Plant Molecular Biology 11:507-515 © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
507
Chromosomal proteins of Arabidopsis thaliana Charles P. Moehs, Elizabeth E McElwain and Steven Spiker*
Genetics Department, North Carolina State University, Raleigh, NC 27695-7614, USA Received 18 May 1988; accepted in revised form 19 July 1988
Key words: Arabidopsis thaliana, histone variants, high mobility group proteins Abstract In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance o f single-copy D N A in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence o f a set of H 2 A and H2B variants. Arabidopsis has at least four H 2 A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat. The high mobility group ( H M G ) non-histone chromatin proteins have attracted considerable attention because o f the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group o f non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as H M G proteins and that cross-react with antisera to H M G proteins of wheat.
Abbreviations." H M G , high mobility group; SDS, sodium dodecyl sulfate
Introduction
Arabidopsis thaliana, a small crucifer, is finding increasing favor with plant molecular biologists as a model organism. A m o n g its advantages for molecular studies is its small genome, which at 7 x 107 base pairs is the smallest higher plant genome known [10]. In addition, its genome consists predominantly of single-copy sequences, facilitating the isolation and cloning of genes [14]. * This is paper 11628of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695 (*author for correspondence; telephone (919)737-2292).
The fact that Arabidopsis possesses a genome with these characteristics has led us to ask if Arabidops& has histone variants. Plants with larger genomes, especially polyploid plants such as wheat, have been shown to have H 2 A and H2B histones that are families of closely related proteins called histone variants [9, 15, 16, 21]. Histone variants are also found in organisms representing all other eukaryotic kingdoms, and there is evidence supporting the idea that histone variants are involved in forming the structure of transcriptionally active or inert chromatin [1, 2, 31]. The evidence is not conclusive, however, and we really do not know if histone variants have distinct physiological roles to play. In fact, evidence
508 against differential functions for histone variants has been presented by Grunstein and co-workers [8, 17] who have shown that yeast can survive as long as they retain the genes for at least one of the two H2A variants and one of the two H2B variants. These observations may not, however, be relevant to multicellular eukaryotes. As single-celled organisms, yeasts do not go through development in the same sense as do multicellular organisms. Furthermore, the yeast genome is uniformly sensitive to DNase I, leading to the speculation that the entire yeast genome is in a transcriptionally poised state [11]. IfArabidopsis, a multicellular plant which goes through all the complex developmental processes of plants with larger genomes, were found to possess no histone variants, the possibility of special physiological roles for histone variants in general would be dealt a severe blow. We thus isolated Arabidopsis histones and analyzed them on four electrophoretic systems. This allowed us to determine the identity of H2A and H2B histones (as well as H1, H3 and H4) and to show that variants of H2A and H2B exist in comparable number to those of plants with larger genomes. Thus, special physiological roles for histone variants are still possible. The major H2A and H2B variants of Arabidopsis differ in molecular weight. This is in accordance with previous findings of molecular weight variants in plants with larger genomes [16, 21], but in contrast to the histone variants of animal systems, most of which do not differ in molecular weight [31]. In addition to this work with the histones, we have studied Arabidopsis chromosomal proteins that can be operationally defined as high mobility group (HMG) chromosomal proteins. H M G proteins have aroused considerable interest because of their putative association with actively transcribing regions of the genome [23, 30]. Because no assay for their function exists, these proteins are defined by their shared characteristics of being extracted from purified chromatin by 0.35 M NaCI and being soluble in 2% trichloroacetic acid [4, 6, 25]. In addition, H M G proteins have highly unusual amino acid compositions: approximately 25 mol% basic and 25 mol% acidic amino acid residues [4, 6]. Proteins that meet this operational definition have been isolated from representatives of all eukaryotic kingdoms [13, 25].
In plants, H M G proteins have been partially characterized only in wheat, barley and maize [22, 23, 28]. We show here that Arabidopsis has chromosomal proteins that are H M G proteins by operational criteria and that cross-react with antisera to wheat H M G proteins.
Materials and methods
Histone and HMG isolation Wheat histones and H M G proteins were isolated as previously described [21, 22]. To isolate Arabidopsis histone and H M G proteins, samples of approximately 200 g of Arabidopsis leaves from 5-week-old seedlings were ground in a Waring blender in a solution containing 0.4 M sucrose, 10 mM Tris-HC1 pH 8, 10 mM MgC12, and 5 mM /3mercaptoethanol. The resulting slurry was filtered through 4 layers of cheesecloth and 2 layers of Miracloth (Calbiochem). The filtrate was centrifuged at 12000xg for 10 minutes. The pelleted material was washed by resuspending it in 0.25 M sucrose, 10 mM Tris-HC1 pH 8, 10 mM MgC12, 1%0 Triton X-100, and 5 mM /3-mercaptoethanol and centrifuging it as above. Following this wash, the pelleted chromatin was homogenized in a medium containing 1.7 M sucrose, 10 mM Tris-HC1 pH 8, 0.15% Triton X-100, 2 mM MgC12, and 5 mM ~mercaptoethanol. The homogenized chromatin was layered over an equal volume of this buffer and centrifuged at 27 000 x g for 30 minutes. H M G proteins were extracted from the resulting purified chromatin with 0.35 M NaC1, and histones were extracted from the chromatin with 0.4 N H2SO 4 by the same procedures used to isolate wheat H M G proteins and histones. All procedures were carried out at 0 - 4 ° C , and all solutions contained 0.1 mM phenylmethanesulfonyl fluoride as a protease inhibitor.
Electroph oresis SDS-polyacrylamide gels and acetic acid-urea gels with stackers were run as previously described [12, 20, 21]. Two-dimensional gels in which the first
509 dimension is an acetic acid-urea-Triton slab and the second dimension is an SDS gel were run according to Spiker et al. [27]. Acetic acid-urea gels with a transverse Triton gradient (0-10 mM) were poured according to Waterborg et al. [29], except that the gels contained 5 M urea and were run for 9 hours at 70 V under constant voltage conditions. All gels were stained with Coomassie Blue [21].
