Chromatin structure from the marine sponge Geodia cydonium


Dawes, K.W.; Bachmann, M.; Zahn, R.K.; Müller, W.E.

Comparative Biochemistry and Physiology. B, Comparative Biochemistry 76(4): 769-775

1983


The histones isolated from the siliceous sponge Geodia cydonium have been separated using two electrophoretic techniques. A comparison of their mobilities with those of calf thymus and rat liver show that some Geodia histone species (H3, H1 and H1(0) exhibit electrophoretic variance. The results show, that as in other eukaryotic systems the sponge chromatin contains the core histones (H2A, H2B, H3 and H4) and the linker histone (H1). ADP-ribosylation of Geodia histones and separation of the individual histones by electrophoresis resulted in four histones being radiolabeled. Digestion of Geodia chromatin with endogenous endonuclease is shown to result in the formation of nucleosome particles containing approximately 200 base pairs of DNA. A major product of endogenous endonuclease digestion is a relatively stable 110 base pair intermediate. Incubation of chromatin with DNase II and separation of the products under denaturing conditions reveals 20 bands migrating at 10 base intervals.

Comp.
Biochem.
Physiol.
Vol.
76B,
No.
4,
pp.
769-775,
1983
0305-0491/83
$3.00
+
0.00
Printed
in
Great
Britain
©
1983
Pergamon
Press
Ltd
CHROMATIN
STRUCTURE
FROM
THE
MARINE
SPONGE
GEODIA
CYDONIUM
KEITH
W.
DAWES,
MICHAEL
BACHMANN,
RUDOLF
K.
ZAHN
and
WERNER
E.
G.
MULLER
Institut
fur
Physiologische
Chemie,
Universitat
Mainz,
Duesbergweg,
6500
Mainz,
F.R.G.
(Received
27
April
1983)
Abstract-1.
The
histones
isolated
from
the
siliceous
sponge
Geodia
cydonium
have
been
separated
using
two
electrophoretic
techniques.
2.
A
comparison
of
their
mobilities
with
those
of
calf
thymus
and
rat
liver
show
that
some
Geodia
histone
species
(H3,
H1
and
H1°)
exhibit
electrophoretic
variance.
3.
The
results
show,
that
as
in
other
eukaryotic
systems
the
sponge
chromatin
contains
the
core
histones
(H2A,
H2B,
H3
and
H4)
and
the
linker
histone
(H1).
4.
ADP-ribosylation
of
Geodia
histones
and
separation
of
the
individual
histones
by
electrophoresis
resulted
in
four
histones
being
radiolabeled.
5.
Digestion
of
Geodia
chromatin
with
endogenous
endonuclease
is
shown
to
result
in
the
formation
of
nucleosome
particles
containing
approximately
200
base
pairs
of
DNA.
6.
A
major
product
of
endogenous
endonuclease
digestion
is
a
relatively
stable
110
base
pair
intermediate.
7.
Incubation
of
chromatin
with
DNase
II
and
separation
of
the
products
under
denaturing
conditions
reveals
20
bands
migrating
at
10
base
intervals.
INTRODUCTION
The
structure
of
eukaryotic
chromatin
is
based
on
a
repeat
unit,
the
nucleosome,
in
which
DNA
is
wrapped
round
and
complexed
with
the
histone
proteins
(Kornberg,
1977
;
McGhee
and
Felsenfeld,
1980).
Nuclease
digestion
studies
have
shown
that
the
nu-
cleosome
comprises
of
approximately
150-200
base
pairs
of
DNA,
depending
on
the
source
(Noll
and
Kornberg,
1977;
Thomas
and
Furber,
1976;
Kreichline
et
al.,
1976;
Simpson
and
Whitlock,
1976).
Each
nucleosome
is
then
complexed
with
an
octomer
of
histones,
comprising
2
each
of
histones
H2A,
H2B,
H3
and
H4.
Histone
H1
is
associated
with
the
region
that
links
the
nucleosomes
together.
The
variation
in
the
length
of
the
nucleosome
in
eukaryotic
chromatin
has
been
shown
to
be
due
to
the
difference
in
the
length
of
the
linker
region.
Invariably
the
stable
intermediate
obtained
after
nuclease
digestion
is
in
the
order
of
145
base
pairs.
This
fragment
termed
the
core
particle
is
associated
with
the
histones
H2A,
H2B,
H3
and
H4
(Shaw
et
al.,
1976).
In
general
the
number
and
type
of
histones
remains
constant
between
species.
However
in
lower
eukaryotes
variants
have
been
detected.
