is equivalent to the molecular weight of calf thymus fl. This indicates that Oxy tricha f l polypeptides are not degraded into proteins the same size or smaller than other Oxytricha histones, which have the following molecular weights: f3 (13 500), f2b (13 500), f2a2 (12 000) and f2al (11 000). The amino acid composition of Oxytricha f l has been determined and in Table I is compared to the composition of calf thymus f l and to f l from the holotrich ciliate, Tetrahymena pyriformis. Oxytricha f l is rich in lysine residues {31.6%) and has a low molar percent of acidic residues. The ratio of basic residues to acidic residues in Oxytricha f l (11.5) is greater than that of calf thymus f l (4.0) or Tetrahymena f l (2.0). Panyim et al. [5] have noted that the mobility of f l increases slightly in the lower classes of vertebrates. The mobility of fish f l > amphibian f l > mammalian fl. F1 from Tetrahymena migrates more rapidly than vertebrate fl, and is in the penultimate position on polyacrylamide-urea gels, behind f2al [6]. The mobility of fl, slightly increased in the lower verte-
112
A
B
C
(+)
41111R~i:~
f 3 "--~..~
•
f 2b ....-.-~Q
~
. ~
~
|
---
fJa flb (-)
Fig. 2. A c o m p a r i s o n o f t h e p o s i t i o n of Oxytricha 51 a n d c a l f t h y m u s f l o n p o l y a c r y l a m i d e - u r e a gels. (A) C a l f t h y m u s h i s t o n e s , (B) Oxytricha m a c r o n u c l e a r h i s t o n e s , (C) Oxytricha m a c r o n u c l e a r f l . C a l f t h y m u s h i s t o n e s w e r e s u p p l i e d b y C a l b i o c h e m , L a J o l l a , Calif. Oxytricha h i s t o n e s w e r e p r e p a r e d as d e s c r i b e d in t h e t e x t a n d t h e f r a c t i o n s w e r e i d e n t i f i e d b y t h e m e t h o d s o f J o h n s [ 9 ] . W i t h t h e e x c e p t i o n o f 51, Oxytricha m a c r o n u c l e a r h i s t o n e f r a c t i o n s c o r r e s p o n d in p o s i t i o n t o t h e f r a c t i o n s o f c a l f t h y m u s . E l e c t r o p h o r e s i s w a s c a r r i e d o u t a t 2 m A / g e l f o r 2 h o n 7 . 5 - c m gels m a d e o f 1 5 % a e r y l a m i d e , 2 . 5 M u r e a ( P H 2 . 7 ) . Gels (B) a n d (C) c o n t a i n b o v i n e s e r u m a l b u m i n as a p o s i t i o n m a r k e r o n t o p o f t h e gel. S l o w m o v i n g , f a i n t b a n d s in ( A ) a n d (B) are f3 a g g r e g a t e s . M i g r a t i o n is f r o m t o p (+) t o b o t t o m (--).
brate classes, is therefore greatly increased in the protozoans, Tetrahymena and Oxytricha. Tetrahymena is a ciliate t h a t does n o t have gene-sized pieces of macronuclear DNA [16]. Tetrahymena f l is n o t as rich in lysine, nor as poor in acidic residues as is Oxytricha f l . The extreme net positive charge on
TABLE I A M I N O A C I D C O M P O S I T I O N O F O X Y T R I C H A SP. M A C R O N U C L E A R f l , T E T R A H Y M E N A P Y R I F O R M I S ( S T R A I N W H - 6 ) M A C R O N U C L E A R f l A N D C A L F T H Y M U S 51 Mol p e r 1 0 0 m o l o f a m i n o a c i d s Amino acid
Oxytricha
Tetrahymena*
Calf t h y m u s * *
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Basic/acidic
31.6 0.5 3.4 1.9 4.1 6.9 1.2 5.1 2.8 29.2 0.0 8.1 0.0 4.3 0.3 0.2 0.2 11.5
29.9 2.3 2.4 9.9 8.6 7.5 7.3 6.1 3.5 14.0 0.0 3.9 Trace 2.3 1.7 0.5 0.4 2.0
27.3 0.0 2.1 2.6 6.0 6.9 4.7 8.4 6.7 23.4 0.0 6.0 Trace 0.8 3.9 0.6 0.6 4.0
* F r o m G o r o v s k y e t al. [ 1 1 ] . ** F r o m P a n y i m e t al. [ 5 ] .