Western blotting Proteins were electrophoretically transferred from SDS gels to nitrocellulose and probed with antibodies to Tetrahymena H 2 A variant, hvl, (kindly supplied by Martin Gorovsky) and to wheat H M G proteins as described in Spiker and Everett [26]. Cross-reacting proteins were detected using horse radish peroxidase-conjugated second antibody (obtained from BioRad) according to supplier's protocol.
Fig. 1. Comparative one-dimensional electrophoresis of wheat and Arabidopsis histones. Histones of Arabidopsis and wheat
Results and discussion
were separated by SDS-polyacrylamide gel electrophoresis (a, left) and by acetic acid-urea-polyacrylamidegel electrophoresis (b, right). Both gels were stained with Coomassie Blue.
Histones o f Arabidopsis When wheat and Arabidopsis histones are compared on SDS-polyacrylamide gels (Fig. la) and acetic acid-urea gels (Fig. lb), it is evident that there are general similarities. Although several non-histone proteins coisolate with the histones o f Arabidopsis, the core histones are the most prominent proteins observed, and are identified on the basis of their comigration with the core histones o f wheat and by other criteria as outlined below. It is not suprising that Arabidopsis H3 and H4 histones comigrate with their wheat counterparts, since these proteins are a m o n g the most evolutionarily conserved known [5]. Chaboute et al. [3] recently cloned two H3 and two H4 genes of Arabidopsis, and found the deduced amino acid sequence of Arabidopsis H3 to be identical to the wheat H3 sequence except for a substitution o f serine by alanine at position 90. They also found that Arabidopsis H4 is identical to one variant of wheat H4. As is with case the histones H3 and H4, there are proteins in Arabidopsis that comi-
grate with the H2 histones of wheat on both SDS and acetic acid-urea gels. On the basis of evidence from these one-dimensional gels alone, it is impossible to identify H 2 A or H2B, or to assess the number of variants o f each. However, the observation of three prominent bands in the H2 region o f the SDS gel (Fig. la) suggests that molecular weight variants o f the H2 histones exist in Arabidopsis. We note that a prominent band in Arabidopsis, midway between H3 and H4 on the SDS gel, comigrates with a protein in wheat that is a minor wheat H 2 A fraction [21]. This A rabidopsis protein appears to be an H 2 A variant based on its detergent-binding properties (see below). The fact that this protein is a m a j o r species in Arabidopsis suggests that one o f the m a j o r H 2 A variants o f Arabidopsis has a lower molecular weight than the m a j o r H 2 A variants of wheat and is of a size more comparable to that of animal H 2 A histones. A prominent band in the Arabidopsis histone preparation migrates to the region o f wheat H I hi-
510 stones on acetic acid-urea gels (Fig. lb). Because of its prominence, electrophoretic mobility and detergent-binding properties (see below), this band is a good candidate for an H1 histone of Arabidopsis. On SDS gels (Fig. la) this same band migrates to approximately the same position relative to the wheat H1 histones, but the Arabidopsis H1 candidate stains much less intensely on the SDS gels. We do not know the reason for the less intense staining, but we do know that it is not due to histone degradation. The proteins in Fig. la and lb came from the same stock solution and were run several times with consistent results and no indication of degradation. The differential staining of H1 is a c o m m o n observation. Note for example that with wheat H1 histones, the higher molecular weight fractions stain much less intensely than the lower molecular weight fractions on SDS gels (Fig. la) while the higher and lower molecular weight fractions stain with approximately equal intensity on acetic acid-urea gels (Fig. lb). In order to gain further insights into the identity of the Arabidopsis histones, we separated these proteins by two-dimensional electrophoresis. A comparison of wheat and Arabidopsis histones separated by this method is presented in Fig. 2a & b. The first dimensions o f these gels (migration from left to
right) are acetic acid-urea-Triton gels. This type of gel separates proteins by differences in size, charge and detergent binding. The second dimensio' :s of these gels (migration from top to bottom) are SDS gels. A m a j o r advantage of this type of twodimensional gel system is that in the first dimension it resolves histones that differ by the extent to which their migration is altered by the presence of Triton. The similarity between the wheat and Arabidopsis histones seen in Fig. 2 is quite striking. Both wheat andArabidopsisH4 histones migrate as single spots. Arabidopsis H3, like its countergart in wheat, is found both in the reduced m o n o m e r and oxidized dimer forms. H 2 A and H2B histones differ considerably in their Triton-binding properties. This not only results in good resolution of the proteins, but also serves as a criterion for their identification [7, 24, 29]. The electrophoretic mobility of H 2 A histones is retarded by Triton to a much greater extent than are the mobilities of any of the other histones [24, 32]. The four spots labeled H 2 A in the twodimensional gel of Arabidopsis histories in Fig. 2b are thus identified as H 2 A histones because of the marked retardation effect of Triton on their electrophoretic mobilities. Based on electrophoretic mobility in SDS gels, two of the four Arabidopsis H 2 A variants have molecular weights that are similar to
Fig. 2. Comparative two-dimensional electrophoresis of wheat and Arabidopsis histones. Histones of wheat (a, left) and Arabidopsis (b, right) were separated by two-dimensionalgels in which the first dimensions (left to right) were Triton-acetic acid-urea gels (8 M urea, 0.38o70 Triton) and the second dimensions (top to bottom) were SDS gels. Note that both wheat and Arabidopsis have H2A and H2B variants of differing mobilities in the SDS dimension of the gel.
