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Transcript
volume 9 Number 121981
Nucleic Acids Research
The characterisation of 1 SF monomer nucleosomes from hen oviduct and the partial characterisation
of a third HMG14/17-like protein in such nucleosomes
Graham H. Goodwin, Carol A. Wright and Ernest W. Johns
Chester Beatty Research Institute, Institute of Cancer Research: Royal Cancer Hospital, Fulham
Road, London SW3 6JB, UK
Received 9 April 1981
ABSTRACT
Nucleosomes released from oviduct nuclei during brief micrococcal nuclease
digestions are enriched in transcribed sequences (Bloom, K.S. and Anderson,
J.N. (1978) Cell, 15, 141-150). Such nucleosomes released into this 1SF supernatant fraction are enriched in proteins HMG14, 17 and a third lower molecular
weight protein which we show in this paper to be related to HMG14 and 17. This
protein, which we call HMGY, runs as a doublet on polyacrylamide gels. A
similar doublet is present in smaller quantities in chicken erythrocyte nuclei.
Monomer nucleosomes in the 1SF supernatant have been separated by polyacrylamide
gel electrophoresis into two main bands. The slower moving band contains the
three HMG proteins HMG14, 17 and Y but lacks histone HI.
INTRODUCTION
Bloom and Anderson have shown that when oviduct nuclei are digested
briefly with micrococcal nuclease and then pelleted by centrifugation the
supernatant (the 1SF fraction) contains monomer nucleosomes which are enriched
5-6 fold in ovalbumen gene sequences [1]. In other words such nucleosomes are
soluble in buffers containing divalent metal ions and low concentrations of
salt (ionic strength~50 mM). These nucleosomes are devoid of H1 but contain
enriched quantities of the proteins HMG14 and 17, the enrichments varying
between 1.5 and 4 fold for these two proteins [2,3]. These results appear to
be in agreement with the findings of Levy and Dixon [4], Bakayev et al. [5],
Weisbrod and Weintraub [6] and Weisbrod et al. [7] that HMG proteins (H6, HMG14
and 17) are specifically associated with transcribed genes. During the course
of this work we noticed a third protein in the 1SF supernatant nucleosomes. This
protein ran ahead of HMG17 on acid gels (band X in ref.3) and was found to be 4fold enriched in the 1SF supernatant when compared to the histones. In this
paper we have carried out a more extensive characterization of this protein in
oviduct and erythrocytes and show that it is very similar to the proteins HMG14
and 17 that we have previously isolated [8]. We have called the protein HMGY
to avoid confusion with the HMGX described by Sterner et al. which is a de© IRL Press Limited. 1 Falconberg Court, London W 1 V 5FG, U.K.
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gradation product of HMG1 or 2 [9].
In our previous study [2] we were unable to fractionate the oviduct lSF
nucleosomes by polyacrylamide gel electrophoresis due to the large amount of
other material in the 1SF supernatant. This difficulty was overcome by first
partially purifying the monomer nucleosomes by gel-filtration. The properties
of the nucleosomes subsequently separated by polyacrylamide gel electrophoresis
is also the subject of this report.
EXPERIMENTAL
1. Isolation and characterization of the chicken erythrocyte and oviduct HMGY
Chicken erythrocyte nuclei were prepared, extracted with 5% perchloric
acid (PCA) and the total PCA-extracted protein was acetone precipitated and
fractionated on a CM-Sephadex column [8,10]. HMGY, plus some HMG17, and another
protein (probably HMG3, a degradation product of HMG1 [11] elute just after the
HMG17 peak (hatched area in Fig. 2 ) . This fraction was concentrated by dialysing versus 60% ethanol containing 10 mM HC1. The protein was then precipitated
by the addition of HC1 to 0.1M and six volumes of acetone. The dried protein
was redissolved in 10 mM sodium acetate pH 5.5 and loaded onto a 1 x 1.5 cm CMcellulose column equilibrated with 10 mM sodium acetate pH 5.5. The column was
eluted with a 20 ml gradient, 0-1M NaCl in the same buffer. HMGY elutes as
marked in Fig.3.
