Vol. 259, No. 19, Issue of October 10,pp. 12007-12013,1984
Printed in U.S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 19% by The American Society of Biological Chemists, Inc.
High-mobility Group Chromosomal Proteins of Wheat*
(Received for publication, April23, 1984)
Steven Spiker
From the Genetics Department, North CarolinaState Uniuersity, Raleigh, North Carolina27695-7614
can selectively retain rnononucleosomes which contain DNA
Fourproteinshavebeen
extracted frompurified
chromatin of wheat embryos
with 0.35 M NaCl. These complementary to messenger RNA. The manner in which
proteins are soluble in 2% (w/v) trichloroacetic acid HMG proteins bindto nucleosomes has been studied inmany
and thus meet the original operational requirements laboratories
to
(see Weisbrod, 1982a and Igo-Kemenes et al.,
be classified as ”high-mobility group”(HMG) chromo- 1982 for review). Because all nucleosomes apparently have
somal proteins. The proteins have been characterized two high-affinity HMG binding sites (Mardian et al., 1980;
by one- and two-dimensional electrophoresis, amino
Sandeen et al., 1980), it is uncertain how the postulated
acid analysis, and peptide mapping. Threeof the pro- specificity in HMG-nucleosome binding might occur. Length
teins (HMGb, c, and d) share the mammalian HMG of DNA in mononucleosomes isolated in vitromay beinvolved
characteristic of being rich in both acidic and basic (Swerdlowand Varshavsky, 1983; Stein andTownsend, 1983)
amino acid residues.
Unlike their putativemammalian but this still leaves open the question ofhow postulated
counterparts, these plant HMG proteins contain less specific binding may occur in uiuo.
than 7 mol % proline. Thefourthwheatprotein
(HMGa) is rich in both proline and in basic amino acidThe fact that we still do not know thetrue biological
residues. This wheat protein, however, contains only functions of HMG proteins has been highlighted by recent
about half the proportion of acidic residues found in reports which question their long-assumed roles as structural
characteristic
also proteins of actively transcribed nucleosomes (Seale et al.,
mammalian HMG proteins-a
found in the trout
testis HMG protein, H6. Comparative 1983; Reeves and Chang, 1983). HMG proteins are, in fact,
peptide maps show that none of the wheat HMG pro- defined on an operational basis, not on a functional basis
teins are degradation products of other
HMG proteins (Johns, 1982). The proteins we now call HMG proteins were
first noticed as impurities in histone H1 preparations from
or the H1 histones. The peptide maps have not, however, been useful in establishing homologies mamwith calf thymus (Johns, 1964). According to the original operamalian HMG proteins. Wheat HMG proteins are re- tional definition presented by Goodwin et al. (1973), HMG
leased from DNase I-treated nuclei and
co-isolatewith proteins are 1) chromatin proteins, 2) extracted from chromicrococcal nuclease-sensitive chromatinfractions.
matin by 0.35 M NaCl, and 3) soluble in 2% (w/v) trichloroHMG proteins of acetic acid. These proteins migrate rapidly in polyacrylamide
Similar observations concerning the
vertebrateanimalshavebeenconsideredconsistent
gel electrophoresis systems; hence, their designation as “highas structural components mobility group” proteins. Further characterization of these
with a role for these proteins
of actively transcribed chromatin.
proteins revealed certain unusual characteristics which Johns
(1982) has added to theoperational definition of calf thymus
HMG proteins.Theseare 4) high in basic amino acidsapproximately 25mol %, 5) high in acidic amino acidsThe HMG’ chromosomal proteins have attracted consid- approximately 25 mol %, and 6) relatively high in proline-7
erable attention inrecentyears because, in part, of their mol % or more. The operational definitionsof HMG proteins
postulated roles as structuralproteins of nucleosomes of ac- are expedient but, as has been stressed by Johns (1982) and
tively transcribed genes (reviewedby Weisbrod, 1982a). Chro- Mayes (1982), care shouldbe taken when such definitions are
matin containing DNA sequences complementary to messen- applied to organisms and tissues other thancalf thymus. The
gerRNA loses its selective sensitivity to DNaseI when operationaldefinitions should aid, nothinder establishing
extracted with 0.35 M NaC1-a concentration which removes functional definitions for the HMG proteins. An illustration
HMG proteins. Moreover, the selective sensitivitycan be of why the operational definitions cannot be strictly applied
restored by reconstituting salt-stripped chromatin with the is provided by the trout testis
protein H6. This protein shows
0.35 M NaC1-extracted proteins or with purified HMG14 or striking sequence homology to calf thymus HMG14 and 17
17 (Weisbrod and Weintraub, 1979; Weisbrod et al., 1980; and is almost certainly the functional equivalent of these
Gazit et al., 1980). Weisbrod (1982b) and Weisbrod and Wein- mammalian HMG proteins (Dixon, 1982). However, the trout
traub (1981) have demonstrated that anHMG affinity column protein does not meet the fifth requirement to be an HMG
* This work was supported in part by National Science Foundation protein. It contains only about 13 mol % glutamic plus asparGrant PCM81-05135 and National Institute
of Health Grant Biomed- tic acid residues.