511 those o f the m a j o r wheat H 2 A variants, a n d two o f the Arabidopsis H 2 A variants have lower m o l e c u l a r weights. H2B histones characteristically migrate m u c h faster t h a n H 2 A in acetic a c i d - u r e a - T r i t o n gels, a n d such is the case for H2B of Arabidopsis. T h e Arabidopsis H2B variants are n o t as well resolved as the H 2 A variants, b u t it is clear that there are at least three m a j o r variants. F u r t h e r m o r e , the three variants have distinct electrophoretic mobilities in the SDS d i m e n s i o n o f the t w o - d i m e n s i o n a l gel shown in Fig. 2b. Thus, Arabidopsis despite its smaller gen o m e has m u l t i p l e variants o f the H 2 A a n d H2B histones, a n d the variants differ in a p p a r e n t m o l e c u l a r weights as has been s h o w n for the histones o f plants with larger g e n o m e s [16, 21]. A n a d d i t i o n a l t e c h n i q u e for c o m p a r i n g o u r
Arabidopsis histone p r e p a r a t i o n with wheat histones also depends o n the differential Triton b i n d ing o f histones. W h e n histones are separated o n a n acetic acid-urea gel that c o n t a i n s a transverse gradient o f Triton, the effect o f Triton o n the m o b i l i t y o f the various histone classes can be clearly observed. T h e results o f such a n experiment are presented in Fig. 3a & b. These gels are similar to Trit o n gradient gels used previously by other investigators [9, 29] to s t u d y barley a n d alfalfa histones. T h e best r e s o l u t i o n ofArabidopsis histones was achieved using a 0 - 1 0 m M gradient o f Triton o n a n acetic acid gel c o n t a i n i n g 5 M urea. Two advantages o f this m e t h o d are i m m e d i a t e l y apparent: (1) c o n t a m i n a t ing n o n h i s t o n e c h r o m o s o m a l proteins o n the Arabidopsis gel do n o t b i n d Triton a n d are unaffect-
Fig. 3. TransverseTriton gradient gel electrophoresis of wheat and Arabidopsishistones. Histones of wheat (a) and Arabidopsis(b) were separated by electrophoresis on acetic acid-urea slab gels which included a gradient from zero Triton on the left to 10 mM Triton on the right. Below the wheat gel is a diagram in which bands corresponding to well-characterized wheat histones are marked. H3ox indicates the oxidized disulfide dimer of H3. In a diagram under the Arabidopsis gel, the histones are marked according to their behavior in the other gel systems and the kinetics of non-ionicdetergent binding in this gel. One band marked "U" has a mobility between HI and H2 in the area of the gel with no Triton. The protein in this band binds Triton with the same kinetics as H2A histones and therefore may be an H2A histone modified by ubiquitin. Another band of unknown identity is marked "?". This band binds Triton with kinetics bet ween those of H2B and H3.
512 ed by the Triton gradient, and (2) differences in Triton binding among the variants within a histone class are observable. Again, the Arabidopsis histones are readily identifiable when compared with their counterparts in wheat. The migration of H1 histones is characteristically unaffected by Triton [32]. In keeping with the generality, the electrophoretic mobilities of the well-characterized H1 histones of wheat remain constant across the Triton gradient (Fig. 3a). A prominent band on the Arabidopsis gel (Fig. 3b) shares this characteristic, and furthermore migrates in the region of H1 histones on both SDS and acetic acid urea gels as illustrated in Fig. 1. This set of observations suggest that it represents an Arabidopsis H1 histone. This gel also confirms the presence of multiple variants of both H 2 A and H2B in Arabidopsis. As was previously found on the twodimensional gel, three major H2B variants are observed on the Triton gradient gel. While two of the variants are less affected by Triton, the mobility of the third decreases across the Triton gradient until, at 10 mM Triton, it comigrates with the putative HI. A similar situation prevails in wheat, except that four bands containing the six wheat H2B variants are observed. In contrast to H2B histones, the migration of H 2 A histones is strongly affected by the Triton gradient. While all the H 2 A variants comigrate at 10 mM Triton, their migration is retarded to different degrees at intermediate concentrations of Triton. Consistent with the previous two-dimensional gel, at least four bands in the Arabidopsis gel have the strong Tritonbinding property characteristic of H 2 A histones. The H3 and H4 bands of wheat and Arabidopsis behave identically in the transverse Triton gradient as expected. Interestingly, an unexplained band (marked " U " ) is found between the putative Arabidopsis H1 and H2 region at the extreme left side of the gel. This band has the Triton-binding property characteristic of H 2 A and migrates just where one would expect to find a ubiquitinated H 2 A [31]. We have not as yet established that this band is a ubiquitinated H 2 A histone, however, a band that migrates between H1 and H2 histones on SDS gels cross-reacts to ubiquitin antibodies (data not shown). It is presently unknown whether the numerous hi-
stone variants which we observed in Arabidops& are functionally distinct. Studies with other organisms have shown, however, that all of the variants are incorporated into chromatin, resulting in considerable nucleosome heterogeneity [19]. The work of Gorovsky and coworkers has provided evidence that suggests that histone variants may have special functions. They have identified a minor H 2 A variant of Tetrahymena that seems to play a role in the functional differentiation of chromatin [1]. They have raised antibodies against this protein, which they call hvl, and have found that it is present only in the transcriptionally active macronucleus of Tetrahymena and not in the transcriptionally inactive micronucleus [2]. Partial sequencing of this protein reveals that it shares some sequence homology with the major H2A of Tetrahymena, but it has diverged sufficiently that antibodies against it do not react with the major H 2 A [1]. Gorovsky has provided us with a sample of anti-
Fig. 4. Cross-reactionof wheat and Arabidopsis histones with antiserum to the putative H2A histone of transcriptionally active Tetrahymenachromatin. One-dimensional SDS polyacrylamide gels were run ofArabidopsis histones (lanes 1 and 3), unfractionated wheat histones (lane 4) and wheat H2A (lane 2). The proteins wereelectrophoreticallytransferred to nitrocelluloseand the nitrocellulose was cut lengthwise, splitting lane 2. Lanes 3, 4 and half of lane 2 were stained with amido black and lane 1 and half of lane 2 were probed with antiserum to the TetrahymenaH2A histone variant, hvl, followed by horseradish peroxidaseconjugated second antibody and visualized by peroxidase activity. A single band ofArabidopsis histone (arrowed) was found to cross-react. This band has mobility on SDS gels corresponding to one of the four major H2A variants of Arabidopsis.