HMGY was prepared from the oviducts of laying hens by preparative gel
electrophoresis. Nuclei were prepared as described by Bloom and Anderson [1]
in the presence of 0.5 mM PMSF and extracted with 5% PCA. The protein in the
PCA extract (30 mg from six oviducts) was acetone precipitated and loaded onto
a 0.3 x 15 x 13 cm 20% polyacrylamide gel containing acetic acid and 2.5M urea
[12]. After electrophoresis the proteins were located by staining with 0.2%
Bromophenol blue in ethanol, acetic acid, water (50:5:45, v/v/v) for 10 minutes.
The HMGY band running ahead of HMG17 was cut out and equilibrated for 7 hours
with 0.1% SDS, 10 mM Tris-HCl (pH 7.5), changing the buffer four times. The
SDS-HMGY complex was then electro-eluted from the gel strip by placing the strip
into a dialysis bag containing the SDS-Tris buffer and immersing the bag in the
buffer in a box with two electrodes. A voltage gradient of 4V/cm was applied for
3 hours. The protein eluted into the buffer in the bag was concentrated by
dialysis versus 60% ethanol, 10 mM HC1 and precipitated with acetone-HCl as
described above.
2. Isolation and fractionation of lSF oviduct nucleosomes
All buffers contained 0.5 mM phenylmethyl sulphonyl fluoride and 5 mM sodium
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butyrate. The ISF supernatant fraction from the oviducts of laying hens was
prepared essentially as described by Bloom and Anderson [ 1 ] . The nuclei were
digested b r i e f l y with micrococcal nuclease (75 units/ml, 2 min at 37°C at a
nucleic acid concentration of 40 absorbance (260 nm) units per ml) in 50 mM
Tris-HCl (pH 7.4), 1 mM CaCl2. After digestion the nuclei were pelleted and
to the supernatant (1SF) was added EDTA (2 mM). The supernatant was concentrated by u l t r a f i l t r a t i o n and loaded onto a Biogel A5m gel f i l t r a t i o n column
(2.5 x 90 cm) equilibrated with 40 mM NaCl ,10 mM TEA-HC1, 2 mM EDTA (pH 7.6).
5 ml fractions were collected. The monomer nucleosome peak, eluting between
fractions 40 and 60, was collected, concentrated, and separated by electrophoresis on a 3 mm thick polyacrylamide 'DNP' slab gel [13] containing 40 mM
NaCl. At the end of the electrophoresis a vertical gel s t r i p was cut o f f and
stained with ethidium bromide to detect the nucleosomes.
3. Protein analysis of the ISF nucleosomes
To analyse the protein composition of the electrophoretically separated
nucleosomes they were either ( i ) extracted from the gel s t r i p with SDS,
purified by phenol extraction [ 2 ] , and analysed on 15% polyacrylamide-SDS slab
gels [14] or on acetic acid-2.5M urea 20% polyacrylamide slab gels [ 1 2 ] , or
( i i ) the proteins in the DNP gel were run into a second dimension electrophoretic
gel (SDS or acid-urea). In the SDS two-dimensional analysis a vertical s t r i p
( 1 cm wide) of the DNP gel was equilibrated for 2 hours in 2.3% SDS, 6.25 mM
Tris-HCl (pH 6.8), 10% glycerol, 5% mercaptoethanol and then placed on top of
the stacking gel of the SDS-slab gel using the system described by O'Farrell
and O'Farrell [15]. In the two-dimensional acid-urea system the DNP s t r i p was
equilibrated for 1 hour in 5% acetic acid, 5% mercaptoethanol, 8M urea and set
with 1% agarose, 0.375M potassium acetate (pH 4) on top of the acid-urea slab
gel system described by Spiker [ 1 6 ] . The separating gel contained 20% polyacrylamide rather than 15%. Protamine sulphate (200 pi of a 10 mg/ml solution
in the acid-urea sample solvent) was layered over the agarose. This displaces
the histones and HMG proteins from the DNA in the DNP s t r i p during the subsequent electrophoresis.
4. DNA analysis of ISF nucleosomes
A s t r i p of the DNP gel was incubated in 1% SDS, 0.18M T r i s , 0.18M boric
acid, 5 mM EDTA (pH 8.3) for 1 hour then in 1% SDS, 18 mM T r i s , 18 mM boric acid,
0.5 mM EDTA, 10 M urea for 1 hour at room temperature and then for 5 min at 85°
to denature the DNA [17].
The s t r i p was loaded onto 12.5% acrylamide slab gel
containing 0.1% SDS, 7M urea, 0.09M T r i s , 0.09M boric acid, 2.5 mM EDTA.