In
yeast
histone
H1
has
evolutionarily
diverged
(Thomas
et
al.,
1976),
and
although
all
5
histones
are
present
only
histone
H4
migrates
comparable
to
its
mam-
malian
counterpart
on
acid/urea
electrophoretic
gels
(Mardian
and
Isenberg,
1978).
In
Tetrahymena
the
micronucleus
lacks
electrophoretic
equivalents
of
his-
tones
HI
and
H3,
but
in
the
macronucleus
all
five
histones
have
been
detected.
Sponges
are
the
first
and
oldest
multicellular
animals
;
marine
species
of
the
order
Triaxonida
are
known
since
the
Proterozoic
(1000
million
years)
(Orlov,
1971).
Up
to
this
contribution,
no
data
on
the
structure
of
the
chromatin
from
sponges
were
available.
Therefore,
it
was
interesting
to
determine,
whether
the
basic
chromatin
elements
were
preserved
over
that
span
of
evolution.
In
the
present
report
we
describe
the
presence
of
five
histones
in
Geodia
cy-
donium
chromatin,
some
of
which
are
electrophoretic
variants.
Four
types
of
histone
are
post-synthetically
modified
by
ADP-ribosylation.
Nuclease
digestion
studies
indicate
that
the
nucleosome
is
structurally
similar
to
that
of
higher
eukaryotes.
MATERIALS
AND
METHODS
Nicotinamide-{U-'
4
C]
adenine
dinucleotide
(274
Ci/
mol)
was
obtained
from
the
Radiochemical
Centre,
Amer-
sham
(England).
ADP-ribose
and
the
double
stranded
DNA
standards
from
Hinc
III
digest
of
¢
X
174RF
DNA
were
purchased
from
P-L
Biochemicals,
Milwaukee
(USA).
The
calf
thymus
histone
fractions
from
Scientific
Research
Committee
Medical
Academy,
Loz
(Poland).
Deoxyribo-
nuclease
I,
ribonuclease
A,
phosphodiesterase
I
(Crotalus
adamanteus),
Staphylococcus
nuclease
and
t-RNAP'
were
purchased
from
Boehringer-Mannheim
(Germany).
Trasylol
was
a
gift
from
Dr
S.
Shiitz,
Bayer,
Leverkusen
(Germany).
The
siliceous
marine
sponge,
Geodia
cydonium
(Tetractinellida),
was
collected
in
the
vicinity
of
Rovinj
(Jugoslavia).
Isolation
of
chromatin
Method
A.
Geodia
tissue
was
cut
into
2
mm
3
cubes,
washed
repeatedly
with
Ca
+
and
Mg+
free
sea
water
(Muller
et
al.,
1977),
and
frozen
rapidly
by
pressing
the
material
between
solid
carbon
dioxide.
To
50
g
of
this
material,
100
ml
of
10
mM
Tris—HC1
buffer
(pH
7.4,
containing
10
mM
NaCI,
1
mM
MgC1
2
,
50%
v/v
glycerol,
1%
v/v
Triton
X-100,
10
µg/ml
of
the
protease
inhibitor
Trasylol)
was
added
and
the
suspen-
sion
homogenised
at
20°C
in
a
cell
homogeniser
(Model
MSK,
B.
Braun,
Melsungen
;
0.5
mm
glass
beads)
for
2
min.
After
removal
of
solid
material
by
filtration
through
cheese
cloth
the
homogenate
was
centrifuged
in
a
Sorvall
HB-4
rotor
at
—15°C
(6000
rev/min
for
20
min).
The
sediment
was
further
769
770
Kean
W.
DAWES
et
al.
homogenised
using
a
Dounce
homogeniser.
This
step
was
repeated
three
times
until
the
resulting
nuclei
were
not
contaminated
with
cytoplasmic
material
;
the
purity
of
nuclei
was
checked
by
fluorescence
microscopy
using
the
acridine
staining
method
(Chayen
et
al.,
1969).
Chromatin
was
isolated
from
the
nuclear
pellet
by
extraction
with
10
mM
Tris—HCI
(pH
7.5,
containing
25
mM
EDTA,
and
74
mM
NaCI)
(Hewish
and
Burgoyne,
1973).
Loosely
bound
non-
histone
proteins
were
removed
by
treatment
with
0.35
M
NaCI
(in
10
mM
Tris-1
-
1C1
buffer,
pH
7.0)
as
described
(Medvedev
et
al.,
1979).
When
Trasylol
was
omitted
10
mM
NaHSO
3
and
0.5
mM
phenylmethylsulphonylfluoride
(PMSF)
were
included
as
protease
inhibitors.
Method
B.
Nuclei
were
isolated
by
the
method
of
Newish
and
Burgoyne
(1973),
but
with
minor
modifications.