513 b o d y against hvl, a n d we have shown that o n l y o n e o f the three m a j o r H 2 A variants o f wheat strongly reacts with this a n t i b o d y [23]. I n Fig. 4, we present a Western blot ofArabidopsis histones p r o b e d with the hvl antibody. It is a p p a r e n t that there is one m a j o r cross-reacting p r o t e i n in the Arabidopsis histone p r e p a r a t i o n . This p r o t e i n migrates j u s t slower t h a n H3 in the H2 region o f the SDS gel. It is likely that the p r o t e i n cross-reacting with the hvl a n t i b o d y represents one o f the higher m o l e c u l a r weight H 2 A variants, similar to o u r results with wheat histones.
teins in Fig. 5. There are at least four distinct b a n d s (arrowed) in the Arabidopsis lane which have mobilities similar to those o f the four wheat H M G proteins. The fastest-migrating, arrowed b a n d comigrates with wheat H M G d . T h e other arrowed b a n d s have electrophoretic mobilities similar to those o f the wheat H M G proteins b u t n o n e co-migrate exactly. To d e t e r m i n e whether the Arabidopsis proteins, which meet the o p e r a t i o n a l criteria for being H M G proteins, share antigenic d e t e r m i n a n t s with proteins isolated similarly from wheat, we p r o b e d the
H M G proteins o f A r a b i d o p s i s We have isolated proteins from Arabidopsis that, acc o r d i n g to the o p e r a t i o n a l d e f i n i t i o n o f J o h n s [6], are H M G proteins. T h a t is, purified Arabidopsis c h r o m a t i n was extracted with 0.35 M NaC1, a n d the 2070 trichloroacetic a c i d - i n s o l u b l e proteins were removed from this extract. Using S D S - p o l y a c r y l a m i d e gel electrophoresis, the soluble proteins (putative H M G proteins) are c o m p a r e d to wheat H M G pro-
Fig. 6. Cross-reaction ofArabidopsis HMG proteins to antisera to wheat HMG proteins. The Arabidopsischromosomal proteins,
Fig. 5. Chromosomal proteins of Arabidopsis that meet operational criteria for HMG proteins. Purified Arabidopsis chromatin was treated with 0.35 M NaCI. The extracted proteins were made 2% (w/v) trichloroacetic acid and the insoluble proteins removed by centrifugation. The soluble proteins isolated in this manner meet the operational criteria for HMG proteins (23, 25). The Arabidopsis and wheat embryo proteins isolated in this way are compared by SDS-polyacrylamide gel electrophoresis. Four bands of Arabidopsis proteins (arrowed) have electrophoretic mobilities in the same range as wheat HMG proteins a-d.
which can be operationally defined as "HMG" proteins (Fig. 5), were separated by SDS polyacrylamide gel electrophoresis alongside wheat HMG proteins. Four lanes of each protein preparation were run. The proteins were electrophoretically transferred to nitrocellulose and probed with antisera to wheat HMGa (panel a), HMGb (panel b), HMGc (panel c) and HMGd (panel d). The wheat proteins are displayed in the right side of each panel, and the Arabidopsis proteins are displayed on the left side of each panel. Antisera to wheat HMGa and HMGb react predominantly to single species of wheat proteins. Anti-HMGc cross-reacts somewhat to HMGd and to some higher molecular weight proteins. Anti-HMGd cross-reacts strongly with HMGa and HMGc and somewhat to higher molecular weightproteins. A singleband of Arabidopsis protein cross-reacts with anti-HMGa. This band (arrowed) has lower electrophoretic mobility than wheat HMGa and is not among the prominent bands detected by Coomassie Blue stain (Fig. 5). A single band of Arabidopsis protein also cross-reacts with anti-HMGb. This band migrates to the position of the second fastest band arrowed in Fig. 5. No Arabidopsis chromosomal proteins have detectable cross-reaction with antiserum to wheat HMGc. A single Arabidopsis protein cross-reacts with antiserum to wheat HMGd. This protein has the same electrophoretic mobility as the protein cross-reacting to antiserum to wheat HMGb (panel b).
514
Arabidopsis proteins with antibodies raised against each of the four wheat H M G proteins [26]. Western blots are presented in Fig. 6. Panel " a " compares wheat and Arabidopsis H M G proteins probed with antibodies to wheat HMGa. This antibody is quite specific for wheat H M G a in that it cross-reacts with none of the other wheat HMGs. This antibody reacts with a single Arabidopsis protein (arrowed), which has a mobility considerably lower than wheat H M G a or any of Arabidopsis bands that were arrowed in Fig. 5. In panel " b " the Arabidopsis protein that cross-reacts with the wheat H M G b antibody migrates just faster than wheat HMGb. This band corresponds to the next to fastest arrowed band on the stained gel in Fig. 5. Panel " c " shows both Arabidopsis and wheat H M G proteins probed with antibodies to wheat HMGc. No Arabidopsis proteins have detectable interaction with the H M G c antibodies. When the Arabidopsis proteins are probed with antibodies to wheat HMGd, a single band appears in the Arabidopsis lane as shown in panel " d " . This band appears to have a molecular weight similar to the Arabidopsis band detected with the wheat H M G b antibody, although it is unknown whether the same Arabidopsis protein is cross-reacting with both wheat H M G b and H M G d antibodies. In conclusion, we have isolated the histones of
Arabidopsis thaliana, a crucifer having the smallest genome of any well-studied plant. Our results indicate that, like plants with much larger genomes such as wheat, Arabidopsis contains several molecular weight variants of both H2A and H2B histones. We thus predict that when the genes for Arabidopsis H2A and H2B histones are isolated, they will constitute multigene families, the members of which code for the individual variants of these histones. We have also shown that some of the Arabidopsis chromosomal proteins that meet the operational criteria to be H M G proteins cross-react with antibodies to wheat H M G proteins.
Acknowledgements This work was supported in part by United States Public Health Service Grant GM37810. We thank
Martin Gorovsky for the generous gift of antiserum to Tetrahymena hvl and Richard Vierstra for the generous gift of antiserum to oat ubiquitin. We also thank General Mills for wheat germ and Mark Conkling for seeds of Arabidopsis.
