Download affinity chromatography of dna-binding proteins from human, murine

J. Cell Sci. 18, 41-65 (1975)
41
Printed in Great Britain
AFFINITY CHROMATOGRAPHY OF
DNA-BINDING PROTEINS FROM HUMAN,
MURINE AND MAN-MOUSE HYBRID
CELL LINES
E. JOST, R. LENNOX AND H. HARRIS
Sir William Diirw School of Pathology, University of Oxford,
Oxford OXi jRE, England
SUMMARY
About 60 proteins from human and murine cell lines were isolated by their ability to bind to
different preparations of DNA. In the intact cell, the majority of these proteins are to be found
in the cell nucleus. The electrophoretic mobilities of the DNA-binding proteins from human,
murine and man-mouse hybrid cell lines were compared in two-dimensional acrylamide gels.
Few, if any, species-specific differences were found. These observations suggest that the
structures of the vast majority of the proteins that interact with DNA are conserved through
evolution. A molecular basis is thus provided for the intracellular compatibility of hybrid cells
derived from different animal species.
INTRODUCTION
Some proteins bind to specific base sequences, whereas others show little preference
for specific base sequences, but bind to single-stranded or double-stranded DNA
(Alberts et al. 1968; von Hippel & McGhee, 1972).* There is evidence for both
regulatory (Paul & Gilmour, 1968; Gilmour & Paul, 1969; Teng, Teng & Allfrey,
1971; Rickwood et al. 1972; Louie & Dixon, 1973; Bradbury, Inglis & Matthews,
1974) and structural (Phillips, 1971; Kornberg, 1974) functions for the latter class of
proteins.
The double helical structure of DNA is common to all species, so severe restrictions
are placed on the variation in the structure of proteins that interact with it. The
primary structure of histones from widely different species is found to be highly conserved (DeLange, Smith, Fambrough& Bonner, 1968; DeLange, Fambrough, Smith
& Bonner, 1969; DeLange & Smith, 1971), and this may also be true for non-histone
proteins that interact with DNA. This might provide a molecular basis for the compatibility of the proteins from cells of different species when they are combined to
form viable interspecific hybrid cells.
Mixing experiments provide a test for heterogeneity amongst proteins from different
sources. Large numbers of cells from one species are mixed with small numbers of
• List of abbreviations: ss-DNA, single-stranded DNA; ds-DNA, double-stranded DNA;
ss-ct DNA, single-stranded DNA from calf thymus; ds-ct DNA, double-stranded DNA from
calf thymus; ss-m DNA, single-stranded DNA from mouse Ehrlich ascites tumour cells.
42
E. Jost, R. Lennox and H. Harris
radioactively labelled cells from the second species, and the pooled proteins are then
subjected to chromatography on appropriate DNA columns. The fraction of the protein
mixture bound to DNA is isolated and then subjected to electrophoresis. Differences
in the mobility of the proteins from the two sources can be detected by comparing the
staining pattern of the proteins with their radioactive content.
The electrophoretic properties of DNA-binding proteins from human and murine
cell lines and from man-mouse hybrids (Allderdice et al. 1973) were found to be
very similar in a two-dimensional gel system resembling that used to separate ribosomal proteins (Kaltschmidt & Wittmann, 1970). Most of the proteins that bound
to DNA were of nuclear origin and some had different affinities for ss-DNA and
ds-DNA. Only a small fraction binds specifically to DNA from the same species.
MATERIALS AND METHODS
Materials
Chemicals were obtained from the following suppliers: agarose and cellulose APX, Serva
(Heidelberg, Germany); calf thymus DNA type V and calf thymus histone type Ha, Sigma (St
Louis, Mo., U.S.A.); deoxyribonuclease I (E.C. 3.1.4.5) and ribonuclease (E.C. 2.7.7.16),
Worthington (Freehold, N.J., U.S.A.); neomycin, Upjohn Ltd. (Crawley, Sussex, G.B.);
kanamycin, Winthrop Lab. (Surbiton-upon-Thames, Surrey, G.B.); streptomycin, Glaxo
Ltd. (Greenford, Essex, G.B.); mycostatia, Squibb and Sons, Inc. (New York, U.S.A.);
foetal calf serum and calf serum, Flow Laboratories (Glasgow, G.B.); media and non-essential
amino acids, Bio-Cult (Glasgow, G.B.); NCS Tissue Solubilizer, Amereham/Searle (Arlington
Heights, 111., U.S.A.); protein hydrolysate-Ci4 (generally labelled) CFB25 (57 mCi/mAtom
carbon, 50 /tCi/ml) and L-[4,s-3H]leucine (53 Ci/mmol), The Radiochemical Centre, Amersham, Bucks, G.B.); L-amino acid mixture (3H(n)) (1 mCi/ml), NET-250, New England
Nuclear (Boston, U.S.A.).
Buffers and media
Extraction buffers: 20 M NaCl or 5-0 M NaCl, 20 mkl Tris, 1 mM EDTA, 1 mM /?-mercaptoethanol, 10 mM Mg2+, 2 mM Caa+, pH7'5- Dialysis buffer I: 600 mM NaCl, 20 mM Tris,
1 mM /?-mercaptoethanol, 10 mM Mgs+, 2 mM Ca1+, pH 75. Dialysis buffer II: 100 mM NaCl,
20 mM Tris, 1 mM EDTA, 1 mM /?-mercaptoethanol, P H 7 5 . Elution buffer: 20 mM Tris,
1 mM EDTA, i mM/?-mercaptoethanol, 10% (w/v) glycerol with varying amounts of NaCl added
as indicated. Polyethylene glycol-buffer (PEG): extraction buffer with 30 % (w/v) polyethylene
glycol 6000. Eagle's Minimal Essential Medium (MEM) was usually supplemented with 10 %
foetal calf serum and Dulbecco's modified Eagle's MEM with 20 % foetal calf serum. Medium
for growing HeLa cells in suspension was Eagle's MEM supplemented with 100 mM sodium
pyruvate, 25 g/1. sodium bicarbonate, non-essential amino acids and 5 % foetal calf serum. All
media contained 3 % (w/v) glutamine, 2'0 mg/1. neomycin, 30 mg/1. kanamycin, 200 mg/1.
streptomycin and 0-17 g/1. mycostatin. HAT medium consisted of the basic media to which
135 mg/1. hypoxanthine, 019 mg/1. aminopterin, and 3-87 mg/1. thymidine had been added.