113
Oxytricha f l may therefore not be typical of protozoan histones, but may be a requisite for f l association with gene-sized DNA pieces. The author thanks Dr D.M. Prescott for his interest and support; and Anders Persson for his operation of the JEOL amino acid analyzer. This work was supported by National Science Foundation grant GB-32232 to D.M. Prescott.
References 1 Wesley, R.D. (1975) Proc. Natl. Acad. Sci. U.S. 72, 678-- 682 2 Prescott, D.M., Bostock, C.J., Mufti, K.G., Lauth, M.R. and Gamow, E. (1971) C h r o m o s o m a (Berl.) 34, 355--366 3 Prescott, D.M., Murti, K,G. and Bostock, C.J. (1973) Nature 242, 576, 597--600 4 Prescott, D.M. an d Murti, K.G. (1973) Cold Spring Harb. Symp. Quant. Biol. 38, 6 0 9 - - 6 1 8 5 Panyim, S., Bflek, D. and Chalkley, R. (1971) J. Biol. Chem. 246, 4 2 0 6 - - 4 2 1 5 6 Gorovsky, M.A., Keevert, J.B. and Pleger0 G.L. (1974) J. Cell Biol. 6 1 , 1 3 4 - - 1 4 5 7 Bustin, M. and Cole, R.D. (1969) J. Biol. Chem. 244; 5 2 8 6 - - 5 2 9 0 8 Rail, S.C. an d Cole, R.D. (1971) J. Biol. Chem. 246, 7 1 7 5 - - 7 1 9 0 9 Johns, E.W. (1964) Biochem. J. 92, 55--59 10 Yabuki, H. and Iwai, K. (1971) J. Bioehem. 70, 731--740 11 Panyinl, S. an d Chalk]ey, R. (1971) J. Biol. Chem. 246, 7 5 5 7 - - 7 5 6 0 12 Blackburn, S. (1968) A m i n o Acid D e t e r m i n a t i o n : Methods and Techniques, pp. 21--22 and pp. 93--96, Marcel Dekker, New Y o r k 13 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337--346 14 Bartley, J. and Chalkley, R. (1970) J. Biol. Chem. 245, 4 2 8 6 - - 4 2 9 2 15 Garrels, J.I., Elgin, S.C.R. and Bonnet, J. (1972) Biochem. Biophys. Res. Commun. 4 6 , 5 4 5 - - 5 5 1 16 Yao, M. an d Gorovsky, M.A. (1974) C h r o m o s o m a (Berl.) 48, 1--18
Biochimica et Biophysica Acta, 407 (1975) 109--113 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 91416
A VERY RAPIDLY MIGRATING f l HISTONE ASSOCIATED WITH GENE-SIZED PIECES OF DNA IN THE MACRONUCLEUS OF OXYTRICHA SP.
ELIZABETH
B. C A P L A N
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo., 80302 (U.S.A.) (Received May 22nd, 1975)
Summary Histones bound to gene-sized pieces of DNA in the macronucleus of
Oxytricha sp. consist of five main fractions: fl, f2al, f2a2, f2b and f3. Although the slightly lysine-rich and the arginine-rich histones in Oxy tricha macronuclei are similar to comparable fractions in vertebrates, the lysine-rich f l fraction differs. Oxytricha f l is unique among eukaryotes in that it migrates faster than f2al and is located nearest the cathode in polyacrylamideurea gels.