The
tissue
was
suspended
in
15
mM
Tris—HCI
(pH
7.4,
containing
60
mM
KCI,
15
mM
NaCI,
0.15
mM
spermine,
0.5
mM
spermidine
and
15
mM
2-mercaptoethanol)
and
hom-
ogenised
for
2
min
with
1
mm
glass
beads
using
the
cell
homogeniser
described
above.
Trasylol
was
added
as
pro-
tease
inhibitor.
Mercaptoethanol
was
omitted
from
this
buffer
when
iodoacetate
and/or
iodosobenzoic
acid
(50
µg,/ml,
end
concentration)
were
added
as
nuclease
inhibitors.
After
isolation
of
the
nuclei
chromatin
was
extracted
as
described
by
Noll
et
al.
(1975).
Histone
modification
and
extraction
Poly(ADP-Rib)
synthesis.
Chromatin,
isolated
as
described
in
"Method
A",
was
suspended
in
50
mM
Tris—HCI
(pH
7.6,
containing
5
mM
2-mercaptoethanol)
at
a
concentration
of
3.9
mg/m1
(
=
chromatin
fraction).
The
poly(ADP-Rib)
polymerase
assay
was
as
described
earlier
(Muller
et
al.,
1975),
but
with
minor
modifications.
Aliquots
(0.45
ml)
of
the
chromatin
preparation
(containing
1.8
mg
protein)
were
incubated
for
4
hr
at
37°C
with
2
ml
salt
mix
which
contained
100
mM
Tris—HCI
pH
8.5,
6
mM
MgCl
2
,
60
mM
KCI,
8
mIVI
KF,
4
mM
dithiothreitol
and
150
pM
4
C]
NAD
+
(535
dpm/pmol).
The
incubated
preparation
was
processed
to
remove
the
non-histone
proteins.
For
time
course
experi-
ments
and
determination
of
the
susceptibility
of
poly(ADP-
Rib)
to
nuclease
digestion
the
reaction
(70
pi
aliquots)
was
terminated
after
incubation
by
addition
of
trichloroacetic
acid.
The
reaction
product
was
then
collected
on
GF/C
filters
and
counted
(Muller
et
al.,
1975).
Extraction
of
histone.
Total
histone
was
extracted
with
0.25
M
HCI
to
give
the
"Histone
fraction"
(Medvedev
et
al.,
1979).
The
sediment
was
also
collected
and
termed
"Residual
protein
fraction".
For
comparison,
as
well
as
for
determi-
nation
of
the
reliability
of
the
isolation
method,
histones
were
isolated
from
adult
rat
livers.
Nuclease
digestions
Chromatin
(approx
1
mg/m1)
was
digested
with
micrococ-
cal
nuclease
(20
units/ml)
or
DNase
I
in
0.25
M
sucrose,
1
mM
Tris—HCI
(pH
8.0,
containing
1
mM
CaCl
2
or
1
mM
MgCl
2
,
respectively).
Endogenous
endonuclease
digestions
were
car-
ried
out
either
in
the
last
extraction
buffer
from
"Method
A"
or
in
the
micrococcal
nuclease
buffer
described
above,
but
without
added
cation.
All
digestions
were
incubated
at
37'C.
Incubations
with
exogenous
nuclease
were
terminated
by
addition
of
EDTA
(25
mM
final
concentration).
DNA
extraction
Digested
chromatin
was
made
1
M
to
NaCI
and
extracted
with
an
equal
volume
of
buffered
phenol.
The
DNA
in
the
aqueous
phase
was
then
precipitated
with
3
vol
of
absolute
ethanol
and
stored
overnight
at
20'
C.
Gel
electrophoresis
Histone
proteins.
Acid/urea
disc
gel
electrophoresis
using
2.5
M
urea/15`,
polyacrylamide
gels
was
performed
accord-
ing
to
Panyim
and
Chalkley
(1969).
For
determination
of
radioactivity
the
gel
was
cut
into
1
mm
slices
and
treated
as
described
previously
(Arendes
etal.,
1980).
Electrophoresis
in
the
presence
of
sodium
dodecyl
sulphate
(SDS)
on
15"
polyacrylamide
gels
was
performed
as
described
by
Laemmli
(1970).
Gels
were
stained
either
with
Coomassie
Blue
(Kish
and
Pederson,
1975),
Amido
Black
(Cohen
and
Gotchel,
1971),
or
periodic
acid
Schiff
(PAS)
(Giirteler
et
al.,
1979)
and
scanned
in
a
Shamatzu
gel
scanner.
DNA
fragments.
Double
stranded
DNA
was
analysed
on
polyacrylamide
slab
gels
using
the
Tris—borate—EDTA
buffer
described
by
Peacock
and
Dingman
(1967).
Electrophoresis
was
for
approximately
3
hr
at
200
V.