Cells
Human HeLa and Daudi cells were grown as suspension cultures in MEM and Dulbecco's
modified MEM, respectively. The mouse lines Ao and A9HT were grown in MEM. Detailed
information about these lines is to be found in previous publications (Engel, McGee & Harris,
1969; Wiener, Klein & Harris, 1973). The man-mouse hybrid lines Pj, P3, P4, OR and Cl,
were derived by Sendai virus-mediated cell fusion (Harris & Watkins, 1965) and have been
described previously (Allderdice et al. 1973). All these cell lines were grown as monolayers in
20-ml flasks or in roller bottles in 120 ml of medium. The malignant mouse tumours were
Mammalian DNA-binding proteins
43
Ehrlich, SEWA and MSWBS. The properties and mode of propagation of these rumours have
been described (Klein, Bregula, Wiener & Harris, 1971). Cells were generally labelled with
either 14 C-protein hydrolysate (4 /tCi/ml) or 3 H-amino acid mixture (4 /tCi/ml) in 50-ml roller
bottles in M E M + 1 0 % foetal calf serum or with 200/iCi (4/iCi/ml) [3H]leucine in MEM
lacking leucine.
DNA-free
extracts
Extracts free from DNA were prepared as follows. Pellets of cells or nuclei were sonicated in
extraction buffer until nuclei were broken. Debris was removed by centrifugation at 1000 g
for 3 min. DNA was precipitated by addition of PEG-buffer to a final concentration of 10 % (w/v)
(Yamamoto et al. 1970). After 1 h at 4 °C, the precipitate was removed by centrifugation at
20000 g for 10 min. Co-precipitation of protein with DNA was usually less than 20 % of the
total soluble protein. The fractions were then dialysed against dialysis buffer I and treated with
DNAase (20 /tg/ml) at 10 °C for 20 min. This treatment removed between 96 and 99 % of the
DNA. Extracts were then dialysed against dialysis buffer II, and a precipitate usually containing
less than 10 % of the total protein was removed by centrifugation for 1 h at 36000 rev/min in a
SW 41 rotor. Electrophoresis on sodium dodecylsulphate (SDS) gels of the redissolved precipitate showed that histones were present among a variety of other proteins. It is not known
whether the non-histone proteins have an affinity for DNA. All steps were carried out in
the cold. Extracts were made 1 0 % (w/v) with respect to glycerol before being loaded on to
the DNA columns.
Preparation of DNA-agarose and DNA-cellulose
For ss-DNA-agarose the procedure described by Bendich & Bolton (1968) with the modifications used by Schaller and co-workers (1972) was followed. Fragmented DNA-agarose was
suspended in elution buffer, packed into a column and washed until no DNA could be detected
in the eluate by its absorption at 260 nm. With about 3-5 mg/ml bed volume, not less than 70 %
of the DNA input was usually retained on the column. Ds-ct DNA-cellulose was prepared by
the method of Litman (1968) with minor variations described elsewhere (Scherzinger, Litfin &
Jost, 1973): 60-70% of the DNA was bound to the cellulose (1-3 mg/ml bed volume).
Operation of the column
Columns were equilibrated with elution buffer. All proteins were passed through agarose or
cellulose columns (1 cm x 10 cm) free of DNA before passage through DNA-containing
columns. Less than 1 % of the protein input was adsorbed non-specificaJly to agarose and less
than 0-3 % to cellulose. Proteins were bound to DNA-containing columns (1 cm x 20 cm) by
passing DNA-free protein extracts through columns at flow rates of about 5 ml/h. Columns
were washed with approximately 4 column volumes of dialysis buffer II until O.D. 280 was
below 005/ml or radioactivity was at background level and then eluted with an elution buffer
containing 0-4 M or 2-0 M NaCl. Fractions of 01-0-2 column volumes were collected; their
absorbancy at 230 and 280 nm and/or their radioactive content were measured. If the material
bound to DNA and eluted from the column was re-chromatographed, it was found that 90 %
of it bound to DNA again. When the eluted proteins from the first and the second cycles of
chromatography were analysed by SDS acrylamide gel electrophoresis, they were found to be
identical. The ratio of the amount of protein bound to DNA, as measured by absorbancy at
280 run, to that of the amount of DNA bound to the column was always less than 1. The
columns were therefore unlikely to be overloaded with protein.
SDS acrylamide gel electrophoresis
Protein from column fractions (peak I and peak II) was dialysed against 10% acetic acid,
divided into units containing o-i O.D. 280 and lyophilized; 25 /tl of buffer (62-5 itM Tris
pH 6 8 , 3 % SDS, 5 % /?-mercaptoethanol, 10 % glycerol (v/v)) was added to these samples. The
apparatus of Studier (1973) and the stacking gel system of Laemmli (1970) were used. The
stacking gel was 5 ' 5 % and the separating gel was 1 2 % acrylamide. Lyophilized samples of
44
E. Jost, R. Lennox and H. Harris
proteins which had been heated to 90 °C for 2 min were layered on the 5-5 % acrylamide gel.
Electrophoresis was carried out at 50 V/slab gel (18 cm x 16 cm x 0-2 cm). Gels were fixed and
stained in 0 1 % Coomassie brilliant blue in 5 0 % methanol, 7 5 % acetic acid and destained in
5 0 % methanol, 7 5 % acetic acid and finally in 7-5 % acetic acid. Densitometer traces of the
stained gels were made directly from the slabs with a Joyce-Loebl microdensitometer.
Two-dimensional electrophoresis
Proteins were prepared for two-dimensional electrophoresis by two methods. Samples of
proteins containing about 5 and 1-5 O.D. 280 units respectively from the first and second peaks
of the eluant from DNA columns were dialysed against 1 0 % acetic acid, concentrated in a
rotory evaporator to a volume of 0-3 ml, and dialysed against several changes of a buffer containing 6 M urea, 2 mM EDTA, 50 mM boric acid, 4 % acrylamide, 0-3 % bisacrylamide adjusted
to pH 8 7 with 5 M KOH. Proteins were also prepared by lyophilizing in 10 % acetic acid. The
dry material was redissolved in 10 % acetic acid and dialysed against the above buffer.
The method described by Kaltschmidt & Wittmann (1970) for ribosomal proteins was used
for electrophoresis in two dimensions. This was possible because the isoelectric points of most
of the proteins binding to DNA were found to be between pH 4-5 and 8-7.
Other procedures
Nuclei were prepared in non-aqueous solvents using minor modifications of the technique of
Kirsch et al. (1970). Cells were lyophilized, resuspended in chilled glycerol and homogenized
in a Polytron homogenizer (15 s, repeated 10-15 times). The homogenate was layered over 1 ml
of 15 % a-chlorohydrin 85 % glycerol and spun in a Beckman SW 50.1 rotor for 30 min at
20000 rev/min at 4 °C. The supernatant was removed and layered over another 1 ml of
a-chlorohydrin-glycerol and centrifuged at 25000 rev/min for 2 h at 4 C C. The resulting pellet
contains the nuclei. The supernatant was respun at 30000 rev/min for 12 h at 4 °C. The supernatant from this spin constituted the cytoplasmic fraction.