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Electrophoresis was carried out f o r 2 hours at 180V and the single-strand DNA
fragments observed by staining with ethidium bromide as described by Todd and
Garrard [ 1 7 ] .
RESULTS
I t can be seen from Fig. 1 that there is a band running ahead of HMG17
in the protein extracted from the unfractionated 1SF supernatant (Fig.ig) and
from monomer nucleosomes isolated from the supernatant by sucrose gradient
centrifugation ( F i g . i f ) (see also Fig. 1 of r e f . 3 ) . This protein is also seen
running i n about the same position in the PCA extract of oviduct nuclei ( F i g .
1e). As w i l l be seen below, this protein running ahead of HMG17, which we
have called HMGY, is in fact a doublet; t h i s is not f u l l y apparent on this
gel because the bands are somewhat diffuse. (The gel of Fig.1 contains 15%
b e d
Fig. 1
15% polyacrylamide acetic acid-urea gel electrophoresis of
(a) Whole histone standard
(b) Whole calf thymus HMG standard
(c) Chicken erythrocyte HMGY and HMG3(?) from the CM-Sephadex
chromatography of Fig. 2 (hatched area)
( d ) , (e) PCA-extracted protein from oviduct nuclei
(f) Proteins extracted from oviduct monomer nucleosomes purified
from the 1SF supernatant fraction by sucrose gradient
centrifugation [2]
(g) Proteins extracted from the oviduct 1SF supernatant
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polyacrylamide; later we found that 20% polyacrylamide gels give much sharper
bands). This protein is also present in chicken erythrocyte nuclei but in
smaller quantities and whilst fractionating erythrocyte proteins by CMSephadex (Fig.2) we found a protein fraction eluting just behind HMG17 which
contained a protein with the same mobility as protein HMGY (Fig.1c). A
partially purified preparation of the erythrocyte HMGY was obtained by a
further CM-cellulose chromatography of this fraction (Fig.3). Table 1 gives
the amino acid composition of the protein and shows that it resembles HM614
and 17 very closely. Fig. 4 shows polyacrylamide gel electrophoretic analyses
of the protein so isolated and is compared with the proteins extracted from
oviduct monomer nucleosomes. The chicken erythrocyte HMGY (which is still
contaminated with some HMG17) runs in very nearly the same position as the
oviduct HMGY on acid-urea gels (Fig.4A). However, a two-dimensional gel
electrophoresis, acid-urea followed by SDS (Fig.4B) showed the erythrocyte
protein splitting into a doublet and it was the minor, faster moving component
with comigrates with the oviduct HMGY in the SDS dimension. It is not certain
from this gel whether the oviduct HMGY has also split into a doublet in the
1.5-
1.0-
CVJ
0.5-
Fraction number
Fig.
2
CM-Sephadex (pH 8.8) chromatography of chicken erythrocyte HMG
proteins. HMGY elutes as shown by the hatched area. 11 ml fractions
collected.
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40
60
Fraction number
Fig.3
CM-celiulose chromatographic p u r i f i c a t i o n of the HMGY f r a c t i o n obtained
from the CM-Sephadex chromatography of Fig. 2. 0.3 ml fractions
collected.
SDS dimension since the HMGY spot is rather f a i n t . Examination of SDS gels
of the t o t a l oviduct nuclear PCA-extract does reveal a protein running in the
same position as the faster moving erythrocyte HMGY component and also a f a i n t e r
(oviduct) component running with the erythrocyte major protein ( F i g . 5 ) . I t
Table 1
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Cys
Met
He
Leu
Tyr
Phe
Lys
His
Arg
2766
Arnino acid composition [% moles) of chicken erythrocyte
HM614, HMG17 and HMGY, and oviduct HMGY
HMG14
HMG17
Erythrocyte
HMGY
Oviduct
HMGY
9.3
4.6
5.2
15.6
10.5
5.6
18.0
0.3
1.1
0.2
0.1
24.0
1.1
4.1
9.1
3.0
4.3
11.7
12.1
10.0
17.2
2.2
1.2
0.2
0.1
23.6
0.2
4.6
8.5
3.8
5.3
12.9
8.2
9.2
16.1
1.1
1.8
0.6
0.7
22.2
0.8
6.3
6.2
5.0
4.8
13.7
10.9
8.2
14.8
1.8
0.3
1.8
0.4
26.2
0.8
4.2
Nucleic Acids Research
B
- i
1417Y -
17 •
a
Fig. 4
< 14
* 17
b
Electrophoretic analysis of (a) proteins extracted from oviduct
monomer nucleosomes and (b) partially purified erythrocyte HMGY
from the CM-cellulose chromatography of Fig. 3. A is a 15% polyacrylamide acetic acid-urea gel. B is a two-dimensional analysis,
acetic acid-urea in the first and 15% polyacrylamide-SDS in the
second.