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515 terminal extension. Eur J Biochem 150:499-506 (1985). 17. Rykowski MC, Wallis JW, Choe J, Grunstein M: Histone H2B subtypes are dispensable during the yeast cell cycle. Cell 25:477-487 (1981). 18. Simon JH, Becker WM: A polyethylene glycol/dextran procedure for the isolation of chromatin proteins (histones and non-histones) from wheat germ. Biochim Biophys Acta 454:154-171 (1976). 19. Simpson RT: Modulation of nucleosome structure by histone subtypes in sea urchin embryos. Proc Natl Acad Sci USA 78: 6803-6807 (1981). 20. Spiker S: A modification of the acetic-acid urea system for use in microslab polyacrylamide gel electrophoresis. Anal Biochem 108:263-265 (1980). 21. Spiker S: Histone variants in plants. J Biol Chem 257: 14250-14255 (1982). 22. Spiker S: High-Mobility Group chromosomal proteins of wheat. J Biol Chem 259:12007-12013 (1984). 23. Spiker S: Histones and HMG proteins of higher plants. In: Kahl G (ed) Architecture of Eukaryotic Genes, pp. 143-162. VCH Verlagsgesellschaft, Weinheim (1988). 24. Spiker S, Key JL, Wakim B: Identification and fractionation of plant histones. Arch Biochem Biophys 176:510-518 (1976).
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507
Chromosomal proteins of Arabidopsis thaliana Charles P. Moehs, Elizabeth E McElwain and Steven Spiker*
Genetics Department, North Carolina State University, Raleigh, NC 27695-7614, USA Received 18 May 1988; accepted in revised form 19 July 1988
Key words: Arabidopsis thaliana, histone variants, high mobility group proteins Abstract In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance o f single-copy D N A in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence o f a set of H 2 A and H2B variants. Arabidopsis has at least four H 2 A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat. The high mobility group ( H M G ) non-histone chromatin proteins have attracted considerable attention because o f the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group o f non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as H M G proteins and that cross-react with antisera to H M G proteins of wheat.
Abbreviations." H M G , high mobility group; SDS, sodium dodecyl sulfate
Introduction
Arabidopsis thaliana, a small crucifer, is finding increasing favor with plant molecular biologists as a model organism. A m o n g its advantages for molecular studies is its small genome, which at 7 x 107 base pairs is the smallest higher plant genome known [10]. In addition, its genome consists predominantly of single-copy sequences, facilitating the isolation and cloning of genes [14]. * This is paper 11628of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695 (*author for correspondence; telephone (919)737-2292).
The fact that Arabidopsis possesses a genome with these characteristics has led us to ask if Arabidops& has histone variants. Plants with larger genomes, especially polyploid plants such as wheat, have been shown to have H 2 A and H2B histones that are families of closely related proteins called histone variants [9, 15, 16, 21]. Histone variants are also found in organisms representing all other eukaryotic kingdoms, and there is evidence supporting the idea that histone variants are involved in forming the structure of transcriptionally active or inert chromatin [1, 2, 31]. The evidence is not conclusive, however, and we really do not know if histone variants have distinct physiological roles to play. In fact, evidence
508 against differential functions for histone variants has been presented by Grunstein and co-workers [8, 17] who have shown that yeast can survive as long as they retain the genes for at least one of the two H2A variants and one of the two H2B variants. These observations may not, however, be relevant to multicellular eukaryotes. As single-celled organisms, yeasts do not go through development in the same sense as do multicellular organisms. Furthermore, the yeast genome is uniformly sensitive to DNase I, leading to the speculation that the entire yeast genome is in a transcriptionally poised state [11]. IfArabidopsis, a multicellular plant which goes through all the complex developmental processes of plants with larger genomes, were found to possess no histone variants, the possibility of special physiological roles for histone variants in general would be dealt a severe blow. We thus isolated Arabidopsis histones and analyzed them on four electrophoretic systems. This allowed us to determine the identity of H2A and H2B histones (as well as H1, H3 and H4) and to show that variants of H2A and H2B exist in comparable number to those of plants with larger genomes. Thus, special physiological roles for histone variants are still possible. The major H2A and H2B variants of Arabidopsis differ in molecular weight. This is in accordance with previous findings of molecular weight variants in plants with larger genomes [16, 21], but in contrast to the histone variants of animal systems, most of which do not differ in molecular weight [31]. In addition to this work with the histones, we have studied Arabidopsis chromosomal proteins that can be operationally defined as high mobility group (HMG) chromosomal proteins. H M G proteins have aroused considerable interest because of their putative association with actively transcribing regions of the genome [23, 30]. Because no assay for their function exists, these proteins are defined by their shared characteristics of being extracted from purified chromatin by 0.35 M NaCI and being soluble in 2% trichloroacetic acid [4, 6, 25]. In addition, H M G proteins have highly unusual amino acid compositions: approximately 25 mol% basic and 25 mol% acidic amino acid residues [4, 6]. Proteins that meet this operational definition have been isolated from representatives of all eukaryotic kingdoms [13, 25].
In plants, H M G proteins have been partially characterized only in wheat, barley and maize [22, 23, 28]. We show here that Arabidopsis has chromosomal proteins that are H M G proteins by operational criteria and that cross-react with antisera to wheat H M G proteins.
Materials and methods
Histone and HMG isolation Wheat histones and H M G proteins were isolated as previously described [21, 22]. To isolate Arabidopsis histone and H M G proteins, samples of approximately 200 g of Arabidopsis leaves from 5-week-old seedlings were ground in a Waring blender in a solution containing 0.4 M sucrose, 10 mM Tris-HC1 pH 8, 10 mM MgC12, and 5 mM /3mercaptoethanol. The resulting slurry was filtered through 4 layers of cheesecloth and 2 layers of Miracloth (Calbiochem). The filtrate was centrifuged at 12000xg for 10 minutes. The pelleted material was washed by resuspending it in 0.25 M sucrose, 10 mM Tris-HC1 pH 8, 10 mM MgC12, 1%0 Triton X-100, and 5 mM /3-mercaptoethanol and centrifuging it as above. Following this wash, the pelleted chromatin was homogenized in a medium containing 1.7 M sucrose, 10 mM Tris-HC1 pH 8, 0.15% Triton X-100, 2 mM MgC12, and 5 mM ~mercaptoethanol. The homogenized chromatin was layered over an equal volume of this buffer and centrifuged at 27 000 x g for 30 minutes. H M G proteins were extracted from the resulting purified chromatin with 0.35 M NaC1, and histones were extracted from the chromatin with 0.4 N H2SO 4 by the same procedures used to isolate wheat H M G proteins and histones. All procedures were carried out at 0 - 4 ° C , and all solutions contained 0.1 mM phenylmethanesulfonyl fluoride as a protease inhibitor.