Partially purified HeLa nuclei were prepared in aqueous solutions by swelling the cells in
o-oi M NaCl, 0-0015 M Mg 3+ , 0 0 1 M Tris p H 7 5 for 15 min, and then breaking the cells in a
Dounce homogenizer. When 90-95 % of the cells were broken to release intact nuclei, the
nuclei were pelleted by a low speed spin at 1000 g for 5 min. Ehrlich nuclei were prepared in a
similar way. They were centrifuged through 2-2 M sucrose at 53000 g for 60 min and then
purified by being layered in 1 volume of 0 3 3 M sucrose over 2 volumes of 0 5 8 M sucrose and
spun at 1100 g for 15 min. DNA from Ehrlich cells was prepared by the method of Marmur
(1961). Metaphase spreads were produced by the air-drying technique of Rothfels & Siminovitch (1958). They were stained by Giemsa or by the Giemsa-C-banding technique (Dev,
Miller, Allderdice & Miller, 1972).
Linear sucrose gradients (25-5 %, w/v) were prepared in 100 mM NaCl elution buffer pH 7-5.
They were spun in a S W 5 0 . 1 rotor for the stated time in a Spinco L2-65 ultracentrifuge.
Fractions (0-2 ml) were collected, their optical density at 260 nm recorded and their radioactive
content measured in a toluene-based scintillant. DNA and protein were reassociated by stepwise removal of salt; final dialysis was done in 100 mM NaCl elution buffer. The radioactivity
in aqueous samples was measured in Bray's scintillator; the radioactivity in gels was measured
by cutting out stained spots from the gel, drying them and then solubilizing them in o- i-o-15 ml
hydrogen peroxide; 0 7 5 ml of NCS solubilizer was added and the samples were then counted
in a toluene-based scintillant.
Protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951) with
bovine serum albumin as a standard. An O . D . 280 of o-i was equivalent to 131 /tg of protein
from peak I and to 170 /tg of protein from peak II.
Mammalian DNA-binding proteins
45
RESULTS
Mammalian DNA-binding proteins from whole cells and nuclei
Proteins were extracted from radioactively labelled mouse A9 cells by treatment
with high salt and applied to a column of ss-ct DNA-agarose. After washing, a fraction
of the proteins remained bound to the column. This fraction was eluted in two steps
with 0-4 M NaCl and 2-0 M NaCl elution buffer (Fig. 1 A). Measurement of the O.D.
at 280 nm indicated that 11 % of the protein input was eluted with 0-4 M NaCl
(peak I) and 2-0% with 2-0 M NaCl (peak II). Radioactivity measurements showed
that 12 and 4% of the input protein was eluted in peaks I and II, respectively.
Nuclei were isolated from cells by a variety of techniques and the DNA-binding
proteins in them were compared with those from whole cells. In the experiment shown
30
40
Fraction no.
50
Fig. 1. Chromatography on ss-ct DNA-agarose of proteins from extracts of whole
cells and nuclei. A, 1 x 108 mouse Ao cells were grown in MEM with 10% FCS and
labelled with 200 /iCi (2 /tCi/ml) 3 H-amino acid mixture for 3 days. They were
then mixed with 15 x io e unlabelled cells and the proteins were prepared and chromatographed as described in Materials and methods. 3'S-ml fractions were collected
and pooled as indicated for electrophoresis. 50 /tl of each fraction was counted for
radioactivity in Bray's scintillator. Protein input was 75 O.D. 280 units. #
0,
O.D. 280 as percentage of input; O
O, ' H radioactivity. B, proteins of 4 x i o '
HeLa cells were prepared and chromatographed as described in Materials and
methods except that 5 M NaCl extraction buffer was used. Protein input was 250 O.D.
280 units. Protein input from aqueous HeLa nuclei was 60 O.D. 280 units. A—-—A,
HeLa cells; A
A, HeLa nuclei.
46
E. Jost, R. Lennox and H. Harris
in Fig. IB, proteins from HeLa nuclei isolated in aqueous solvents were bound to
ss-ct DNA-agarose and subsequently eluted with high salt. Measurements of the O.D.
at 280 nm indicated that in this experiment 13 and 12 % of the nuclear proteins eluted
in peaks I and II, respectively. This compares with the figures 10 and 2-2 % in peaks I
and II when proteins from whole cell extracts were applied to the column. Ten and
14% were retained in peaks I and II when nuclear proteins were derived from Ehrlich
cells by a slightly different procedure.
HeLa and Ehrlich nuclei were also isolated using non-aqueous solvents. Binding
was 10% (peak I) and 15% (peak II) in extracts from HeLa nuclei and 14 and 13%
in extracts from Ehrlich nuclei. When proteins from Ehrlich cytoplasm derived by the
non-aqueous separation procedure were analysed, less than 1 % of them were found to
bind in peak I and less than 0-3% in peak II in ss-ct DNA-agarose.
Protein distribution in SDS-acrylamide gels
The patterns of DNA-binding proteins obtained from cells and nuclei prepared in
aqueous and non-aqueous solvents were compared. About 30 proteins could be
resolved by SDS acrylamide electrophoresis in the pooled fraction from whole HeLa
cells eluting in peak I (Fig. 5 A). Similar patterns were obtained from the human line
Daudi and from the mouse lines A9, A9HT and the mouse tumours Ehrlich, SEWA
and MSWBS. The distribution of proteins derived from isolated HeLa nuclei prepared in aqueous solvents was compared with that of proteins from whole cells (Fig.
5 A, B). The two patterns were found to be approximately the same except that bands
no. 11, 12, 13 and 14 were reduced in quantity in the nuclear preparation.
The proteins from HeLa cells eluting in 2-0 M NaCl from ss-ct DNA-agarose
could also be separated into about 30 bands in an SDS acrylamide gel (Fig. 5 c, D).
Some of the more heavily staining bands (e.g. no. 20, 21, 27, 28 and 29) were histones
as judged by co-electrophoresis with calf thymus histones. The histones dissociate from
chromatin at above 0-4 M NaCl(Ohlenbusch, Olivera, Tuan & Davidson, 1967; Tuan
6 Bonner, 1969). This pattern is similar to that of the human Daudi line and also to
those of the mouse lines A9, A9HT, Ehrlich, SEWA and MSWBS. When this pattern
was compared with that of the DNA-binding proteins derived from HeLa nuclei prepared in aqueous solvents it was found that the patterns were essentially the same.
However, bands no. 19-23 and 27-29 were more heavily stained in the nuclear
preparation.
When protein patterns derived from nuclei prepared in aqueous solvents were
compared with those from nuclei prepared in non-aqueous solvents, it was found that
aqueous isolation of the nuclei did not result in loss of DNA-binding proteins. These
results indicate that the DNA-binding proteins are present in the nucleus of the intact
cell and that few were lost during the isolation procedure. Nuclear extracts from
Ehrlich cells and HeLa cells contain a larger amount of histones that bind to DNA
than do the corresponding whole-cell extracts. This suggests that the cytoplasm may
contain the enzymes that degrade histones.