Proteins thathave properties similar to mammalian HMG
ical Research Support Group No. RR07071. This is Paper 9332 of the
Journal Series of the North Carolina Agricultural Research Service, proteins have been found in many organisms (Mayes, 1982;
Raleigh, NC27695. The costs of publication of this article were Bassuk and Mayfield, 1982; Hamana and Iwai, 1979; Katula,
defrayed in part by the payment of page charges. This article must 1983; Marquez et al., 1982; Spiker et al., 1978; Weber and
therefore be hereby marked ‘‘aduertkernent” in accordance with 18 Isenberg, 1980). However, the properties of these proteins
U.S.C. Section 1734 solely to indicate this fact.
‘The abbreviations used are: HMG, high-mobility group; Ph- correspond in varying degrees to those of calf thymus HMG
MeS02F, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sul- proteins and it is open to question whether they should be
fate; HPLC, high-performance liquid chromatography.
considered HMG proteins. We have previously reported the
12007
Wheat HMG Proteins
12008
existence of four chromosomal proteins from wheat embryos
which meet the salt extractability and acid solubility requirements to be termed “HMG” proteins (Spiker et al., 1978).
One of these (wheat HMGb) was additionally shown to be
HMG-like initscontent
of basic and acidic amino acid
residues. We have now further characterized the wheat HMG
proteins by amino acid composition, molecular weight, peptide
maps, and association with putatively transcriptionallyactive
regions of the genome. Comparative amino acid compositions
and peptide maps indicate that each of the four plant HMG
proteins are genuine proteins and not degradation artifacts of
other HMG proteins or H1histones. Amino acid compositions
and peptide maps were not conclusive indicators of which of
the plantHMG proteins might correspond to thehigh-molecular-weight mammalian HMGl and 2 or the low-molecularweight mammalian HMG 14 and 17. Wheat HMG proteins b,
c, and d meet the requirements for being termed HMG proteins in that they are
high in both acidic and basic amino acid
residues. However, the proline contents of these are all less
than 7 mol %. Wheat HMGa meets the requirement of being
relatively high in proline (12 mol %) but, like trout H6, it is
relatively low in acidic amino acid residues-about 12 mol %.
The plant HMG proteins share the characteristics of vertebrate HMG proteins of being released from nuclei treated
with DNase I and co-isolating with the susceptible fraction of
chromatin obtained by micrococcal nuclease treatment.
EXPERIMENTALPROCEDURES
Source of Tissue-Wheat embryos were obtained from General
Mills (Minneapolis, MN) or were mechanically isolated from dry,
mature grains (Triplett, 1979). The embryos from both sources
yielded chromatin with indistinguishable properties. Thymus glands
were obtained from 5-week-old pigs and placed on dry ice within 5
min after slaughter.
Isolation of Chromatin-Chromatin was isolated from wheat embryos by the method of Simon and Becker (1976) with the following
modifications. All solutions contained 0.1 mM PhMeS02F and12 mM
NaHS03. A Polytron homogenizer (Brinkmann) was used to disrupt
the tissue. The final step inthe purification was centrifugation of the
chromatin through 1.7 M sucrose, 15 mM 2-mercaptoethanol, 10 mM
Tris-HC1 (pH8), 0.5% Triton X-100 (Rohm andHaas), 12 mM
NaHS03,O.l mM PhMeS02Ffor 30 min at 16,000 X g. All procedures
were carried out a t 0-2 “C.