Electroph oresis SDS-polyacrylamide gels and acetic acid-urea gels with stackers were run as previously described [12, 20, 21]. Two-dimensional gels in which the first
509 dimension is an acetic acid-urea-Triton slab and the second dimension is an SDS gel were run according to Spiker et al. [27]. Acetic acid-urea gels with a transverse Triton gradient (0-10 mM) were poured according to Waterborg et al. [29], except that the gels contained 5 M urea and were run for 9 hours at 70 V under constant voltage conditions. All gels were stained with Coomassie Blue [21].
Western blotting Proteins were electrophoretically transferred from SDS gels to nitrocellulose and probed with antibodies to Tetrahymena H 2 A variant, hvl, (kindly supplied by Martin Gorovsky) and to wheat H M G proteins as described in Spiker and Everett [26]. Cross-reacting proteins were detected using horse radish peroxidase-conjugated second antibody (obtained from BioRad) according to supplier's protocol.
Fig. 1. Comparative one-dimensional electrophoresis of wheat and Arabidopsis histones. Histones of Arabidopsis and wheat
Results and discussion
were separated by SDS-polyacrylamide gel electrophoresis (a, left) and by acetic acid-urea-polyacrylamidegel electrophoresis (b, right). Both gels were stained with Coomassie Blue.
Histones o f Arabidopsis When wheat and Arabidopsis histones are compared on SDS-polyacrylamide gels (Fig. la) and acetic acid-urea gels (Fig. lb), it is evident that there are general similarities. Although several non-histone proteins coisolate with the histones o f Arabidopsis, the core histones are the most prominent proteins observed, and are identified on the basis of their comigration with the core histones o f wheat and by other criteria as outlined below. It is not suprising that Arabidopsis H3 and H4 histones comigrate with their wheat counterparts, since these proteins are a m o n g the most evolutionarily conserved known [5]. Chaboute et al. [3] recently cloned two H3 and two H4 genes of Arabidopsis, and found the deduced amino acid sequence of Arabidopsis H3 to be identical to the wheat H3 sequence except for a substitution o f serine by alanine at position 90. They also found that Arabidopsis H4 is identical to one variant of wheat H4. As is with case the histones H3 and H4, there are proteins in Arabidopsis that comi-
grate with the H2 histones of wheat on both SDS and acetic acid-urea gels. On the basis of evidence from these one-dimensional gels alone, it is impossible to identify H 2 A or H2B, or to assess the number of variants o f each. However, the observation of three prominent bands in the H2 region o f the SDS gel (Fig. la) suggests that molecular weight variants o f the H2 histones exist in Arabidopsis. We note that a prominent band in Arabidopsis, midway between H3 and H4 on the SDS gel, comigrates with a protein in wheat that is a minor wheat H 2 A fraction [21]. This A rabidopsis protein appears to be an H 2 A variant based on its detergent-binding properties (see below). The fact that this protein is a m a j o r species in Arabidopsis suggests that one o f the m a j o r H 2 A variants o f Arabidopsis has a lower molecular weight than the m a j o r H 2 A variants of wheat and is of a size more comparable to that of animal H 2 A histones. A prominent band in the Arabidopsis histone preparation migrates to the region o f wheat H I hi-
510 stones on acetic acid-urea gels (Fig. lb). Because of its prominence, electrophoretic mobility and detergent-binding properties (see below), this band is a good candidate for an H1 histone of Arabidopsis. On SDS gels (Fig. la) this same band migrates to approximately the same position relative to the wheat H1 histones, but the Arabidopsis H1 candidate stains much less intensely on the SDS gels. We do not know the reason for the less intense staining, but we do know that it is not due to histone degradation. The proteins in Fig. la and lb came from the same stock solution and were run several times with consistent results and no indication of degradation. The differential staining of H1 is a c o m m o n observation. Note for example that with wheat H1 histones, the higher molecular weight fractions stain much less intensely than the lower molecular weight fractions on SDS gels (Fig. la) while the higher and lower molecular weight fractions stain with approximately equal intensity on acetic acid-urea gels (Fig. lb). In order to gain further insights into the identity of the Arabidopsis histones, we separated these proteins by two-dimensional electrophoresis. A comparison of wheat and Arabidopsis histones separated by this method is presented in Fig. 2a & b. The first dimensions o f these gels (migration from left to
right) are acetic acid-urea-Triton gels. This type of gel separates proteins by differences in size, charge and detergent binding. The second dimensio' :s of these gels (migration from top to bottom) are SDS gels. A m a j o r advantage of this type of twodimensional gel system is that in the first dimension it resolves histones that differ by the extent to which their migration is altered by the presence of Triton. The similarity between the wheat and Arabidopsis histones seen in Fig. 2 is quite striking. Both wheat andArabidopsisH4 histones migrate as single spots. Arabidopsis H3, like its countergart in wheat, is found both in the reduced m o n o m e r and oxidized dimer forms. H 2 A and H2B histones differ considerably in their Triton-binding properties. This not only results in good resolution of the proteins, but also serves as a criterion for their identification [7, 24, 29]. The electrophoretic mobility of H 2 A histones is retarded by Triton to a much greater extent than are the mobilities of any of the other histones [24, 32]. The four spots labeled H 2 A in the twodimensional gel of Arabidopsis histories in Fig. 2b are thus identified as H 2 A histones because of the marked retardation effect of Triton on their electrophoretic mobilities. Based on electrophoretic mobility in SDS gels, two of the four Arabidopsis H 2 A variants have molecular weights that are similar to
Fig. 2. Comparative two-dimensional electrophoresis of wheat and Arabidopsis histones. Histones of wheat (a, left) and Arabidopsis (b, right) were separated by two-dimensionalgels in which the first dimensions (left to right) were Triton-acetic acid-urea gels (8 M urea, 0.38o70 Triton) and the second dimensions (top to bottom) were SDS gels. Note that both wheat and Arabidopsis have H2A and H2B variants of differing mobilities in the SDS dimension of the gel.