Mammalian DNA-binding proteins
47
Fractionation of mouse proteins on single-stranded and double-stranded DNA from calf
thymus and single-stranded DNA from Ehrlich cells
Extracts from the mouse Ehrlich and A9 cells were bound to single-stranded and
double-stranded calf thymus DNA and single-stranded mouse DNA. Extracts of the
Ehrlich cells were first applied to ss-ct DNA attached to agarose, and the unabsorbed
material was applied directly to a column which contained ds-ct DNA attached to
cellulose. The two columns were mounted in series. The columns were then detached
and the proteins absorbing to them eluted separately with salt. On average, 8 and
i-6% of the protein eluted from the ss-ct DNA in peaks I and II respectively;
0-3% eluted from the ds-ct DNA-cellulose with 2-0 M NaCl (Table 1).
Table 1. Binding of proteins prepared from mouse cells to single-stranded and doublestranded calf thymus DNA and to single-stranded mouse DNA
Recovery as a percentage of the input
DNA
Calf thymus
ss-DNA
ds-DNA
Mouse
ss-DNA
Calf thymus
ds-DNA
ss-DNA
Eluted in:
Mean
S.D.
No. of expts
0 4 M NaCl
2-0 M NaCl
2-0 M NaCl
8
i-6
03
3
07
26
22
o-i
13
2-0 M NaCl
0-5-1-0
0 4 M NaCl
2 0 M NaCl
2 0 M NaCl
56
I-I
2-4
3
2-5
o-i
i-o
6
5
6
On reversing the sequence of chromatography by using ds-ct DNA in the first
column and ss-ct DNA in the second column, 5-6 and I - I % of the protein input
eluted from ds-ct DNA as peak I and II and 2-4% from the column containing ss-ct
DNA. The proteins from A9 cells that did not bind to ss-ct DNA and therefore
eluted in the void column were also applied to a ss-m DNA column; 0-5-1-0% of the
protein eluted with 2-0 M salt. If Ehrlich nuclei prepared by the aqueous technique
were used as starting material instead of whole cells, then 1-5-2-0 % of the total protein
eluted from the last column containing mouse ss-DNA.
Sucrose density gradient analysts
DNA-protein interactions in the absence of a matrix immobilizing the DNA were
investigated by sucrose density centrifugation. Radioactively labelled mouse A9 proteins eluted from ss-ct DNA-agarose with 0-4 and 2-0 M salt were mixed with either
ss-ct DNA or ds-ct DNA and sedimented in sucrose gradients (25-5 %, w/v) (Fig. 2).
The extent of binding was determined by the amount of labelled protein that cosedimented with the DNA. When proteins from peak I or peak II eluted from a
ss-ct DNA column were mixed with ss-ct DNA and the salt concentration reduced
48
E. Jost, R. Lennox and H. Harris
stepwise, about 90% of the protein from each peak co-sedimented with the DNA
(Fig. 2 A and c). However, when proteins in peak I or peak II were mixed with ds-ct
DNA, part of the material did not sediment with the DNA (Fig. 2B and D). This
shows that ss-ct DNA has a higher binding capacity than ds-ct DNA for these proteins. The mouse proteins that eluted from ss-m DNA agarose were also reassociated with ss-ct DNA and studied in sucrose gradients. It was found that about 50% of
the protein label co-sedimented with the DNA.
A
02
0-1
V
I \
Top -
B
A
0-2
0-1
<\1
-
Top
gfy
I
0
10
20
0
Fraction number
Fig. 2. Analysis of complex formation between DNA and proteins by sucrose density
gradient centrifugation. Labelled protein fractions from mouse A9 cells isolated on
ss-ct DNA-agarose were mixed with single-stranded or double-stranded calf thymus
DNA and dialysed stepwise against 0-3 M NaCl, 0-15 M NaCl and o-i M NaCl elution
buffer, to allow complex formation. Samples were layered on top of a 25 % to 5 %
sucrose gradient (w/v) in o-i M NaCl elution buffer and centrifuged in a SW 50.1
rotor. # — 0 , O.D. 260; O—O, radioactivity.
A, 50 fd (2 mg/ml) of heat-denatured and sheared calf thymus DNA were mixed
with 100 fil (18 fig) of A9 protein from peak I eluted from ss-ct DNA and were spun
at 45000 rev/min for 4-5 h.
B, 50 fi\ (2 mg/ml) of native and sheared calf thymus DNA were mixed with 200 fi\
(36 fig) of A9 protein from peak I eluted from ss-ct DNA and spun at 45000 rev/min
for 3 5 h.
C, 200 fil (4 mg/ml) of heat-denatured and sheared calf thymus DNA were mixed with
100 fi\ (54 fig) of A9 protein from peak II eluted from ss-ct DNA and spun at 46000
rev/min for 4 h.
D, 200 fi\ (2 mg/ml) of native and sheared calf thymus DNA were mixed with 100 fil
(80 fig) of A9 protein from peak II eluted from ss-ct DNA and spun at 45000 rev/min
for 4 h.
Electrophoresis in SDS acrylamide gels and 2-D acrylamide gels
The proteins eluting from ss-ct DNA, ds-ct DNA and ss-m DNA columns were
compared electrophoretically. The Ehrlich cell extracts of peak I from ss-ct DNA and
ds-ct DNA show a great similarity (Fig. 6 A and B). Only about 7 out of 30 proteins
Mammalian DNA-binding proteins
49
seem to have different affinities for the two types of DNA. Proteins no. 3, 4, 7 and
11.1 were preferentially bound by ss-ct DNA and no. 6.1, 14.1 and 15.1 seem to have
higher affinity for ds-DNA. Amongst the proteins eluted from DNA columns with
2-0 M salt, some components were also preferentially retained on ss-DNA or on dsDNA (Fig. 6c and D): these were no. 2, 7, 15, 17, 18, 19, 25.2 and no. 7.1, 24, 25.1
respectively. The proteins not retained on ss-ct DNA were chromatographed on
ds-ct DNA and vice versa, eluted in one step with 2-0 M salt and analysed in SDS
acrylamide gels (Fig. 6E and F). Only one major and a few minor bands were found in
the eluant from the ds-DNA column, whereas about 20 bands were found in that from
the ss-DNA column. The mouse proteins that bind specifically to mouse DNA were
obtained by passing Ehrlich or A9 extracts that did not bind to ss-ct DNA through an
agarose column containing ss-m DNA, and then eluting with 2-0 M salt. There were
between 10 and 15 proteins in the eluate (Fig. 6G).
Mixtures of proteins obtained by elution of the various columns were analysed to a
high resolution by 2-D acrylamide gel electrophoresis. Extracts made by mixing
2xio° unlabelled Ehrlich cells with 2x10® labelled A9 cells were applied to an
ss-ct DNA column and eluted as peak I and peak II. Peak I could be resolved by
staining the electropherogram into about 30 heavily stained spots and about 40-50
additional spots detectable when the gel was overloaded. (For purposes of identification spots in peak I are given numbers A1-A41.) Peaks I eluted from ss-ct DNA
(Fig. 7 A) and ds-ct DNA (Fig. 8 A) show an identical pattern for the heavily stained
spots (no. A5, A12, A12.1, A12.2, A12.3, A13, A14, A15, A16, A16.1, A17, A17.1,
A18, A19, A20, A39, A40). Spots no. A30, A32, A32.1, A33, A33.1, A37, A38, which
include proteins moving to the anode, were more prominent in eluates from
ss-DNA, and spots no. A5.1, A7.3 and A7.5 were more prominent in those from
ds-DNA.