would therefore appear on the basis of these results that there are a pair of
very closely related HMG-like proteins in erythrocytes and oviduct but the
proportions of the two components of this doublet differ in the two tissues,
or rather in the nuclei from the two tissues. To check that the oviduct
protein was indeed the same or at least related to the erythrocyte protein,
oviduct HMGY was isolated from the nuclear PCA-extract by preparative gel
electrophoresis. The amino acid composition of the protein is given in Table 1
showing that it is very similar to the erythrocyte HMGY and to HMG14 and HMG17.
Also, the oviduct HMGY elutes together with HMG17 when the oviduct PCA extract
is fractionated by CM-Sephadex chromatography (data not shown). To check that
HMGY was not a degradation product, oviduct tissue was extracted directly with
PCA and the proteins analysed on a polyacrylamide gel (Fig. 6 ) . The HMGY
doublet is present, suggesting that the protein is not a degradation product,
but the proportions of two components are reversed when compared with oviduct
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Nucleic Acids Research
—
Fig. 5
SDS-gel electrophoresis of (a) erythrocyte HMGY
fraction (b) oviduct nuclei PCA-extracted protein.
I
nuclear HMGY. This suggests that during nuclear isolation some of the slower
component may be converted to the faster during nuclear isolation or that some
of i t is lost during the nuclear i s o l a t i o n . From the relative intensities of
these bands and the HMG17 band i t would appear that the l a t t e r could be happen-
Fig. 6
a b e d
2768
20% polyacrylamide acetic
acid-urea gel electrophoresis
of: ( a ) , (b), ( c ) , increasing
loadings of a PCA-extract of
oviduct tissue, (d) PCAextract of oviduct nuclei and
(e) c a l f thymus HMG standard.
Nucleic Acids Research
ing.
In our previous papers [2,3] we showed that the 1SF supernatant was enriched in HMG14, 17 and Y when quantified relative to histones. In order to
further characterise the nucleosomes in this supernatant fraction the monomer
nucleosomes were first purified by gel filtration and then analysed by polyacrylamide gel electrophoresis (Fig.7). The nucleosomes separate into two
main bands as we have found before for thymus and erythrocyte nucleosomes
soluble in 0.1M NaCl - 2 mM EDTA [2,3]. Analysis of the proteins associated
with electrophoretically separated nucleosomes by a second dimension electroSMI
SM2
= H1
ZHMG1.2
1417*
Fig. 7
>«•
Two-dimensional analysis of electrophoretically separated 1SF monomer
nucleosomes.
1SF nucleosomes were separated in the first-dimension by electrophoresis on DNP gels (right to left) as shown in the ethidium stained
gel and then dissociated and the proteins run into an SOS-slab gel
with markers:
(a) Total PCA-extract of chicken erythrocyte nuclei, (b) mixture of
calf HMG and whole histone. C H s stands for core histones.
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Nucleic Acids Research
phoresis into an SDS gel (Fig.7) or an acid-urea gel (Fig.8) reveals that the
faster moving nucleosome component SM1 (SM standing for salt-soluble monomer)
is the core particle containing DNA associated with just the four core histones
whilst the slower moving nucleosome SM2 contains HMG14, 17, HMGY, the four core
histones but no histone H1. (In this particular SDS gel HMG17 and HMGY have
co-migrated with core histones and cannot be seen). Fig. 8B, an acid-urea polyacrylamide gel of the proteins extracted from the SM2 nucleosome band, shows
HMGY s p l i t t i n g into a doublet. A fainter nucleosome band is also seen running
j u s t behind the SM2 nucleosome in the DNP gel (Fig. 7, Fig. 8A). This nucleosome may be associated with other non-histone proteins seen in the SDS gel of
Fig.7. There is no indication of highly acetylated histones in any of these
nucleosomes despite the inclusion of sodium butyrate in the buffers used in the
preparation.