511 those o f the m a j o r wheat H 2 A variants, a n d two o f the Arabidopsis H 2 A variants have lower m o l e c u l a r weights. H2B histones characteristically migrate m u c h faster t h a n H 2 A in acetic a c i d - u r e a - T r i t o n gels, a n d such is the case for H2B of Arabidopsis. T h e Arabidopsis H2B variants are n o t as well resolved as the H 2 A variants, b u t it is clear that there are at least three m a j o r variants. F u r t h e r m o r e , the three variants have distinct electrophoretic mobilities in the SDS d i m e n s i o n o f the t w o - d i m e n s i o n a l gel shown in Fig. 2b. Thus, Arabidopsis despite its smaller gen o m e has m u l t i p l e variants o f the H 2 A a n d H2B histones, a n d the variants differ in a p p a r e n t m o l e c u l a r weights as has been s h o w n for the histones o f plants with larger g e n o m e s [16, 21]. A n a d d i t i o n a l t e c h n i q u e for c o m p a r i n g o u r
Arabidopsis histone p r e p a r a t i o n with wheat histones also depends o n the differential Triton b i n d ing o f histones. W h e n histones are separated o n a n acetic acid-urea gel that c o n t a i n s a transverse gradient o f Triton, the effect o f Triton o n the m o b i l i t y o f the various histone classes can be clearly observed. T h e results o f such a n experiment are presented in Fig. 3a & b. These gels are similar to Trit o n gradient gels used previously by other investigators [9, 29] to s t u d y barley a n d alfalfa histones. T h e best r e s o l u t i o n ofArabidopsis histones was achieved using a 0 - 1 0 m M gradient o f Triton o n a n acetic acid gel c o n t a i n i n g 5 M urea. Two advantages o f this m e t h o d are i m m e d i a t e l y apparent: (1) c o n t a m i n a t ing n o n h i s t o n e c h r o m o s o m a l proteins o n the Arabidopsis gel do n o t b i n d Triton a n d are unaffect-
Fig. 3. TransverseTriton gradient gel electrophoresis of wheat and Arabidopsishistones. Histones of wheat (a) and Arabidopsis(b) were separated by electrophoresis on acetic acid-urea slab gels which included a gradient from zero Triton on the left to 10 mM Triton on the right. Below the wheat gel is a diagram in which bands corresponding to well-characterized wheat histones are marked. H3ox indicates the oxidized disulfide dimer of H3. In a diagram under the Arabidopsis gel, the histones are marked according to their behavior in the other gel systems and the kinetics of non-ionicdetergent binding in this gel. One band marked "U" has a mobility between HI and H2 in the area of the gel with no Triton. The protein in this band binds Triton with the same kinetics as H2A histones and therefore may be an H2A histone modified by ubiquitin. Another band of unknown identity is marked "?". This band binds Triton with kinetics bet ween those of H2B and H3.
512 ed by the Triton gradient, and (2) differences in Triton binding among the variants within a histone class are observable. Again, the Arabidopsis histones are readily identifiable when compared with their counterparts in wheat. The migration of H1 histones is characteristically unaffected by Triton [32]. In keeping with the generality, the electrophoretic mobilities of the well-characterized H1 histones of wheat remain constant across the Triton gradient (Fig. 3a). A prominent band on the Arabidopsis gel (Fig. 3b) shares this characteristic, and furthermore migrates in the region of H1 histones on both SDS and acetic acid urea gels as illustrated in Fig. 1. This set of observations suggest that it represents an Arabidopsis H1 histone. This gel also confirms the presence of multiple variants of both H 2 A and H2B in Arabidopsis. As was previously found on the twodimensional gel, three major H2B variants are observed on the Triton gradient gel. While two of the variants are less affected by Triton, the mobility of the third decreases across the Triton gradient until, at 10 mM Triton, it comigrates with the putative HI. A similar situation prevails in wheat, except that four bands containing the six wheat H2B variants are observed. In contrast to H2B histones, the migration of H 2 A histones is strongly affected by the Triton gradient. While all the H 2 A variants comigrate at 10 mM Triton, their migration is retarded to different degrees at intermediate concentrations of Triton. Consistent with the previous two-dimensional gel, at least four bands in the Arabidopsis gel have the strong Tritonbinding property characteristic of H 2 A histones. The H3 and H4 bands of wheat and Arabidopsis behave identically in the transverse Triton gradient as expected. Interestingly, an unexplained band (marked " U " ) is found between the putative Arabidopsis H1 and H2 region at the extreme left side of the gel. This band has the Triton-binding property characteristic of H 2 A and migrates just where one would expect to find a ubiquitinated H 2 A [31]. We have not as yet established that this band is a ubiquitinated H 2 A histone, however, a band that migrates between H1 and H2 histones on SDS gels cross-reacts to ubiquitin antibodies (data not shown). It is presently unknown whether the numerous hi-
stone variants which we observed in Arabidops& are functionally distinct. Studies with other organisms have shown, however, that all of the variants are incorporated into chromatin, resulting in considerable nucleosome heterogeneity [19]. The work of Gorovsky and coworkers has provided evidence that suggests that histone variants may have special functions. They have identified a minor H 2 A variant of Tetrahymena that seems to play a role in the functional differentiation of chromatin [1]. They have raised antibodies against this protein, which they call hvl, and have found that it is present only in the transcriptionally active macronucleus of Tetrahymena and not in the transcriptionally inactive micronucleus [2]. Partial sequencing of this protein reveals that it shares some sequence homology with the major H2A of Tetrahymena, but it has diverged sufficiently that antibodies against it do not react with the major H 2 A [1]. Gorovsky has provided us with a sample of anti-
Fig. 4. Cross-reactionof wheat and Arabidopsis histones with antiserum to the putative H2A histone of transcriptionally active Tetrahymenachromatin. One-dimensional SDS polyacrylamide gels were run ofArabidopsis histones (lanes 1 and 3), unfractionated wheat histones (lane 4) and wheat H2A (lane 2). The proteins wereelectrophoreticallytransferred to nitrocelluloseand the nitrocellulose was cut lengthwise, splitting lane 2. Lanes 3, 4 and half of lane 2 were stained with amido black and lane 1 and half of lane 2 were probed with antiserum to the TetrahymenaH2A histone variant, hvl, followed by horseradish peroxidaseconjugated second antibody and visualized by peroxidase activity. A single band ofArabidopsis histone (arrowed) was found to cross-react. This band has mobility on SDS gels corresponding to one of the four major H2A variants of Arabidopsis.