Analysis
of
DNA
under
denaturing
conditions
was
per-
formed
using
6
M
urea--6"„
polyacrylamide
slab
gels
(Maniatis
et
al.,
1975).
Samples
were
dissolved
in
10
M
urea-
diluted
buffer
prior
to
loading.
Electrophoresis
was
as
described
earlier
(Maniatis
et
al.,
1975).
Usually
samples
were
dissolved
at
a
concentration
of
2-3
mg/m1
based
on
the
starting
concentration.
Gels
were
stained
overnight
with
0.005%;
Stains
All
in
50"
formamide,
destained
under
running
tap
water
and
scanned
at
550
nm.
Analytical
methods
Protein
was
determined
by
the
u.v.
absorbance
method
described
by
Kalb
and
Bernlohr
(1977).
Chromatin
was
solubilised
by
extraction
with
0.2%;
SDS
and
DNA
measured
using
an
absorption
coefficient
at
260
nm
of
20
ml/mg/cm.
RESULTS
AND
DISCUSSION
Histone
characterisation
A.
Acid/urea
electrophoresis.
Electrophoresis
of
ex-
tracted
Geodia
histone
proteins
on
gels
containing
urea
and
acetic
acid
results
in
the
separation
of
8
stained
protein
bands
(Fig.
1).
Only
4
bands
migrate
with
equivalent
mobilities
to
the
histones
from
rat
liver
(not
shown)
on
calf
thymus.
These
bands
correspond
to
mammalian
histones
H1,
H2B,
H2A
and
H4.
The
presence
of
other
strongly
stained
protein
bands
is
indicative
that
Geodia
histones
may
exert
electropho-
retic
variance.
After
PAS
staining
of
one
gel
the
fast
migratory
band
seen
in
Fig.
1
stained
positive
indicat-
ing
it
to
be
a
glycoprotein.
It
has
been
reported
that
histones
H2B
and
H
I
show
higher
increased
absorp-
tion
at
450
nm
than
at
700
nm
after
staining
with
Amido
Black
(Cohen
and
Gotchel,
1971).
With
Geodia
histones
2
bands
show
this
property
corresponding
to
equivalent
histone
H2B
and
a
low
mobility
protein
as
shown
in
Fig.
2.
This
data,
therefore,
suggests
that
Geodia
histones
H3,
HI
and
H1`
are
electrophoretic
variants.
No
change
in
the
histone
pattern
was
ob-
served
when
chromatin
was
extracted
in
the
presence
of
other
protease
inhibitors.
B.
SDS
electrophoresis.
Eukaryotic
histones
show
a
good
resemblance
in
their
mobilities
on
gels
contain-
ing
SDS.
The
method
is
therefore
a
reliable
way
in
which
to
determine
if
a
full
complement
of
histones
are
present
in
chromatin.
As
shown
in
Fig.
3,
Geodia
histones
are
resolved
into
6
bands
with
similar
mobi-
lities
to
those
from
rat
liver.
This
clearly
indicates
that
the
same
number
of
histones
exist
in
Geodia
chromatin
and
that
at
least
2
histones
are
electrophoretic
variants
as
suggested
above.
The
densiometric
scan
of
the
rat
liver
histones
confirms
an
approximate
molar
ratio
of
2:1
for
the
inner
histones
to
histone
HI,
but
a
much
lower value
is
demonstrated
by
the
Geodia
histones.
Additional
evidence
that
histones
H
I
and
H
I
°
are
present
in
Geodia
chromatin
was
demonstrated
by
their
solubility
in
5%
perchloric
acid.
After
extraction
771
Chromatin
structure
from
the
marine
sponge
Geodia
cydonium
H2B
HI
H3
H2B
H2A
H4
-
4,
HI
1
,13
H2A
HI
°
H4
1
HI
°
O
Op
t
ica
l
de
ns
ity
(
650
nm
)
E
c,
in
a
0
Jr
AA/
k,
O
Migration
J
Migration
Fig.
1.
Acid/urea
electrophoresis
of
Geodia
histones
(A)
and
calf
thymus
histones
(B).
Gels
were
stained
with
Coomassie
Blue
and
scanned
at
650
nm.
The
origin
of
the
gels
is
on
the
right.
Arrows
mark
the
position
of
authentic
markers.
Band
X
corresponds
to
the
positively
stained
glycoprotein
band.
E
0
0
1
E
0
Migration
Fig.
2.
Comparison
of
the
absorption
of
Geodia
histones
at
two
wavelengths.
Histones
were
separated
by
acid/urea
electrophoresis,
stained
with
Amido
Black,
and
scanned
at
700
nm
(A)
or
450
nm
(B).
X
marks
the
position
of
the
2
bands
which
show
different
absorption
at
the
two
wavelengths.