Isolation of HMG Proteins-HMG proteins were isolated from
wheat embryos essentially by the methods used by Goodwin et al.
(1973) to isolate calf thymus HMG proteins. These methods are the
basis of the operational definition of HMG proteins, i.e. nonhistone
proteins extracted from purified chromatin with 0.35 M NaCl and
soluble in 2% trichloroacetic cid. Purified wheat embryo chromatin
was washed twice in 75 mM NaCl, 25 mM Na-EDTA (pH 7.5), 12 mM
NaHS03, 0.1 mM PhMeSOzF. The chromatin pellet was then homogenized in 0.35 M NaC1, 1 mM Tris-HC1 (pH 8). 0.1 mM PhMeS02F
using a glass-Teflon homogenizer. Pellet and supernatant fractions
were obtained by centrifugation at 12,000 X g for 10 min. The
extraction procedure was repeated and the supernatants combined.
The combined supernatants were made 2% (w/v) trichloroacetic acid
by slow addition of 100% (w/v) trichloroacetic acid. After standing
on ice for 30 min, the solution was centrifuged (12,000 X g, 10 min)
to remove insoluble material. The supernatant fraction was made 10
mM 2-mercaptoethanol and 0.015 volume of 30% NH,OH was added.
All operations to this point were carried out at 0-2 “C. After adding
3 volumes of acetone to thesolution and allowing it to stand
overnight
a t 5 ‘C, the precipitated HMG proteins were removed by centrifugation, washed once in acetone, 0.1 N HCl (6:1), twice in acetone, and
dried in ~ ( I C U Oa t room temperature. Typical yields were 10 mgof
HMG proteins/300 g of dry embryos (2-5% of histone yield).
HMG proteins were extracted from frozen pig thymus essentially
by the method of Sanders (1977). Tissue washomogenized in 10
volumes of0.75 N HCIO,,0.1
mM PhMeS02F using a Polytron
homogenizer. The homogenate was centrifuged (12,000 X g, 10 min)
and thepellet fraction re-extracted with an additional 10 volumes of
0.75 N HClO,. The combined supernatants were filtered, 3.5 volumes
of acetone was added, and the solution was made 0.07 N HCl by
adding concentrated HC1. The resulting H1 histone precipitate was
removed by centrifugation and an additional 2.5 volumes of acetone
(based on supernatant volumebefore first acetone addition) was
added to the resulting supernatant. The HMG precipitate (contaminated by some H1 histone) was allowed to form overnight at 5 “C,
collected by centrifugation, then washed and dried in the same
manner as were the wheat HMG proteins.
Polyacrylamide Gel Electrophmesis-SDS-l8% polyacrylamide gels
were run as previously described (Spiker, 1982). Two-dimensional
electrophoresis of wheat HMG proteins was carried out essentially
according to O’Farrell et al. (1977) using nonequilibrium pH gradient
electrophoresis in the first dimension and SDS gels (as above) in the
second dimension.
Isolation of HMG Fractions-Wheat HMG fractions a, b, c, and d
were isolated by preparative electrophoresis (Spiker and Isenberg,
1983) or by HPLC. HPLC was carried out using a combination of
reverse-phase (Waters Microbondapak C-18) and cation exchange
(Brownlee Labs CX-300). Details will be presented elsewhere.
Amino Acid Analysis-Amino acid analysis of purified HMG fractions was carried out using the continuous gradient elution method
of Klapper (1982).
Peptide Mapping-Peptide maps were obtained by hydrolysis of
proteins in gel slices by the method of Cleveland et al. (1977) as
modified by Luna et al. (1979). A further modification of this system
(Spiker, 1980) allowed 3-mm-thick gel slices to be placed in 3-mm
wells of a stacking gel along with the proteolytic enzymes and the
resulting peptides to be separated on a 1.5-mm-thick running gel. In
the work presented in this paper, a similar modification was used
except that the 3-mm-thick stacking gel was poured over a 0.5-mm
running gel and a “mini gel” apparatus as described by Matsudaira
and Burgess (1978) was used.