Histones found in the macronucleus of the hypotrich ciliate, Oxytricha sp., are associated with DNA that has an average molecular weight of 2.08 • 106 [1] and a mean length of 1.1/~m (Wesley, R.D., personal communication). These short DNA segments are not artifacts caused by mechanical shearing or enzymatic digestion, but are small DNA duplexes that occur naturally in hypotrichs and provide all genetic instructions for vegetative growth [2--4]. The number of nucleotide bases of hypotrich macronuclear DNA segments has been estimated, and is the equivalent of one to a few structural genes of average size [3,4]. With the exception of fraction fl, the histones bound to gene-sized pieces of DNA in Oxytricha do not differ significantly from vertebrate histones in electrophoretic mobility or molecular weight. It has been established that histones f2al, f2a2, f2b and f3 have been conserved in structure during the evolution of eukaryotes, but that the lysine-rich f l fraction has become diversified. The heterogeneity of the f l fraction has been demonstrated by varied f l electrophoretic mobility patterns among vertebrates and invertebrates [5,6], by unlike chromatographic patterns of f l extracted from tissues of the same organism [7], and by microheterogeneity in partial
110
primary sequences of f l polypeptides [8]. In Oxytricha macronuclei, the f l fraction is similar to vertebrate f l in that it is soluble in 5% HC104 [9] and rich in lysine residues. Unlike vertebrate f l , however, Oxytricha macronuclear f l migrates ahead of all other histone fractions toward the cathode in polyacrylamide-urea gels. Histones were isolated from Oxytricha that were grown in sterile cultures of 0.2% cerophyl and maintained on the food organism, Tetrahymena pyriformis. Tetrahymena were grown axenically in 2% proteose peptone. All isolation procedures were carried o u t at 4°C. Cells were collected by sedimentation and lyzed in 0.05% Triton X-100, 0.02% spermidine-HC1, 3% sucrose, pH 6.8 (approximately 0.5 ml packed cells/10 ml Triton solution). Macronuclei were separated as a pellet from cytoplasmic elements and from micronuclei by centrifuging the lysate 10 min at 90 × g through 40 ml of 0.05% Triton, 0.02% spermidine-HC1, 0.05% CaC12, 10% sucrose (pH 6.8). Macronuclei were further purified by centrifugation for 10 min at 130 X g through 40 ml of 0.05% Triton, 0.02% spermidine-HC1, 0.05% CaCl~, 30% sucrose (pH 6.8). The last step was repeated 3 times. Chromatin was prepared in the following manner by a modification of the procedure of Yabuki and Iwai [10]. The nuclear pellet was suspended in 3 ml of 0.14 M NaC1 / 0.05 M NaHSO3 and the suspension was stirred gently for 15 min. A chromatin pellet was collected after centrifugation of the suspension 10 min at 3300 × g. The removal of R N A and soluble non-histone proteins from chromatin was monitored by measuring the absorbance of the supernatant fraction at 260 and 280 nm, and the saline washes were repeated a total of 4 times. Histones were extracted from the chromatin pellet with 1.5 ml of 0.4 M HC1 by gentle stirring for 20 min. After the suspension was centrifuged 20 min at 3300 × g, supernatant histone chloride was decanted. The extraction was repeated twice more with 0.75 ml of 0.4 M HC1. Cold acetone (10 vol./volume) was added to the pooled supernatant fractions and histones were allowed to precipitate overnight at -20°C. Histones were fractioned by the methods of Johns [9], and f l , f 2 a l , f2a2, f2b and f3 were identified. The molecular weights of Oxytricha histones were determined b y the m e t h o d Of Panyim and Chalkley [11]. Amino acid analysis was carried o u t according to the