The
origin
is
on
the
right.
Fig.
3.
Sodium
dodecyl
sulphate
electrophoresis
of
calf
thymus
(A)
and
Geodia
histones
(B).
Gels
were
stained
with
Coomassie
Blue
and
scanned
at
650
nm.
X
shows
the
position
of
bovine
serum
albumin
marker.
Mobility
is
from
right
to
left.
the
2
bands
equivalent
to
histones
H1
and
Hl°
were
not
detected
on
an
SDS—polyacrylamide
gel.
Poly
ADP-ribosylation
of
extracted
chromatin
Under
standard
assay
conditions
ADP-ribosylation
of
extracted
chromatin
results
in
the
incorporation
of
1.21
pmol
ADP-Rib
per
10µg
protein
after
incubation
for
60
min
at
37°C.
The
reaction
showed
a
dependence
on
temperature
with
62%
of
that
amount
at
37°C
incorporated
at
22°C.
During
the
incubation
the
degree
of
ribosylation
varied
proportionally
with
time.
To
clarify
that
[
14
c]
NAD
+
was
assembled
as
poly(ADP-Rib)
in
the
chromatin
digestion
experi-
ments
were
carried
out
as
described
earlier
(Muller
et
al.,
1976).
After
incubation
of
a
probe
for
60
min
(incorporation
rate
0.90
pmol/8
tig
chromatin
protein)
aliquots
were
supplemented
with
either
DNase
I,
RNase
A,
micrococcal
nuclease
or
phosphodiesterase
I
(final
concentration
150
µg/ml)
and
incubated
further
for
100
min
at
37°C.
In
the
nuclease
treated
prepar-
ation
the
amount
of
radioactivity
decreased
by
20%
while
in
the
phosphodiesterase
treated
probe
only
residual
acid
soluble
activity
remained
(25%).
From
these
results
we
therefore
assume
that
the
chromatin
was
poly
ADP-ribosylated
(Hilz
and
Stone,
1976).
After
ribosylation
the
chromatin
showed
a
slight
shift
in
absorption
maximum
from
258
to
264
nm.
Poly
ADP-ribosylation
of
histone
fraction
The
radiolabelled
Geodia
chromatin
was
separated
into
3
fractions
and
the
amount
of
radioactivity
in
each
determined
as
described
in
"Materials
and
Methods".
As
shown
in
Table
1
a
major
amount
of
radioactivity
is
incorporated
in
the
"Histone
fraction",
327.8
pmol/mg
protein,
with
295.2
and
21.3
pmol/mg
protein
in-
ADP-ribose
H4
H2B
H1
-600
500
-400
-300
200
100
Ra
d
ioac
t
iv
ity
(dp
m
/slice
)
I
140
120
100
80
60
40
20
Slice
number
0
Aci
d
s
o
lu
bi
li
ty
(
%)
50-
40-
30-
20-
10
-
x
l
0
(
,
I
"
30
1 I
60
772
KEITFI
W.
DAWES
et
al.
Table
1.
Association
of
radioactive
ADP-Rib
units
with
various
nuclear
proteins
Acid
insoluble
Protein
radioactivity
Protein
fraction
(mg)
(pmol,fmg)
Chromatin
1.82
220.2
Non-histone
protein
0.61
21.3
Histones
0.94
327.8
Residual
protein
fraction
0.27
295.2
Chromatin
was
incubated
in
the
standard
poly(ADA-Rib)
assay
for
4
hr
and
subsequently
fractionated
as
described
under
"Materials
and
Methods".
Seventy
microliter
aliquots
were
assayed
for
acid-insoluble
radioactivity.
corporated
in
the
"Residual
protein
fraction"
and
"Non-histone
fraction",
respectively.
The
extent
of
radiolabelling
in
individual
histones
was
determined
by
analysis
of
the
histone
fraction
on
a
urea/acetic
acid
polyacrylamide
gel.
The
results
show
that
91%
of
the
radioactivity
is
associated
with
4
separated
bands
(Fig.
4).
These
bands
migrate
equivalent
to
histones
HI,
H2B,
H2A
and
H4
from
calf
thymus
with
1658
dpm
(29%),
1273
dpm
(22%),
2373
dpm
(41%)
and
446
dpm
(8%),
respectively.
A
small
amount
of
radioactivity
corresponds
in
mobility
(R
1
:
0.28)
to
the
band
which
has
the
property
of
showing
decreased
absorption
at
700
nm
after
Amido
Black
staining.
Chromatin
bound
endonuclease
activity
Incubation
of
Geodia
chromatin,
without
added
nuclease,
activates
an
endogenous
endonuclease(s)
which
releases
acid
soluble
oligonucleotides.