The proteins present in peak II could be resolved into about 30 spots (Figs. 7B and
8 B). The major proteins are given the numbers Bi and B27.1. This peak includes the
histones, since when calf thymus histones (Sigma II a fraction) were treated in the
same way, Fig. 8D, they had the same RF as the proteins designated B6.1, B9, B12.3,
B13, B14 and B16. The different histones were not present in the same amount.
Differences in affinity for ss-DNA were observed in the group of proteins in spots no.
B19 and B26.
Proteins not retained by ss-ct DNA were chromatographed on ds-ct DNA and vice
versa, eluted in one step with 2-0 M salt and analysed in 2-D acrylamide gels. The
eluate from ds-ct DNA contained 2 major and 9 minor proteins, whereas that from
ss-ct DNA contained about 20 (Figs. 7 c and 8 c). Most of the proteins are at positions
corresponding to those of proteins in the extracts eluted from the first column.
Differences between species
Human (HeLa or Daudi) and mouse (A9) proteins that bound to calf thymus DNA
were compared. Man-mouse hybrid lines containing different numbers of human
chromosomes were also examined. Clones P4, P3, Pi, CI9 and OR contained, on
average, 2, 10, 12, 14 and 22 human chromosomes respectively in addition to the
50
E.Jost, R. Lennox and H. Harris
mouse chromosomal set. Clone OR had a high number of cells with two mouse
chromosome sets. The chromosome analyses had been done before the chemical
studies. Cell extracts were applied to ss-ct DNA-agarose; the peak I and peak II
fractions were obtained and subjected to electrophoresis on SDS and 2-D acrylamide
gels. Fig. 3A-D'shows the densitometer traces made from the SDS acrylamide gels of
the proteins in peak I. The traces for man, mouse and hybrid cell extracts are very
90
70
40
20
10
Mol.
Fig. 3. Densitometer tracings of Coomassie blue-stained SDS acrylamide slab gels of
DNA-binding proteins of human, murine and man-mouse hybrid cell lines. Proteins
are from the pooled peak I fractions eluted from ss-ct DNA-agarose by 0-4 M NaCl.
o-i O.D. 280 units of protein from, each line were loaded on to the gels. Peaks are
numbered for easier identification. The approximate molecular weights of the polypeptides are given as estimates from standard runs on comparable gels. The origin is
at the left, the front at the right, A, proteins from mouse Ao cells; B, proteins from
human Daudi cells; c, proteins from the man-mouse hybrid line P4 with an average
of 2 human chromosomes per cell; D, proteins from the man-mouse hybrid line P i with
an average of 12 human chromosomes per cell.
similar. Densitometer traces of SDS acrylamide gels of the proteins in peak II are
given in Fig. 4A-D. They, too, were similar. These proteins were further resolved in
the two-dimensional gel system which separates proteins according to charge and
molecular weight. Bulk quantities of human cells were mixed with trace quantities of
labelled mouse cells and vice versa. In some experiments double labelling techniques
were used; for example, 3H-labelled hybrid clones were mixed with 14C-labelled
parental A9 cells. These techniques provide a very sensitive method for detecting
differences in the RF of individual spots in the 2-D gels. The reference pattern
Mammalian DNA-binding proteins
51
from which the numbers for the individual spots listed in Tables 2 and 3 can be
derived is given for mouse extracts (Fig. 7 A, B). The pattern derived from human
cells and from the hybrid cell lines proved to be essentially similar, so that the same
numbers could be given to the spots. The results of the labelling experiments are
shown in Tables 2 and 3. For example, in 31 of the densely stained spots obtained
from extracts of unlabelled HeLa cells mixed with traces of labelled A9 cells, eluted
in peak I from ss-ct DNA, label derived from the mouse cells could be detected in the
2.
B
D
90
70
40
20
Mol. wtxiO' 3
10
Fig. 4. Densitometer tracings of Coomassie blue-stained SDS acrylamide slab gels of
DNA-binding proteins of human, murine and man-mouse hybrid cell lines. Proteins
are from the pooled peak II fractions eluted from ss-ct DNA-agarose by 2-0 M NaCl.
Protein loading was o-i O.D. 280 units, A, proteins from mouse A9 cells; B, proteins
from human Daudi cells; C, proteins from the man—mouse hybrid line P4; D, proteins
from the man-mouse hybrid line Pi.
stained area of the spot (Table 2, column A). Similarly, in 29 spots examined, the
normalized ratios of 3H label derived from clone P4 to that of 14C label derived from
the parental A9 cells were unity (ra + zcr = 0-99 ± 0-42). This indicates that no protein
was present in only one of the two cell types mixed together. This type of experiment
was repeated with the proteins in peak II eluted from ss-ct DNA. Again few, if any,
differences between human, mouse and hybrid cells could be found (Table 3). The
major histones eluted in peak II were found in similar positions in the gel for the
different cell types, as expected.
4-2
52
E.jfost, R. Lennox and H. Harris
Table 2. Distribution of radioactivity in major DNA-binding proteins
of human, murine and man-mouse hybrid cells
Percentage of radioactiv ity (A-C) or normalized ratios of 3 H/ 14 C (D-F)
in stained spots
Spot
no.*
A
B
C
D
E
F
1-28
1-25
087
2-14
1-52
078
063
1-13
099
O73
—
i-oo
1-47
i-o6
i-86
0-91
1049
O79
I-I2
078
—
098
1-08
O-8 3
0-69
1-46
1-16
i-39
1-18
i
067
2
0-58
3
4
5
239
064
6
7
10-44
—
0-36
0-44
—
028
IO
O-2O
050
0-36
II
O-78
2-77
3-34
i-43
0-84
0-32
027
O-93
—
—
5-14
2-38
i'54
072
0-94
089
I-2O
O79
O79
098
077
i-oo
0-77
12
13
14
15
16
310
172
17
065
18
2-37
19
—
—
22
1-24
1-49
0-64
8-78
226
0-58
0-41
491
146
—
—
2-44
2-53
0-19
0-27
279
0-62
1-38
079
—
1-29
065
1-47
o-88
5-17
I-IO
—
208
4-02
1-09
313
O99
176
1-05
—
24
25
28
29
279
178
3°
1 89
32
359
32-1
33
3-06
3'57
290
050
—
—
35
36
161
1-06
14-01
167
119
3-52
o-8o
o-57
o-35
37
38
39
2-52
236
030
070
087
098
i-oo
40
237
0-70
082
118
41
399
030
098
174
7-21
7-26
204
o-8o
0-87
0-58
017
—
—
8
1-
55
i-oo
0-91
1 26
077
—
1-06
096
i-39
—
—
—
0-92
085
o-6o
I-OI
1-25
I-OI
1-02
o-66
1-17
i'37
1-25
1-07
0-98
071
0-51
073
113
—
o-86
—
0-58
o-6o
O75
096
098
083
—
084
095
0-70
1-08
0-64
o-53
1-54
o-8o
088
(A) 3 x 10' HeLa cells mixed with 1 x io A9 cells labelled with 300 /tCi (6 /tCi/ml) of
PHjleucine (53 Ci/mmol) for 2 days. Total radioactivity in stained spots: 40606 dpm. Counting
efficiency 28 %. Label at starting position 15 %. 70 % of the label was in 31 spots and 30 % in
35 spots not listed.