Salt Extraction of HMG Proteins-The amount of wheat HMG
proteins extracted by various concentrations of NaCl wasdetermined
as follows. Wheat embryo chromatin purified by centrifugation
through 1.7 M sucrose as described above was washed with 75 mM
NaCI, 1mM Tris-HC1 (pH 7), 0.1 mM PhMeS02F.An even suspension
of chromatin in this buffer was divided into measured aliquots and
adjusted to the desired NaCl concentration. Each suspension was
then layered over a 15%sucrose solution containing the desired NaCl
concentration and centrifuged a t 47,000 rpm in a Ti 60 rotor (Beckman) for 22 h. The pellets were suspended in water, extracted with
0.4 N H~SOI and the acid-soluble proteins precipitated by dialysis
against ethanol. The supernatants were exhaustively dialyzed against
water containing 0.1 mM PhMeS02F,made 0.4 N &SO4, and further
dialyzed against ethanol to precipitate the proteins. PolyacrylamideSDS gels were then run, stained with Coomassie Blue and scanned
to quantitate DNA-bound protein from the pellets and free proteins
from the supernatants.
RESULTSANDDISCUSSION
Electrophoretic Mobility and MolecularWeight Estimution-The electrophoretic mobilities of the wheat HMG proteins arecompared to those of mammalian (pig thymus) HMG
proteins in the SDS gel in Fig. 1. In general, the electrophoretic mobilities are similar but no wheat HMG matches any
pig HMG in mobility.
In Fig. 2 the mobilities of wheat HMG proteins on SDS
gels are compared to those of ovalbumin, carbonic anhydrase,
myoglobin, cytochrome c, and ubiquitin. Assuming molecular
weights of 45,000, 29,000, 17,200, 11,700, and 8,500, respectively, for these standards,the molecular weights of the wheat
HMG proteins are calculated to be a = 24,300, b = 20,400, c
= 17,400, and d = 14,500. Histones, which have a high
proportion of basic residues, migrate anomalously on SDS
gels (Panyim and Chalkley, 1971). This anomalous migration
leads to an overestimate of their molecular weights when
typical molecular weight standards are used for comparison.
Like the histones, HMG proteins have a high proportion of
basic amino acid residues; but, because they are also high in
acidic residues, it is uncertain what standards are appropriate
for molecular weight estimation.When pig thymus HMG
proteins are used for standards, the molecular weights of the
Wheat HMG Proteins
.-m
c
12009
b
a
U
a l
“ r ”
H1
1 2’
“a
14 \
“b
17-
“c
d
-
C
“d
FIG.1. Sodium dodecyl sulfate-polyacrylamide gels of
wheat embryo and pig thymus HMG proteins. Wheat embryo
and pig thymus HMG proteinswere isolated and separated by SDSpolyacrylamide gel electrophoresis as described in the text and stained
with Coomassie Blue. Pig HMG proteins 1, 2, 14, and 17 are labeled
and co-migrate with their counterparts from calf thymus (Goodwin
FIG.3. Wheat HMG fractions purified by high-performance
et al., 1978). Contaminating pig thymusH1 histones are also labeled.
liquid chromatography. Wheat HMG proteins a, b, c, and d were
Wheat HMG proteins a, b, c, and d are labeled.
purified by HPLC asdescribed in the text. Inpan& a-d, respectively,
the purified proteins were separated by SDS electrophoresis along
with an unfractionated HMG preparation. Scans of the gels showed
all preparations to be more than95% pure.
TABLE
I
- 1
Amino acid content of HMG proteins fromcalf thymus and wheat
embryos
-2
Residue
c”
d’
“ d
3
.4
”5
FIG.2. Molecular-weight estimation ofwheat HMG protein. Sodium dodecyl sulfate-polyacrylamide gels were used as described in the text to electrophoretically separate wheat HMG proof known molecular
teins (lefttwo lanes) andstandardproteins
weights (right two lanes). 1 = ovalbumin, 45,000; 2 = carbonic anhydrase, 29,000; 3 = myoglobin, 17,200; 4 = cytochrome c, 11,700; 5 =
ubiquitin, 8,500.