513 b o d y against hvl, a n d we have shown that o n l y o n e o f the three m a j o r H 2 A variants o f wheat strongly reacts with this a n t i b o d y [23]. I n Fig. 4, we present a Western blot ofArabidopsis histones p r o b e d with the hvl antibody. It is a p p a r e n t that there is one m a j o r cross-reacting p r o t e i n in the Arabidopsis histone p r e p a r a t i o n . This p r o t e i n migrates j u s t slower t h a n H3 in the H2 region o f the SDS gel. It is likely that the p r o t e i n cross-reacting with the hvl a n t i b o d y represents one o f the higher m o l e c u l a r weight H 2 A variants, similar to o u r results with wheat histones.
teins in Fig. 5. There are at least four distinct b a n d s (arrowed) in the Arabidopsis lane which have mobilities similar to those o f the four wheat H M G proteins. The fastest-migrating, arrowed b a n d comigrates with wheat H M G d . T h e other arrowed b a n d s have electrophoretic mobilities similar to those o f the wheat H M G proteins b u t n o n e co-migrate exactly. To d e t e r m i n e whether the Arabidopsis proteins, which meet the o p e r a t i o n a l criteria for being H M G proteins, share antigenic d e t e r m i n a n t s with proteins isolated similarly from wheat, we p r o b e d the
H M G proteins o f A r a b i d o p s i s We have isolated proteins from Arabidopsis that, acc o r d i n g to the o p e r a t i o n a l d e f i n i t i o n o f J o h n s [6], are H M G proteins. T h a t is, purified Arabidopsis c h r o m a t i n was extracted with 0.35 M NaC1, a n d the 2070 trichloroacetic a c i d - i n s o l u b l e proteins were removed from this extract. Using S D S - p o l y a c r y l a m i d e gel electrophoresis, the soluble proteins (putative H M G proteins) are c o m p a r e d to wheat H M G pro-
Fig. 6. Cross-reaction ofArabidopsis HMG proteins to antisera to wheat HMG proteins. The Arabidopsischromosomal proteins,
Fig. 5. Chromosomal proteins of Arabidopsis that meet operational criteria for HMG proteins. Purified Arabidopsis chromatin was treated with 0.35 M NaCI. The extracted proteins were made 2% (w/v) trichloroacetic acid and the insoluble proteins removed by centrifugation. The soluble proteins isolated in this manner meet the operational criteria for HMG proteins (23, 25). The Arabidopsis and wheat embryo proteins isolated in this way are compared by SDS-polyacrylamide gel electrophoresis. Four bands of Arabidopsis proteins (arrowed) have electrophoretic mobilities in the same range as wheat HMG proteins a-d.
which can be operationally defined as "HMG" proteins (Fig. 5), were separated by SDS polyacrylamide gel electrophoresis alongside wheat HMG proteins. Four lanes of each protein preparation were run. The proteins were electrophoretically transferred to nitrocellulose and probed with antisera to wheat HMGa (panel a), HMGb (panel b), HMGc (panel c) and HMGd (panel d). The wheat proteins are displayed in the right side of each panel, and the Arabidopsis proteins are displayed on the left side of each panel. Antisera to wheat HMGa and HMGb react predominantly to single species of wheat proteins. Anti-HMGc cross-reacts somewhat to HMGd and to some higher molecular weight proteins. Anti-HMGd cross-reacts strongly with HMGa and HMGc and somewhat to higher molecular weightproteins. A singleband of Arabidopsis protein cross-reacts with anti-HMGa. This band (arrowed) has lower electrophoretic mobility than wheat HMGa and is not among the prominent bands detected by Coomassie Blue stain (Fig. 5). A single band of Arabidopsis protein also cross-reacts with anti-HMGb. This band migrates to the position of the second fastest band arrowed in Fig. 5. No Arabidopsis chromosomal proteins have detectable cross-reaction with antiserum to wheat HMGc. A single Arabidopsis protein cross-reacts with antiserum to wheat HMGd. This protein has the same electrophoretic mobility as the protein cross-reacting to antiserum to wheat HMGb (panel b).
514
Arabidopsis proteins with antibodies raised against each of the four wheat H M G proteins [26]. Western blots are presented in Fig. 6. Panel " a " compares wheat and Arabidopsis H M G proteins probed with antibodies to wheat HMGa. This antibody is quite specific for wheat H M G a in that it cross-reacts with none of the other wheat HMGs. This antibody reacts with a single Arabidopsis protein (arrowed), which has a mobility considerably lower than wheat H M G a or any of Arabidopsis bands that were arrowed in Fig. 5. In panel " b " the Arabidopsis protein that cross-reacts with the wheat H M G b antibody migrates just faster than wheat HMGb. This band corresponds to the next to fastest arrowed band on the stained gel in Fig. 5. Panel " c " shows both Arabidopsis and wheat H M G proteins probed with antibodies to wheat HMGc. No Arabidopsis proteins have detectable interaction with the H M G c antibodies. When the Arabidopsis proteins are probed with antibodies to wheat HMGd, a single band appears in the Arabidopsis lane as shown in panel " d " . This band appears to have a molecular weight similar to the Arabidopsis band detected with the wheat H M G b antibody, although it is unknown whether the same Arabidopsis protein is cross-reacting with both wheat H M G b and H M G d antibodies. In conclusion, we have isolated the histones of
Arabidopsis thaliana, a crucifer having the smallest genome of any well-studied plant. Our results indicate that, like plants with much larger genomes such as wheat, Arabidopsis contains several molecular weight variants of both H2A and H2B histones. We thus predict that when the genes for Arabidopsis H2A and H2B histones are isolated, they will constitute multigene families, the members of which code for the individual variants of these histones. We have also shown that some of the Arabidopsis chromosomal proteins that meet the operational criteria to be H M G proteins cross-react with antibodies to wheat H M G proteins.
Acknowledgements This work was supported in part by United States Public Health Service Grant GM37810. We thank
Martin Gorovsky for the generous gift of antiserum to Tetrahymena hvl and Richard Vierstra for the generous gift of antiserum to oat ubiquitin. We also thank General Mills for wheat germ and Mark Conkling for seeds of Arabidopsis.
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