methods outlined by Blackburn [12]. Short (7.5 cm) and long (25 cm) polyacrylamide-urea gels were made according to the methods of Panyim and Chalkley [13] with a final pH of 2.7 and a concentration of 15% acrylamide and 2.5 M urea. 11 cm gels containing sodium dodecyl sulfate were made according to the m e t h o d of Panyim and Chalkley [11] with a final pH of 10 and a concentration of 15% acrylamide and 0.5% sodium dodecyl sulfate. Histones were reduced with fresh 0.5 M g-mercaptoethanol before electrophoresis. Pre-electrophoresis was carried o u t for 3 h (7.5-cm gels) or 17 h (25-cm gels), and electrophoresis was performed at 2 mA/gel for 2 h on 7.5-cm gels and at 200 V for 16 h on 25-cm gels. Electrophoresis was performed at 1 mA/gel for 5.5 h on l l - c m (sodium dodecyl sulfate) gels. Gels were stained with 1% Amido Schwartz, 7% acetic acid, 40% ethanol, and destained in the acid-ethanol solvent. Oxytricha macronuclear histones separated in 25-cm polyacrylamideurea gels are shown in Fig. 1. Fractions f3, f2b, f2a2, f 2 a l , f l a and f l b are
111
(,)
(-)
!liiI~!i~llI!~ ~ ! ~III1~IllIil Jl~lltllii ~1111 IIIHIHIIllll:l~lll filllll~]ll]lllll IIIilHii lll!lll~l lil!]ll*I !lllilil!~l~lll!II !111]11!I !liii!ll~ ~II~II
"":' ~ ~.~S.,.!,"!.,.,.: ~.. ~] ..~*~,..~.,'__~' 'I° ' z ~
, ~Is , ,i,~ ~ ~
, ~ ,:,IB,:~ , ~
~ ,I~ ' . . ~
Fig. I, High resolution eleetaophoresis of Oxytricha macronueleax histones on polyaerylamlde-urea gels ( I ) f3 s u b f r a e t i o n s , (2) f2b f r a c t i o n , (3) f2a2 s u b f r a c t i o n s , (4) f 2 a l s u b f r a c t i o n s , (5) f l a subf r a c t i o n (5') f l b s u b f r a c t i o n . Oxytricha m a c r o n u c l e a r h i s t o n e s w e r e p r e p a r e d as d e s c r i b e d in the t e x t . E l e c t r o p h o r e s i s was c a r r i e d o u t at 2 0 0 V for 16 h o n 25 c m gels m a d e o f 15% a c r y l a m i d e , 2.5 M u r e a (pH 2.7). M i g r a t i o n is f r o m l e f t (+) t o right (--).
designated 1, 2, 3, 4, 5 and 5', respectively. Fig. 2 shows the position of Oxytricha f l compared to the position of calf thymus f l in 7.5 cm polyacrylamide-urea gels. In vertebrates, f l histone migrates slowest in polyacrylamide-urea gels, and is found nearer the anode than other histone fractions. In Oxytricha, however, f l migrates ahead of f2al and is found nearer the cathode than other histone fraction3. The position of a histone in polyacrylamide-urea gels is a function of the size and the net positive charge of the molecule. For the following reasons, it is not likely that the position of Oxytricha f l in polyacrylamide-urea gels is a result of the degradation of f l into smaller molecules that run more rapidly than an undegraded species. First, precautions were taken to avoid proteolysis by using 0.05 M NaHSO3 during chromatin isolation. 0.05 M NaHSO3 inhibits the activity of a protease that attacks nucleohistone and histone in both calf thymus and rat liver [14,15]. According to Bartley and Chalkley [14], the earliest sign of proteolysis is a degradation of f l with the resultant production of small components that appear as faint bands migrating faster than f l histone in electrophoresis. Oxytricha fl, extracted with 5% HC104 or with whole histone in 0.4 M HC1, exhibits identical patterns in electrophoresis that do not include minor components migrating faster than the major f l subfractions. Secondly, the molecular weight of Oxytricha f l has been estimated as 21 000 by the method of Panyim and Chalkley [11] and is equivalent to the molecular weight of calf thymus fl. This indicates that Oxy tricha f l polypeptides are not degraded into proteins the same size or smaller than other