Calf
Wheat
thymus”
9.3
10.7
8.1
12.0
Asx
5.2 2.7
3.22.51.4 3.7 1.2 4.2
Thr
5.0
7.4
7.8
2.3
Ser
12.0
15.3
12.7
5.3
10.5
17.1
Glx
18.1 17.5
7.05.83.6
8.9
4.7
12.6
12.9
8.5
Pro
8.6
11.2
6.5
5.3
6.513.7
GIY
11.3
15.9
20.3
18.4
14.5
9.0
8.1 14.8
Ala
5.42.3
4.71.93.6 3.2 2.0 4.2
Val
Trace Trace 0.7
CYS
2.3
1.5 0.4
Met
0.5
Ile
1.8
1.3
2.0 2.22.2 4.5 1.0 2.0
Leu
TYr
2.0 2.9
0.4
3.1
0.6
3.8
2.7
Phe
3.03.6
15.9
15.9
17.0
13.4
24.3
19.0
21.3
19.4
LYS
0.2
1.3
1.7
0.3
2.0
0.3
His 0.9
2.74.7
4.23.92.5 7.1 4.1 5.6
‘4%
24.1
19.5
20.5
28.4
24.6
Lys + Arg 25.2
22.5
Asx + Glx 28.8 26.8 25.2
“Goodwin et al. (1978).
wheat HMG proteins are calculated
to be a = 23,100, b =
14,500, c = 10,000, and d = 6,300.
Purification of Fractions-Wheat HMG fractions were purified by preparative electrophoresis (Spiker and
Isenberg,
1983) or by HPLC. In the SDS gels in Fig. 3, the purity of
the fractions obtainedby HPLC is demonstrated.A two-step
procedure is used. In the first step,
a cation-exchange column
at pH 6 and a NaCl gradient separates HMGa and b from
HMGc andd. In thesecond step, a reverse-phase C-18 column
separates HMGa from HMGb and HMGc from HMGd. In
most cases, no impurities are detected even on overloaded
gels. Some contaminationof HMGb with HMGc can
be noted
in panel b of Fig. 3. Scans of Coomassie Blue-stained gels
show that HMGb is more than 95% pure. Different preparations of mammalian HMG proteins are contaminated with
H 1 histones to various degrees (Nicolas and Goodwin, 1982).
Such is also the case with wheat HMG preparations which
embryo
HMGl HMG2 HMG14 HMG17 HMGa HMGb HMGc HMGd
mol %
mol %
7.2
13.9
11.7
7.0
8.3
1.7
2.7
1.9
0.7
10.7
8.2
7.6
9.2
11.8
1.8
0.4
1.5
0.8
0.2
1.1
2.3
1.4
1.2
20.1
18.6
12.5 22.7
29.2
24.4
can be seen as thehigh-molecular-weight band in the unfractionated HMG standards inFig. 3.
Amino Acid Compositions and Comparison with Calf HMG
Proteins-The amino acid compositions of the four wheat
HMG proteins and the four calf thymus HMG proteins are
shown in TableI. The mole per centof lysine plus arginine is
about 20% for each of the wheat HMGproteins. These values
are certainlyhigh but not ashigh as thosefor the calf thymus
HMG proteins. The mole percentages for the acidic residues
Wheat HMG Proteins
12010
obviously related
for wheat HMGb,c, and d are well within the rangesof those even the “weak” test for relatedness, they are
of the calf thymus HMG proteins. Wheat HMGa, however, by sequence. The sequences of these two proteins can be
eachproteinresultingin
a44%
has only 12.5% acidic residues. This low percentage of acidic aligned with twogapsin
residues does not conform to the definitions
of HMG proteins sequence homology. This homology includesa stretch in
proposed by Johns (1982) for calf thymus. However, in light which no gaps are necessary to generate 12 consecutive and
of a similar low percentage of acidic residues in trout testis 19 of 22 sequence identities. Conversely, calf thymus HMG2
H6, which is almost certainly an HMG (Dixon,
1982), the and HMG14 meet the“weak” test for relatedness even though
possibility that this wheat protein should be considered an there is little sequence homology between the two proteins.
HMG protein cannot be ruled out. In fact, if the last charac- Calf HMG2 (Walker, 1982) and HMG14 contain only two
teristic of calf thymus HMG proteins, proline content, is an common sequences with as many as four consecutive amino
important diagnostic, HMGa is the only wheat HMG which acid residues.
meets the criterion of a t least 7 mol % set by Johns (1982).
As can be seen in Fig. 4, wheat HMGb and HMGd meet
Wheat HMGa has 12.6 mol % proline. The others have less the “strong” test criterion for being related. Wheat HMGc
than 7%.
meets the “weak” test criterion for being related to wheat
In order to makea comparison of amino acid compositions HMGbandHMGd.WheatHMGcandHMGd
meet the
of HMG proteins which would take all the amino acids into “weak” test for beingrelated tocalf thymus HMG14. No other
account, we have used the indexof Marchalonis and Weltman relationships are evident using the criteria
of Cornish-Bowden
(1971) and the critical values of Cornish-Bowden (1980) for (1980).
testing the significance of amino acid composition indexes.