The Procion navy stained gel ( F i g . 8A) was scanned and the peak areas of
HMG14, 17, Y and H4 in the SM2 band measured. From graphs of Procion navy uptake versus gm mols of pure protein loaded onto gels the molar ratios of the
B
SM2
SM1
f
JCHs
-14
-17
-Y
a b
Fig. 8
Analysis of electrophoretically separated 1SF monomers on acetic
acid-urea polyacrylamide gels.
A. Two dimensional analysis as in Fig.7, except proteins were run
into acetic acid-urea gels and stained with Procion navy [2].
B. (a) Whole histone standard
(b) Proteins extracted from SM2 band
Stained with Coomassie blue.
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proteins in SM2 were calculated. (Since a standard graph for HMGY was not
available it was assumed that it bound the same amount of Procion navy as
HMG17). The H4:HMG14:HMG17:HMGY ratios was found to be 2.0/0.9/0.5/0.5,
i.e., nearly two molecules of HMG per nucleosome. This may be an overestimate
since we are not sure that all the histone is displaced from the DNA by the
protamine sulphate, i.e. there could be preferential dissociation of HMG
proteins since they are the least strongly bound. Analysis of the DNA of the
electrophoretically separated nucleosomes (Fig.9) shows that the HMG-containing
nucleosomes (SM2) have DNA of somewhat longer lengths than that of the core
particle (SM1), as we have shown previously [2,3].
DISCUSSION
In addition to the well characterised HMG14 and 17 proteins [18] avian
nuclei contain a pair of very closely related proteins which we have called
HMGY. This doublet of proteins is soluble in perchloric acid, run together
on SDS and acid-urea gels, elute together from CM-Sephadex at pH 8.8 and CMcellulose pH 5.5 and are both present on nucleosomes. HMGY from erythrocytes
and oviduct have been partially characterised by amino acid analysis and this
has confirmed their similarity to HMG14 and HMG17. HMG14 and 17 are characterised by their high contents of basic (lysine) and acidic amino acids and by
SM2
Fig. 9
SMI
Two-dimensional analysis of the DNA of the electrophoretically separated 1SF nucleosomes. 1SF nucleosomes were
separated by electrophoresis on DNP gels in the first dimension
(left to right) and then run into a denaturing 12% polyacrylamide gel (top to b o t t o m ) .
2771
Nucleic Acids Research
their lack of aromatics, cysteine and methionine and low content of hydrophobic amino acids. HMG14 and 17 have fairly high contents of proline,
glycine and alanine. HMGY has all these properties. Except for being smaller,
HMGY is very similar to HMG17 in that it elutes just after the main peak of
HMG17 on the CM-Sephadex column and elutes with HMG17 on the CM-cellulose
column. HMGY does not appear to be a degradation product of HMG14 or HMG17
but this cannot be ruled out until the protein has been sequenced. It is of
interest to note that HMGY has a mobility on SDS polyacrylamide gels very close
to that of the trout protein H6 and also there is a pair of proteins in calf
thymus, HMG19A and 19B which are similar to, but not identical to the chicken
HMGY in having similar chromatographic and electrophoretic properties and
similar amino acid compositions [19]. Oviduct nuclei have possibly an order of
magnitude more HMGY than erythrocyte nuclei. Also the proportions of the two
components of the HMGY doublet are different in the nuclei of these two cells;
but this may simply reflect differential losses during the isolation of nuclei
since PCA extracts of oviduct cells appear to have a good deal more of the lower
mobility component as compared with the PCA-extract of nuclei.
The nucleosomes released into the 1SF supernatant fraction are composed of
mainly two types of nucleosomes, core particles and HMG-containing nucleosomes.