(B) 2-6 X io 9 Daudi cells mixed with 1 x io 8 A9 cells labelled with 200 /iCi 14 C-amino acid
hydrolysate (4/tCi/ml). Total radioactivity in stained spots: 37060 dpm. Counting efficiency
60%. 6 0 % of the label was in 28 spots. Total number of spots visible was 88.
(C) 2Xio*A9 cells mixed with 1-5 x 10s Daudi cells labelled with 400 ftCi (4/iCi/ml)
3
H-amino acids mixture. Total radioactivity in stained spots: 22000 dpm. 57% of the label was
in 27 spots. Total number of spots visible was 69.
(D) 1 x 10' P4 cells were mixed with 1 x io 8 P4 cells labelled with 400/iCi (4/tCi/ml) of
'H-amino acid mixture and 1 x 10' A9 cells labelled with 200 /tCi (4 /tCi/ml) of 14 C-amino acid
hydrolysate. The total 3 H radioactivity in stained spots was 49100 dpm. Counting efficiency
28 %. The total 14C radioactivity in the stained spots was 17 202 dpm. Counting efficiency, 60 %.
73 % of 3 H label and 72 % of the 14C label were in the 30 spots recorded. The total no. of spots
visible was 56.
Mammalian DNA-binding proteins
53
Table 3. Distribution of radioactivity in major DNA-binding proteins
of human, murine and man-mouse hybrid cells
Percentage of radioactivity (A-C) or normalized ratios of 3 H/ 14 C (D-F)
in stained spots
Spot
no.*
f
A
B
C
D
4
S
030
0-40
o-6i
i-88
1-12
I 04
6
100
022
o-75
—
—
7
8
9
390
032
0-20
2480
0-74
0-48
58-7O
O58
10
11
12
—
•
46-1
074
F
—
i-oo
0-74
o-54
0-87
0-65
0-98
118
1-22
099
070
I'OO
o-88
091
090
1-02
I'll
1-30
1-26
o-86
074
070
TOO
113
085
i-45
—
—
191
i-6o
o-53
5-84
4-85
17-03
20-53
047
141
—
2-37
i-88
o-39
—
o-43
o-88
0-78
2'79
4-50
482
Il6
093
093
081
1 07
076
0-70
342
—
2-26
069
064
030
1-28
o-8o
0-30
4'34
13
22-41
»4
965
t5
100
19
119
21
i-43
22
089
23
092
25
26
o-6o
040
—
0'75
i-39
0-74
E
I-l8
1-09
1-48
1-44
089
—
—
o-8o
(A) Experimental conditions as for column A in Table 2. Total radioactivity in stained spots:
41 358 dpm. 75 % of the label was in 18 spots. The total number of spots was 48.
(B) Experimental conditions as for column B in Table 2. Total radioactivity in stained spots:
17500 dpm. 93 % of the label was in 16 spots. The total number of spots was 18.
(C) Experimental conditions as for column C in Table 2. The total radioactivity in stained
spots was 10500 dpm. 95 % of the label was in 13 spots.
(D) Experimental conditions as for column D in Table 2. The total radioactivity in stained
spots was 24200 dpm 3 H and 8748 dpm 14 C. 78 % of the 3 H label and 80 % of the 14C label was
in 18 spots. The total number of spots was 30.
(E) Experimental conditions as for column E in Table 2. The total radioactivity in stained
spots was 29180 dpm a H and 18928 dpm 14 C. 76 % of the 3 H label and 74 % of the 14C label
was in 18 spots. The total no. of spots was 30.
(F) Experimental conditions as for column F in Table 2. The total radioactivity in stained
spots was 19650 dpm 3 H and 6904 dpm l 4 C. 67 % of the 3 H label and 66 % of the 14C label was
in 13 spots. The total number of spots was 32.
• For key see Fig. 70.
Table 2 (cont.)
(E) 1 x io f P3 cells were mixed with 1 x 108 P3 cells labelled with 475 fiCi (10/iCi/ml) of
3
[ H]leucine and 1 x io 8 A9 cells labelled with 200 /tCi (4/iCi/ml) 14 C-amino acid hydrolysate.
The total 3 H radioactivity in stained spots was 49095 dpm. Counting efficiency 28 %. The total
14
C radioactivity in the stained spots was 35000 dpm. Counting efficiency 6 0 % . 7 4 % of the
3
H label and 76 % of the 14C label was in the 28 spots recorded. Total number of spots visible
was 49.
(F) 1 x 10' Pi cells were mixed with 1 x io 9 Pi cells labelled with 200 /iCi (5 /*Ci/ml)
3
H-amino acid mixture and 1 x io 8 A9 cells labelled with 200 /tCi (4 /tCi/ml) 14 C-amino acid
hydrolysate. The total *H radioactivity in stained spots was 32450. Counting efficiency 28 %.
The total 14C radioactivity in the stained spots was 13500 dpm. Counting efficiency 60%.
83 % of the 3 H label and 86 % of the 14C label was in 29 spots. The total number of spots
visible was 52.
• For key see Fig. 7 A.
54
E. Jost, R. Lennox and H. Harris
DISCUSSION
Binding capacity of DNA for cellular proteins
The proteins in chromatin are usually isolated as an acid-soluble fraction containing
histones (Bonner et al. 1968), and the residue is generally referred to as non-histone
chromatin protein (Marushige, Brutlag & Bonner, 1968). The non-histone fraction is
very heterogeneous (Teng et al. 1971; Marushige, Brutlag & Bonnet, 1968; Elgin &
Bonner, 1970, 1972; Seale & Aronson, 1973; McGillivray & Rickwood, 1974). Teng
and co-workers (Teng et al. 1971) found that 12% of the nuclear proteins could be
separated from the rest by phenol extraction. This 12% contained the non-histone
proteins. Between 3 and 8% of soluble cellular protein has been found by various
authors (Salas & Green, 1971; van der Vliet & Levine, 1973) to bind to DNA attached
to columns. The present work shows that, on average, about 10% of the soluble
cellular proteins and 25 % of the nuclear proteins bind to DNA, as measured by
absorbancy at 280 nm. The amounts of non-histone protein isolated from cells by
various authors using different extraction procedures or by the affinity of these proteins for DNA-containing columns are therefore quite similar. Recovery of histones
by their affinity for DNA is, however, lower than expected: 40 % of the nuclear protein fraction is acid-soluble and is therefore denned as histone (Allfrey et al. 1973),
but only 15 % of the nuclear proteins and about 2 % of the total cellular proteins elute
from DNA-containing columns in the histone-containing peak II. This discrepancy
may be due partially to the low absorbancy of histones at O.D. 280 nm and to differences in the isolation procedures.