Wheat HMGa has some characteristics reminiscent of H 1
The index, SAQ is definedas: SAQ = lo4 ( X A - X,#, where histones. It hashigh proline and alanine content.However, it
XiAis the mole fraction of amino acid residues of the ith type is much lower in lysine and higher in arginine than wheat H1
in protein A and X,- is the mole fraction of residues of the histones (Spiker et al., 1976). No sequences are available for
ith type in protein B. Cornish-Bowden (1980) has developed wheat H 1 histones. However, we have used the partial setables of “critical values” for predicting “relatedness” of pro- quence information and amino acid compositions of aminoteins based on amino acid composition comparison by SAQ. and carboxy-terminal regions of maize H1 histone (Hurley
The values are dependent upon the total number of amino and Stout, 1980) to determine if the amino acid composition
acid residues in the larger of the two proteins (N). For each of HMGa could be consistent with that of a simple degradaN , critical values for a “strong” test and for a “weakn test tion product of H1 histone. Using this approach, we could
have been calculated. According to Cornish-Bowden, the cal- find noevidence that HMGa isderived from H1. Comparative
culated value of SAQ nearly always will be greater than the peptide maps of wheat HMGa and total H1 histones (data
critical value forthe “strong” test
if the proteins are unrelated.not shown) also provide no evidence that HMGa is derived
Calculated values of SAQ will almost always be smaller than from H1. When the amino acid compositions of HMGa and
the criticalvalue forthe “weak” test if the proteins are
related. total H1 histone are compared by the method of CornishWe have calculated SAQ for all pairs
of wheat and calf Bowden (1980), the two proteins do not meet the
“weak” test
HMG proteins and compared the
values to the criticalvalues for being related. By the sameprocedures, none of the wheat
of Cornish-Bowden (1980) in an attempt to determine
if any HMG proteins have aminoacid compositions which are “reof the wheat HMG proteins are more closely related to the lated” to anyof the wheat H2 histones.
larger calf HMG proteins (1 and 2) or to the smaller calf
Two-dimemiowl Electrophoresis-Commercially available
HMG proteins (14 and17). The results of these calculations
ampholines have not proved usefulin characterizing the wheat
are summarized in Fig. 4. In this figure if two proteins meet
HMG proteins by isoelectric focusing. Thus, we have used
the “weak” test requirement for being related, they areconnonequilibrium
pH gradient electrophoresis(O’Farrell et al.,
nected by a dotted line. If they meet the “strong” test
for being
1977).
In
Fig.
5,
a two-dimensional gel is shown in which a
related,theyareconnected
bya solid line. It shouldbe
emphasized that we make no claims that proteins arenecessarily related by sequence if they meet the“weak” or “strong”
test for relatedness. Nor do we claim that proteins which do
not meet the “weak” test are necessarily unrelated. For example, calf HMG14 and HMG17 have
been entirely sequenced
(Walker etal., 1977,1979), and even though they do not meet
HMG a
__,,(.._...
/’’_
...’
I
HMG &____._....._........
HMG d
HMG 14
HMG I
::
HMG 17
-HMG 2
]
]
H1
WHEAT
CALF
FIG. 4. Similarities ofamino acid composition of wheat and
calf thymus HMG proteins. The index of Marchalonis and Weltman (1971) was calculated for all pairs of calf and wheat HMG
proteins. If a pair meets the“weak” test requirementfor being related
(Cornish-Bowden, 1980), the proteins are connectedby a dotted line.
If a pair also meets the “strong” test requirement for being related,
the proteins are connected witha solid line.
a
FIG.5. Two-dimensional electrophoresis of wheat HMG
proteins. A preparation of wheat HMG proteins was separated by
two-dimensional electrophoresis essentially according to O’Farrell et
al. (1977) using nonequilibrium pH gradient electrophoresis in the
first dimension and NaDodSO, electrophoresis in thesecond dimension. An aliquot of the same HMG preparation which had not been
separated in the first dimensionwas separated in the second dimension for comparison. Wheat HMGa (labeled a) co-migrates with the
wheat H1 contaminant in the firstdimension. Wheat HMG proteins
b, c, and d (unlabeled in figure) are each resolved into two species in
the first dimension.