In
the
previous
contribution
(Dawes
et
al.,
in
press)
it
is
4-
-
H
2A
Fig.
4.
Distribution
of
["C]
poly(ADP-Rib)
on
acid/
urea/polyacrylamide
gels.
Geodia
chromatin
was
incu-
bated
in
the
standard
poly(ADP-Rib)
polymerase
assay.
Subsequently
the
histone
fraction
was
isolated,
and
36
mg
of
protein,
containing
6300
dpm,
was
applied
to
the
gel
and
electrophoresis
carried
out
as
described
under
"Materials
and
Methods".
The
arrows
mark
the
positions
of
authentic
histone
markers.
The
origin
of
the
gels
is
on
the
right.
The
radioactivity
of
1
mm
slices
was
determined.
1
I [
90
120
Incubation
time
(min)
Fig.
5.
Release
of
acid
soluble
products
from
endonuclease
degraded
Geodia
chromatin.
Method
A
extracted
chromatin
(1
mg/ml)
was
digested
with
endogenous
endonuclease
in
10
mM
Tris-HC1
pH
7.0
containing
350
mM
NaCI
and
25
mM
EDTA
(0-0).
Method
B
extracted
chromatin
was
digested
with
endogenous
endonuclease
(0
-
-
0),
or
partially
purified
Geodia
endonuclease
(30
LI/m1)(x
-
-
x)
in
1
mM
Tris-HCI
pH
8.0
containing
250
mM
sucrose
and
10
mM
EDTA
at
a
concentration
of
1
mg/ml
and
350
µg/ml,
respectively.
Aliquots
were
removed
at
the
indicated
times
and
the
amount
of
product
soluble
in
0.6
N
HC10
4
deter-
mined.
reported
that
two
nucleases
are
bound
to
the
extracted
Geodia
chromatin,
the
first
is
inhibited
by
iodosoben-
zoic
acid
and
not
by
EDTA
(enzyme
B),
the
second
by
EDTA
or
iodoacetate
(enzyme
A).
The
results
imply
that
enzyme
A
is
cation
activated.
While,
during
the
extraction
procedure
enzyme
B
remains
firmly
bound
to
the
chromatin,
there
is
some
evidence
which
suggests
that
enzyme
A
is
susceptible
to
extrac-
tion
with
buffers
containing
NaCl.
Endonuclease
digestion
of
Geodia
chromatin
Endogenous
endonuclease
digestion.
Chromatin
iso-
lated
by
Method
A
is
capable
of
undergoing
self-
digestion
as
described
above.
In
the
presence
of
EDTA
to
inhibit
enzyme
A
digestion
proceeds
to
a
limit
of
46%
acid
solubility
(Fig.
5).
The
double
stranded
products
of
the
reaction
were
analysed
on
a
6%
polyacrylamide
slab
gel
as
shown
in
Fig.
6.
For
the
determination
of
the
sizes
of
the
released
fragments
a
comparison
was
made
to
a
calibration
curve
con-
structed
with
the
aid
of
Hinc
III
digestion
fragments
of
X
174
DNA.
At
5%
acid
solubility
a
series
of
bands
are
present
which
correspond
to
fragments
of
320-400
base
pairs
(bp),
200-175
bp
and
a
band
at
147
bp.
Some
smaller
fragments
are
also
present.
As
digestion
pro-
ceeds,
at
12.5%
acid
solubility
a
band
of
260
bp
is
released
and
the
147
bp
fragment
undergoes
further
degradation.
It
is
apparent
at
the
limit
of
digestion
(46%
acid
solubility)
that
only
smaller
fragments
are
the
products
with
95,
110
and
126
bp.
Analysis
of
the
degradation
products
from
the
limit
digest
in
a
sucrose
gradient
separates
two
peaks
as
shown
in
Fig.
7.
The
second
peak
has
a
sedimentation
coefficient
of
9.9
±
0.2
S
relative
to
16
and
5
S
ribosomal
subunits
from
E.
coli.
Values
of
10.6
and
11.2
S
have
been
reported
for
a
115
bp
fragment
from
micrococcal
nuclease
treated
Chromatin
structure
from
the
marine
sponge
Geodia
cydonium
773
I
I
II
100
150
200
300
500
400
600
Number
of
base
pairs
Fig.
6.
Native
6%
polyacrylamide
electrophoresis
of
DNA
fragments
from
endogenous
endonuclease
digested
chro-
matin.
Method
A
extracted
chromatin
(1
mg/m1)
was
digested
in
10
mM
Tris—HC1(pH
7.0,
containing
350
mM
NaCI
and
25
mM
EDTA).
The
products
were
isolated,
submitted
to
electrophoresis
and
stained
as
described
in
"Materials
and
Methods".