Many factors affect the amount of protein retained on DNA columns. (1) Between
1 and 4% of the DNA is not removed from cells by polyethylene glycol treatment
and DNAase degradation, so that some proteins may bind to this DNA at low salt and
be removed as an aggregate before application of the proteins to the column. (2) Depending on pH and salt concentration, histones tend to aggregate. SDS gel electrophoresis of these aggregates and of DNA-containing aggregates removed at the same
time showed that histones were present together with other proteins. (3) Histones are
degraded enzymically (Bartley & Chalkley, 1970; Kleiman & Huang, 1972) in the
absence of inhibitors. This degradation seems to be a major reason for the low yield
of histone in the cellular extracts studied in the present experiments. When the
extract in peak II is derived from isolated nuclei, 5-6 times more histone is obtained,
indicating that the degradative enzymes are probably localized in the cytoplasm. (4)
The protein/DNA ratio in chromosomes is not greater than 2 (van den Broek, Nooden,
Sevall & Bonner, 1973; Bhorjee & Pederson, 1972). In our experiments the ratio of
protein applied to the DNA on the column was less than 1. Assuming that the
protein-DNA complexes on the column are similar to those in the chromosomes,
the present experiments were therefore done under conditions of DNA excess, so
that the proteins applied to the column were unlikely to saturate all DNA-binding
sites. On the other hand, conditions of DNA excess might preclude some interactions
between protein and DNA that are dependent on localized high concentrations of protein.
Mammalian DNA-bindijig proteins
55
A large number of proteins bind to DNA. At a concentration that gives an O.D. 280
of o-i in peak I and peak II, some 60 proteins may be resolved on SDS acrylamide
gels. Two-dimensional electrophoresis resolves about 100 proteins, of which 60 are
major components. Under similar conditions of fractionation the ratio of DNAbinding proteins to soluble cellular protein in E. coliis about 5 times lower than in mammalian cells; the E. coli DNA-binding proteins can be separated into about 30 major
fractions in a two-dimensional system (Jost, Schuster & Messer, unpublished results).
In bacteriophage T7, however, 20-25 % °f t n e t o t a l phage protein binds to DNA.
This may reflect the larger proportion of the genome coding for proteins concerned in
replication and transcription (Scherzinger et al. 1973). There are many times more
replicative and transcriptional units in higher cells than in E. coli, so that on a per cell
basis the amount of each protein involved in these processes should be greater. This
difference should be detectable in our electrophoretic system, since it can be estimated that the proteins from extracts of peak I in the stained bands in an SDS gel are
present in IO 6 -IO 8 copies/cell. If it is assumed that approximately the same numbers
of different proteins are required for replicative and transcriptional functions in E.
coli as in higher cells, then the additional complexity of the mammalian proteins that
bind to DNA must reflect other functions, perhaps those involved in maintaining
chromosomal structure.
Nuclear origin of DNA-binding proteins
A comparison of the yield of DNA-binding proteins from nuclear and cytoplasmic
extracts should indicate whether there are large pools of these proteins in the cytoplasm (Vaughan & Comings, 1973). Cytoplasmic fractions were prepared in both
aqueous and non-aqueous solvents; in both cases the yields of proteins binding to
DNA were at least 25-fold smaller than those obtained from nuclear fractions. During
the preparation of cytoplasmic fractions, contaminating nuclear debris was removed
under conditions of low ionic strength; this might have permitted proteins initially
present in the cytoplasm to bind to the nuclear DNA and be removed with it. Nevertheless, the results show that nuclear proteins are preferentially retained on columns
containing DNA, thus reflecting the relationship found in vivo.
Strand-specific binding
We have confirmed previous observations (Salas & Green, 1971; Fox & Pardee,
1971) that denatured DNA has a higher binding capacity than native DNA. On
average, 9-6% of the input was bound to ss-DNA and 6-7% to ds-DNA. Only 0-3 %
of the proteins not binding to ss-DNA were retained by ds-DNA, thus showing that
ss-DNA effectively removes nearly all non-specific DNA-binding proteins. On the
other hand, much more of the protein not bound to ds-DNA was bound by ss-DNA
(2-4%). Histones, which form the major components of peak II, are always retained
on the first column containing ds-DNA. Overloading was therefore an unlikely
explanation of these findings. Although the majority of proteins showed no strandspecificity, two seemed to prefer native DNA and between 15 and 18 seemed to prefer
denatured DNA. A preference for denatured DNA is shown by the T4, T7 and E.
56
E.Jost, R. Lennox and H. Harris
coli unwinding proteins (Alberts & Frey, 1970; Scherzinger et at. 1973; Sigal et al.
1972), by the proteins specified by bacteriophage fd gene 5 (Pratt, Laws & Griffith,
1974), by the proteins specified by Mi3 gene 5 (Alberts et al. 1971) and by E. coli RNA
polymerase (Niisslein & Heyden, 1972). Whether or not the strand specificity shown
by the proteins in vitro is related to their function in vivo remains open to discussion,
but theoretical considerations suggest that DNA is forced into the single-stranded
form during replication and transcription.
Conservation of structure in DNA-binding proteins
The original purpose of these experiments was to compare the molecular weights
and net charges of DNA-binding proteins from human and mouse cells in the hope
that differences in the RF of individual proteins might be exploited to facilitate
genetic analysis with interspecific hybrid cells. Contrary to expectation, the pattern
shown by the mouse proteins that bind to calf thymus DNA was very similar to that
of the human proteins that bind to calf thymus DNA. Other studies have been interpreted to show that, although the non-histone proteins from different species have very
similar molecular weights (Elgin & Bonner, 1970), they are nevertheless different
(Elgin et al. 1973).
A number of studies have shown that not only the molecular weights and charges of
individual histones from different species are similar, but also their amino acid
sequences (DeLange et al. 1968; 1969; DeLange& Smith, 1971). There are no data on
the primary structure of the major non-histone proteins, but the results reported here
indicate that there are few differences between man and mouse in the molecular
weight or net charge of homologous DNA-binding proteins. Convergent evolution
might have selected for this similarity, but a more likely explanation is that the primary
structures have remained very similar. Since the general structure of DNA has remained unchanged in evolution, there must be severe restrictions on evolutionary
variation in the proteins, whether histones or non-histones, that bind to it.
Additional arguments for the similarity of the DNA-binding proteins from different
species may be derived from the fact that interspecific hybrid cells are viable. In a
hypothetical man-mouse hybrid cell containing the complete chromosomal complements of both parents, three kinds of DNA—protein interaction may be envisaged.