Wheat HMG Proteins
a
b
b d
a c cd b d
12011
c d
1
2
-.
.IC*-.)
I__._
FIG. 6. Comparative one-dimensional peptide maps of wheat and pig thymus HMG proteins. Peptide
maps of wheat and pig thymus HMG proteins were made using Staphylococcal V8 protease by the method of
Cleveland et al. (1977) modified as described in the text. The l a n e s in the five panels in this figure are labeled with
the proteins mapped ( a = HMGa; b = HMGb; c = HMGc; d = HMGd; I = HMG1; 2 = HMGZ). The peptides are
stained with Coomassie Blue. The two, common, high-molecular-weight bands in each lane are due to theproteolytic
enzyme.
Salt Extraction of Wheat HMG Proteins-We have used
extraction of purified wheat chromatin with 0.35 M NaCl in
order to establish that the proteinswe have studied meet the
original operational criteria for HMG proteins. However, we
notice that when such procedures are used, some of the HMG
proteinsarenotextracted
from chromatin. In Fig.7, the
percentages of HMG proteins extractedby various concentrations of NaCl are shown. Well over half of the total HMG
proteins are extracted by 0.35 M NaCl. However, about 10%
of the HMG proteins remained bound to chromatin at 0.4 M
.2 .3 4 .5
NaCl and all detectableHMGproteins were not removed
until
0.45 M NaCl.
NaCl (MI
Association with Putatiuely Actiue Chromatin-Some of the
FIG. 7. Salt extraction of wheat HMG protein. The percent- first, but certainly not conclusive, indications that vertebrate
ageof total chromatin-bound HMG proteins extracted from chromatin by various concentrations of NaCl was determined as described HMG proteins are structural proteins of active chromatin
were the observations that these proteins are released from
in the text.
DNase I-treated chromatin (Vidali et al., 1977; Levy-Wilson
et
al., 1977). We have previously shown (Spiker et al. 1983)
wheat HMG preparation is separated by nonequilibrium pH
gradient electrophoresisin the firstdimension and SDSelec- that DNase I treatment of nuclei releases the wheat proteins
trophoresis in the second dimension. HMGa and contaminat- which we have called HMG proteins. Micrococcal nuclease
ing histone H1 are labeled in the figure. Calf thymus HMG also more readily attacks chromatin containing transcribed
proteins have secondary modifications and thus are separatedsequences than it attacks bulk DNA (Bloom and Anderson,
into multiple forms by isoelectric focusing (Nicolas and Good- 1978). Jackson etal. (1979) have developed a procedure which
uses micrococcal nuclease to obtainachromatinfraction
win, 1982). Similarly, wheat HMGbhas two forms-one
which migrates slowly in the first dimension and one which which contains HMG proteins inamounts stoichiometric with
migrates nearly as rapidly as HMGa. HMGc and d also have the histone fractions. When the procedure of Jackson et al.
(1979) is used on wheat chromatin, fractions similarly highly
two forms each when separated by this procedure.
Peptide Mapping-We have used the procedures of Cleve- enriched in the proteinswe call HMG proteins are obtained.
land et al. (1977) to compare the peptide maps of each of the Fig. 8 shows SDS gels of proteins of the wheat chromatin
wheat HMG proteins with the others, with each of the pig fractions prepared by the procedures of Jackson etal. (1979).
thymus HMG proteins, and with wheat H1 histones. Some of In lane 1 of this figure are the proteins of the micrococcal
the maps are shown in Fig. 6. When the maps of the wheat nuclease-resistant fraction. The only prominent proteins in
HMG proteins are compared, a few bands of common mobility this fraction are the histones. In lane 2 of this figure are the
can be demonstrated. However, there is no indication that proteins of the micrococcal nuclease-susceptible fraction. This
any of the lower-molecular-weight HMG proteins arederived fraction contains histones but italso contains very prominent
from any of the higher-molecular-weight ones or that exten- proteins which co-migrate with the wheat proteins we have
sive sequence homology exists in the wheat HMG proteins. called HMG proteins. This fraction also contains a number
Similarly, in comparison of wheat HMG maps with those of of higher-molecular-weight proteins which may be analogous
the pig thymus HMG proteins, no evidence of extensive to the proteins studied by Prior et al. (1983) or by Emerson
and Felsenfeld (1984).
homology can be found.