Oxytricha histones, which have the following molecular weights: f3 (13 500), f2b (13 500), f2a2 (12 000) and f2al (11 000). The amino acid composition of Oxytricha f l has been determined and in Table I is compared to the composition of calf thymus f l and to f l from the holotrich ciliate, Tetrahymena pyriformis. Oxytricha f l is rich in lysine residues {31.6%) and has a low molar percent of acidic residues. The ratio of basic residues to acidic residues in Oxytricha f l (11.5) is greater than that of calf thymus f l (4.0) or Tetrahymena f l (2.0). Panyim et al. [5] have noted that the mobility of f l increases slightly in the lower classes of vertebrates. The mobility of fish f l > amphibian f l > mammalian fl. F1 from Tetrahymena migrates more rapidly than vertebrate fl, and is in the penultimate position on polyacrylamide-urea gels, behind f2al [6]. The mobility of fl, slightly increased in the lower verte-
112
A
B
C
(+)
41111R~i:~
f 3 "--~..~
•
f 2b ....-.-~Q
~
. ~
~
|
---
fJa flb (-)
Fig. 2. A c o m p a r i s o n o f t h e p o s i t i o n of Oxytricha 51 a n d c a l f t h y m u s f l o n p o l y a c r y l a m i d e - u r e a gels. (A) C a l f t h y m u s h i s t o n e s , (B) Oxytricha m a c r o n u c l e a r h i s t o n e s , (C) Oxytricha m a c r o n u c l e a r f l . C a l f t h y m u s h i s t o n e s w e r e s u p p l i e d b y C a l b i o c h e m , L a J o l l a , Calif. Oxytricha h i s t o n e s w e r e p r e p a r e d as d e s c r i b e d in t h e t e x t a n d t h e f r a c t i o n s w e r e i d e n t i f i e d b y t h e m e t h o d s o f J o h n s [ 9 ] . W i t h t h e e x c e p t i o n o f 51, Oxytricha m a c r o n u c l e a r h i s t o n e f r a c t i o n s c o r r e s p o n d in p o s i t i o n t o t h e f r a c t i o n s o f c a l f t h y m u s . E l e c t r o p h o r e s i s w a s c a r r i e d o u t a t 2 m A / g e l f o r 2 h o n 7 . 5 - c m gels m a d e o f 1 5 % a e r y l a m i d e , 2 . 5 M u r e a ( P H 2 . 7 ) . Gels (B) a n d (C) c o n t a i n b o v i n e s e r u m a l b u m i n as a p o s i t i o n m a r k e r o n t o p o f t h e gel. S l o w m o v i n g , f a i n t b a n d s in ( A ) a n d (B) are f3 a g g r e g a t e s . M i g r a t i o n is f r o m t o p (+) t o b o t t o m (--).
brate classes, is therefore greatly increased in the protozoans, Tetrahymena and Oxytricha. Tetrahymena is a ciliate t h a t does n o t have gene-sized pieces of macronuclear DNA [16]. Tetrahymena f l is n o t as rich in lysine, nor as poor in acidic residues as is Oxytricha f l . The extreme net positive charge on
TABLE I A M I N O A C I D C O M P O S I T I O N O F O X Y T R I C H A SP. M A C R O N U C L E A R f l , T E T R A H Y M E N A P Y R I F O R M I S ( S T R A I N W H - 6 ) M A C R O N U C L E A R f l A N D C A L F T H Y M U S 51 Mol p e r 1 0 0 m o l o f a m i n o a c i d s Amino acid
Oxytricha
Tetrahymena*
Calf t h y m u s * *
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Basic/acidic
31.6 0.5 3.4 1.9 4.1 6.9 1.2 5.1 2.8 29.2 0.0 8.1 0.0 4.3 0.3 0.2 0.2 11.5
29.9 2.3 2.4 9.9 8.6 7.5 7.3 6.1 3.5 14.0 0.0 3.9 Trace 2.3 1.7 0.5 0.4 2.0
27.3 0.0 2.1 2.6 6.0 6.9 4.7 8.4 6.7 23.4 0.0 6.0 Trace 0.8 3.9 0.6 0.6 4.0
* F r o m G o r o v s k y e t al. [ 1 1 ] . ** F r o m P a n y i m e t al. [ 5 ] .
113
Oxytricha f l may therefore not be typical of protozoan histones, but may be a requisite for f l association with gene-sized DNA pieces. The author thanks Dr D.M. Prescott for his interest and support; and Anders Persson for his operation of the JEOL amino acid analyzer. This work was supported by National Science Foundation grant GB-32232 to D.M. Prescott.
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