First, there may be absolute species-specificity of the proteins that bind to DNA,
mouse proteins binding to mouse DNA and human proteins to human DNA. This
possibility can be eliminated unless it is assumed that each chromosome codes for all
the proteins that bind to it. Since viable lines of man-mouse hybrid cells exist that
contain only one, or even a part of one human chromosome, and since genes on this
chromosome are expressed, it is reasonable to suppose that the human DNA functions
normally when mouse proteins are attached to it. Secondly, one could argue that
mouse proteins, although they might show a higher affinity for mouse DNA, bind well
enough to human DNA to permit it to function. This seems improbable, since cells
are likely to be sensitive to such variations in binding affinity, so that, if they existed, it is
difficult to see how rapidly dividing interspecific hybrid cells could be generated at all.
The third possibility is that the binding sites of homologous mouse and human
Mammalian DNA-binding proteins
57
DNA-binding proteins are so similar that one may replace the other. This possibility
is consistent with the results reported here. A molecular basis for the compatibility of
interspecific hybrid cells is thus provided.
This work was carried out while one of us (E. J.) held a Long Term EMBO Fellowship.
We thank Dr Peter Cook for suggestions and help with the manuscript and Mr Stephen Clark
for excellent technical assistance.
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DNA-agarose
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59
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{Received 30 November 1974)
WIENER,
E. Jost, R. Lennox and H. Harris
90
70
10
11
13
40
15
16
1
18
18-1
19
30
20
21
22
23
23-1
24
25
25-1
26
25-2
26i
28
27
29
283
30
10
29
B
D
Fig. 5. SDS acrylamide gel electrophoresis of DNA-binding proteins from whole
cells and nuclei isolated by the aqueous technique. Proteins were chromatographed
on ss-ct DNA-agarose and eluted with 0-4 M NaCl (peak I) and 2-0 M NaCl (peak II).
Fractions were pooled as indicated in Fig. i, and dialysed against 10% acetic acid.
Samples containing 0 1 O.D. 280 units were lyophilized to dryness, resuspended in
25 fil of a buffer containing 65-5 mM Tris pH 6-8, 3 % SDS, 5 % /?-mercaptoethanol
and 10 % glycerol (w/v). The samples were heated for 2 min at 90 °C before loading
and run at 70 V in a 12 % slab gel with a 5-5 % spacer gel. The vertical scale indicates
approximate mol. wt x io~ J . A, HeLa whole cell proteins eluted with 0-4 M NaCl
from ss-ct DNA-agarose; B, HeLa nuclear proteins eluted with 0-4 M NaCl from
ss-ct DNA-agarose; C, HeLa whole cell proteins eluted with 2 0 M NaCl from ss-ct
DNA-agarose; D, HeLa nuclear proteins eluted with 2'0 M NaCl from ss-ct DNAagarose.
Mammalian DNA-binding proteins
61
90
68
7-1
10
1
13
14
14-1
15
18
40
17
19
20
21
22
20
20
23
23-1
22
22-1
23
24
24
25
25-1
25-2
25
26
26
27
27
28
28
29
29
30
B
D
Fig. 6. SDS acrylamide gel electrophoresis of DNA-binding proteins from whole
mouse cells fractionated on ss-ct DNA, ds-ct DNA and ss-m DNA. For further
details see legend to Fig. 5. A, O-I O.D. 280 units of protein eluted from ss-ct DNAagarose with 0-4 M NaCl; B, O-I O.D. 280 units of protein eluted from ds-ct DNAcellulose with 0-4 M NaCl; c, o-i O.D. 280 units of protein eluted from ss-ct DNAagarose with 2-0 M NaCl; D, O-I O.D. 280 units of protein eluted from ds-ct DNAcellulose with 2-0 M NaCl; E, 002 O.D. 280 units of protein eluted from ds-ct DNAcellulose with 20 M NaCl after the extracts had been chromatographed on ss-ct DNA;
F, 005 O.D. 280 units of protein eluted from ss-ct DNA-agarose with 20 M NaCl
after the extracts had been chromatographed on ds-ct DNA; G, 005 O.D. 280 units of
protein from ss-m DNA-agarose eluted with 2-0 M NaCl after the extracts had been
chromatographed on ss-ct DNA-agarose.
10
62
E. Jost, R. Lennox and H. Harris
Fig. 7. Two-dimensional acrylamide gel electrophoresis of proteins from unlabelled
Ehrlich cells mixed with labelled mouse A9 cells. The extracts were eluted from an
ss-ct DNA-agarose column by 0-4 M NaCl (A) and 2-0 M NaCl (B). The distribution of
the proteins released from ds-ct DNA after passage through ss-ct DNA is also shown
(c). Pooled fractions from the peaks were dialysed against 10 % acetic acid and samples
were concentrated in a rotary evaporator to a volume of 0-3 ml. The proteins were
dialysed for 12 h against 3 changes of gel containing 6 M urea, 2 mM EDTA, 50 mM
boric acid, 4 % acrylamide, and 0-2 % n-^n' methylene bisacrylamide pH 86. Samples
were polymerized in the middle of the second dimension gel by the method of
Kaltschmidt & Wittmann (1970). The first dimension gel wag 8 % at pH 8-7. Electrophoresis in the first dimension was for 60 h at a constant voltage of 70 V. The seconddimension gel was 14 % at pH 4-6. Gels were run in the second dimension for 24 h at
105 V. All other procedures were as described in Materials and methods, A, proteins
binding to ss-ct DNA, peak I (approx. 7-5 O.D. 280 units); B, proteins binding to
ss-ct DNA, peak II (approx. 2-5 O.D. 280 units); c, proteins eluted with 2-0 M NaCl
from ds-ct DNA after the extracts had been chromatographed on ss-ct DNA (total
proteins from 4-0 x 10* cells).
Mammalian DNA-binding proteins
1-D
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12-20
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2-D
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rv
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or
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B
64
E. Jost, R. Lennox and H. Harris
Fig. 8. Two-dimensional acrylamide gel electrophoresis of proteins from unlabelled
Ehrlich cells mixed with labelled mouse A9 cells. The proteins were eluted from a
ds-ct DNA-cellulose column by 0-4 M NaCl (A) and 2-0 M NaCl (B). The distribution
of the proteins released from ss-ct DNA after passage through ds-ct DNA is also
shown (c). Purified calf thymus histone (D) is shown to serve as a histone standard.
Pooled fractions were treated as stated in the legend to Fig. 7 and in Materials and
methods. A, proteins binding to ds-ct DNA, peak I (approx. 7-5 O.D. 280 units); B,
proteins binding to ds-ct DNA, peak II (approx. 2-5 O.D. 280 units); C, proteins
eluted with 20 M NaCl from ss-ct DNA after the extracts had been chromatographed
on ds-ct DNA (approx. 5-0 O.D. 280 units); D, calf thymus histone (Sigmafraction II a).
Mammalian DNA-lnnding proteins
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