Wheat HMG Proteins
12012
1
2
3
+a
4-b
4 - C
3+
4-d
FIG. 8. Wheat HMG proteins co-isolate with micrococcal
nuclease-sensitive chromatin.Wheat embryo chromatin was separated into micrococcal nuclease-sensitive andmicrococcal nucleaseresistant fractions by the method of Jackson et al. (1979). The
proteins of each fraction werethen separated by SDS-polyacrylamide
gel electrophoresis. Lane 1 is the proteins from the micrococcal
nuclease-resistant fraction.The only prominent proteinsin this fraction are the histones. Histones H3 and H4 (labeled 3 and 4 ) appear
as single bands. Histones HI (labeled I ) and H2A and H2B (labeled
2) appear as multiple bands. Lane 2 is the proteins from the micrococcal nuclease-sensitive fraction. The histones are also present in
this fraction but they are accompaniedby prominent proteinswhich
co-migrate with the HMG proteins in lane 3. Bands corresponding in
mobility to HMGa, b, and d can be seen in this gel. Two-dimensional
gels (data not shown) indicate that HMGc is also in the nucleasesensitive fraction. In addition to the histones and HMG proteins, two
prominent groups of high-molecular-weight proteins
appear (arrowed)
with the micrococcal nuclease-sensitive chromatin.
Summary-Four proteins are extracted
from purifiedwheat
embryo chromatin with 0.35 M NaCl and are soluble in 2%
(w/v) trichloroacetic acid. Thus,theseproteinsmeetthe
original operational criteria for being “HMG” proteins. Because the true biological roles of mammalian HMG proteins
have not been established, we cannot as yet determineif the
plant HMG proteins are the functional equivalentsof mammalian HMG proteins. The plant HMG proteins do
have
several characteristics, however, which are consistent with
the idea that they do have the samerole in chromatin structure as mammalian HMG proteins. They are
rich in basic
amino acid residues-approximately 20 mol %. Three arerich
in acidic amino acid residues-approximately
23-29 mol %.
The fourth wheat HMG has only about
12 mol % asparate
asparagine and glutamate
glutamine residues. Thus, this
protein is uncharacteristicall; low in acidic residues.. The
”
+
+
weight of this observation must beconsidered in light of
similar acidic amino acid residue content of trout testis protein H6which, on the basisof amino acid sequence, is almost
certainly an HMG protein (Dixon, 1982). The wheat HMG
which is uncharacteristicallylow in acidic amino acidresidues,
HMGa, is the only wheat HMG
which meets the prolinecontent criterion suggested by Johns (1982) for calf thymus
HMGproteins.WheatHMGacontainsabout13
mol %
proline residues. The other three wheat HMG proteins contain 4-6 mol %. We were unable to assign any of the plant
HMG proteins to the
larger HMG class (corresponding
to calf
thymus HMGl and 2) or to the smaller HMG class (corresponding to calf thymus HMG14 and 17) either by simple
inspection of the amino acid contents or by using the procedures of Cornish-Bowden (1980). Wheat HMGc and HMGd
do, however, meet the“weak” requirement of Cornish-Bowden
forbeing “related” to calf thymus HMG14. Comparative
peptide mapping also failed to establish extensive homology
between any of the plant and vertebrate HMG proteins. The
results of the mapping studies did,
however, indicate that the
wheat HMG proteins
were not degradation products
of highermolecular-weight HMG proteins orof the H1histones.
The same HMG proteins which are extracted from wheat
embryo purified chromatincan be extracted from various
differentiated tissues of wheat with 0.5 N HClO, and these
proteins are associated with mononucleosomes separated on
sucrosegradients(datanotshown).Theobservationthat
wheat HMG proteins are associated with DNase I- and micrococcal nuclease-sensitive chromatin is consistent with the
idea that they are the functional equivalents of mammalian
HMG proteins. Complete sequence data on the plant HMG
proteins would be helpful in establishing such a correspondence. However, it is obvious that correspondence in function
cannot be reliably established until the truein vivo functions
of mammalian HMG proteins and plant HMG proteins are
established.
Acknowledgments-Thanks are due to Marcia Bates and Laura
Arwood for technical assistance.
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