(A)
and
(B)
46%
acid
soluble
digestion.
The
arrow
marks
the
position
of
the
Bromophenol
Blue
marker
dye.
The
Hinc
III
DNA
fragments
have
been
used
for
cali-
bration.
erythrocyte
chromatin
(Shaw
et
al.,
1976)
and
for
a
146
bp
fragment
from
rat
liver
chromatin
(Noll,
1974)
respectively.
The
amount
of
acid
soluble
products
released
on
incubation
of
chromatin
isolated
by
Method
B
is
shown
in
Fig.
5.
As
can
be
seen
a
degradation
limit
of
43%
is
achieved
which
compares
quite
well
with
the
result
for
the
chromatin
isolated
by
the
other
method.
It
is
of
interest
to
note
that
Method
A
isolated
chromatin
prior
to
removal
of
non-histone
proteins
gave
a
digest
limit
of
only
20%.
This
result
is
in
agreement
with
other
authors
who
observed
that
removal
of
non-histone
proteins
enhances
the
suscep-
tibility
of
chromatin
to
endonuclease
action
(Billings
and
Bonner,
1972).
The
products
of
endogenous
endonuclease
degraded
chromatin
were
analysed
by
electrophoresis
on
a
6%
polyacrylamide
slab
gel
(not
shown).
Here
a
slightly
different
profile
to
the
one
shown
previously
is
seen.
At
the
beginning
of
the
incubation
the
147
bp
fragment
is
again
present
and
a
band
corresponding
to
200
bp.
As
degradation
pro-
ceeds
the
band
at
200
bp
broadens
out
considerably
and
is
progressively
shortened.
The
147
bp
fragment
is
further
degraded
to
yield
a
110
bp
fragment.
At
the
digestion
limit
bands
corresponding
to
110,
126, 138,
146
and
a
broad
band
centred
on
185
bp
are
the
observed
products.
It
is
obvious
from
both
sets
of
results
that
certain
bands
ate
not
exclusively
derived
from
the
well
ordered
degradation
of
nucleosome
oligomers
(e.g.
not
all
of
the
bands
seen
in
Fig.
6
can
be
explained
by
either
the
release
of
nucleosome
oligomers
or
the
gradual
degradation
of
"free
linkers").
This,
therefore
suggests
that
shearing
had
occurred
during
the
extraction.
Micrococcal
nuclease.
Geodia
chromatin
was
iso-
lated
by
Method
B
as
described
in
"Materials
and
Methods".
To
prevent
any
endogenous
endonuclease
activity
against
chromatin,
inhibitors
were
added
to
the
extraction
buffers.
Digestion
of
Geodia
chromatin
with
micrococcal
nuclease
reaches
a
limit
at
5%
acid
solubility.
This
indicates
that
the
chromatin
is
more
susceptible
to
attack
with
micrococcal
nuclease
than
with
endogenous
nuclease.
Chromatin
preparations
(1
mg/ml)
were
incubated
under
standard
conditions
with
20
units/ml
of
micrococcal
nuclease
and
analyzed
on
a
6%
polyacrylamide
slab
gel
(not
shown).
As
digestion
proceeds,
2
high
molecular
weight
bands
centred
on
300
and
260
bp
are
liberated.
On
further
digestion
the
300
bp
fragment
is
gradually
shortened
to
yield
a
broad
band
at
260
bp.
The
147
bp
fragment,
initially
present,
gradually
disappears
and
coincides
with
the
build
up
of
a
fragment
at
95
bp.
At
the
same
time
a
band
at
130
bp
increases
in
intensity
but,
is
progressively
digested
to
yield
a
band
at
120
bp.
The
reaction
is
terminated
by
minor
bands
with
fragment
lengths
of
147,
120,
110
and
a
major
band
with
95
bp.
Purified
Geodia
endonuclease.
In
the
previous
publi-
cation
(Dawes
et
al.,
in
press)
we
have
described
the
purification
and
properties
of
a
chromatin
bound
endonuclease.
It
was,
therefore,
of
interest
to
determine
if
this
enzyme
retained
an
ability
to
digest
isolated
chromatin.
As
shown
in
Fig.
5
the
enzyme
is
capable
of
5S
165
9
9t0
2S
sedimentation
30
20
10
Fraction
number
Fig.
7.
Sucrose
gradient
centrifugation
of
endogenous
en-
donuclease
degraded
chromatin.
Method
A
extracted
chro-
matin
(1
mg/ml,
0.2
ml)
was
incubated
for
2
hr
and
applied
to
an
11
ml
sucrose
gradient
(5-25%
in
10
mM
Tris—HCI
pH
7.0
containing
350
mM
NaCI
and
25
mM
EDTA).