Other non-histone proteins are evident in the two-dimensional analysis of Fig.7
but it is not clear whether they are also nucleosome associated; they could
be derived from ribonucleoprotein particles or even subunits of oligomeric
enzymes etc. It is apparent though from our analysis that the SM2 particle
is composed primarily of histones plus HMG proteins and a smaller amount of
other uncharacterised high molecular weight proteins. Although the nucleosomes in the 1SF fraction have been shown to be enriched in ovalbumen
sequences we have no convincing evidence that the HMG-containing nucleosomes
are truly derived solely from active chromatin. In fact we have found from
some preliminary (unpublished) experiments that SM2 nucleosomes are no more
sensitive to DNAse I digestion than the SM1 nucleosomes which is not in accordance with the recent results of Weisbrod et al .[7], who have shown that DNAse
I sensitivity of expressed sequences in isolated nucleosomes is induced by
HMG14 or HMG17. This apparent lack of DNAse I sensitivity of the SM2 nucleosomes is not due to deacetylation of the histones since 5 mM sodium butyrate
was included in all the buffers from the initial tissue homogenization to the
final incubation with the DNAse I. It is worth recalling that Garel and Axel
[20] also found that oviduct monomer nucleosomes once isolated from oviduct
nuclei no longer exhibit the DNAse I sensitivity of the ovalbumen gene. This
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Nucleic Acids Research
suggests that rearrangement of HMG protein may be occurring. This is a very
real danger in view of the recent findings that all (or most) of the nucleosomes in the cell nucleus have potential binding sites for HMG proteins though
in vivo only about 10% are occupied by HMG14 and 17 (and Y) proteins [21, 22,
23]. Thus it is probable that there are low and high affinity binding sites
in the cell nucleus; the latter are on transcribed sequences and result in
DNAse I sensitivity when complexed with HMG proteins. That rearrangement
could be occurring comes from several other studies. In a previous report it
was shown that chicken erythrocyte nucleosomes enriched 10-15 fold in HMG14
and 17 could be isolated by salt fractionation and preparative gel electrophoresis [3]. The DNA of these nucleosomes hybridized to globin cDNA at the
same rate as total sheared chicken DNA averaging 550 base pairs in length.
After correcting for the molecular weight differences in the two samples
of DNA the enrichment of globin sequences was thus at most 2 fold. These
nucleosomes are also not DNAse I sensitive (R. Nicolas and G. Goodwin,
unpublished results). Also we have found that isolated satellite chromatin
contains HMG14 and 17 (albeit in smaller quantities) [24]. As a corollary
to this Levinger et al. [25] found that HMG14(17)-containing nucleosomes
have satellite DNA sequences in about the same proportion as total DNA.
Although there are reports of transcription of satellite sequences (see for
example [26] and references therein) it is probably true to say that the bulk
of the satellite chromatin is transcriptionally inert, and therefore one would
not expect HMG proteins to be bound to it if the HMGs are specifically associated with transcribed genes. From the results of this paper and others on
the topic it would seem that the structure of nucleosomes from expressed
sequences could be labile and that some randomization of HMG proteins between
active and inactive nucleosomes could be occurring during lengthy nucleosome
isolation procedures. Nevertheless, Albanese and Weintraub [27] have shown
that using a similar fractionation of erythrocyte nucleosomes on polyacrylamide gels that most of the globin genes are present in the SM2 region of
the gel whilst most of the non-expressed ovalbumen gene sequences are in the
faster moving core particles. Thus, the loss of DNAse I sensitivity of HMGnucleosomes may simply be due to a partial depletion of HMG proteins from
these nucleosomes; SM2 nucleosomes may have only one HMG molecule per nucleosome whereas two may be required for DNAse I sensitivity. Finally it is
possible that HMG proteins are not exclusively located on transcribed
sequences in vivo. Thus, for example, half of the HMG14/17/Y may be bound
to expressed sequences which are DNAse I sensitive and the other half may
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be distributed throughout the rest of the genome, accounting for their
presence in satellite chromatin.
This distribution could then be correlated
(inversely) with levels of DNA methylation.
Thus in transcriptionally active
chromatin where expressed sequences and flanking sequences are generally
undermethylated there would be a higher concentration of HMG proteins whilst
in satellite chromatin where the DNA is twice as methylated as main band
DNA [28] there would be less HMG protein as we have found [24].
ABBREVIATIONS
C.Hs
DNP
:
:
Core Histones
HMG
:
Deoxyribonucleoprotein
High Mobility Group
PCA
TEA
:
:
Triethanolamine
Perchloric Acid
ACKNOWLEDGEMENTS
The a u t h o r s t h a n k J . H a s t i n g s f o r t h e a m i n o a c i d a n a l y s e s .
Financial
support from t h e Medical Research Council i s g r a t e f u l l y acknowledged.
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