Centrifugation
was
for
16
hr
at
33,000
rev/min
(4°C)
in
a
Beckmann
SW
40
rotor.
Fractions
(0.4
ml)
were
collected
using
an
ISCO
density
gradient
fractionator.
Sedimentation
is
from
right
to
left.
Op
ti
ca
l
de
ns
i
ty
{
550
nm
)
Op
t
ica
l
dens
ity
(
254
nm
)
774
KEITH
W.
DAWES
el
al.
0
0.5
I
1 1
0
T
Relative
migration
Fig.
8.
Single
stranded
fragments
from
endonuclease
di-
gestion
of
Geodia
chromatin
analysed
on
a
urea
6°/„
poly-
acrylamide
gel.
Method
B
extracted
chromatin
was
incubated
with
(A)
DNase
I
(500
U)
in
1
mM
Tris—HCI
pH
8.0
containing
250
mM
sucrose
and
1
mM
MgCl
2
for
5
mM
or
with
42
U
of
partially
purified
Geodia
endonuclease
(B)
for
10
mM
at
37°C.
The
position
of
t-RNAP
h
'
is
indicated
by
the
arrow.
degrading
Geodia
chromatin
and
reaches
a
limit
at
40%
acid
solubility.
Electrophoresis
of
the
released
fragments
reveals
a
variety
of
bands
which
compare
quite
well
with
those
observed
from
micrococcal
nuclease
digested
chromatin.
That
is,
with
major
bands
at
147,
130,
110,
95
bp
and
minor
bands
corresponding
to
189,
230
and
340
bp.
Analysis
of
single
stranded
fragments.
DNase
I
has
been
shown
to
cleave
chromatin
to
give
single
stranded
cuts
at
10
base
intervals
(Prunell
et
al.,
1979).
The
analyses
on
native
gels
indicate
that
exogenous
and
endogenous
endonucleases
exhibit
different
properties
towards
the
cleavage
of
Geodia
chromatin.
This
is
also
demonstrated
by
the
analysis
of
the
products
on
a
urea
polyacrylamide
gel.
Geodia
chromatin
on
incubation
with
DNase
I
gives
20
bands
when
analysed
on
a
denaturing
gel
(Fig.
8A).
By
plotting
the
log
of
band
number
versus
mobility
of
each
band
a
straight
line
is
obtained.
Co-
electrophoresis
with
t-RNAP'
(77
bases)
suggests
that
these
bands
migrate
as
multiples
of
10
bases.
Identical
results
are
obtained
with
the
purified
Geodia
endo-
nuclease
treated
chromatin
as
shown
in
Fig.
8(B).
This
suggests
that
the
core
regions
are
nicked
at
10
base
intervals.
The
results
presented
demonstrate
that
chromatin
from
the
sponge
Geodia
cydonium
is
structurally
related
to
that
of
higher
eukaryotes.
It
contains
all
of
the
core
histones
H2A,
H2B,
H3
and
H4,
together
with
a
histone
similar
to
H1
from
higher
eukaryotes.
The
nucleosome
is
characterised
by
a
fragment
length
of
approximately
200
base
pairs,
which
on
further
endonuclease
digestion
yields
a
147
base
pair
core
intermediate.
The
presence
in
Geodia
chromatin
of
histones
showing
electrophoretic
variance,
in
comparison
to
histones
from
higher
animals,
confirms
that
such
a
property
is
associated
with
nuclear
extracts
from
lower
animals.
This
would
be
expected
as
the
sponges
(Parazoa)
are
considered
to
be
the
most
primitive
and
isolated
branch
of
the
multicellular
organisms
(Hyman,
1940).
Moreover
post-synthetic
modification
of
histones
by
ADP-ribosylation
is
not
only
a
property
of
histones
from
higher
animals
but
also
occurs
in
sponges.
However,
we
cannot
from
our
results
describe
conclusively
which
histones
are
ribosylated
;
either
all
of
the
core
histones,
or
histone
H
1
and
three
core
histones
are
post-synthetically
modified.
Histone
111
is
apparently
widely
ribosylated
in
other
systems
(Riquelme
et
al.,
1979),
while
rat
liver
histone
112B,
is
by
far
more
highly
ribosylated
than
the
other
core
histones
(Burzio
et
al.,
1979).
The
ability
of
poly(ADP-
Rib)
polymerase
to
ribosylate
histones
is
influenced
by
the
distribution
of
non-histone
proteins,
the
size
of
the
oligonucleotides,
and
the
stoichiometry
of
the
enzyme
associated
with
isolated
chromatin
fragments
(Jump
and
Smulson,
1980).
Removal
of
non-histone
proteins
from
Geodia
chromatin
prior
to
ribosylation
tends
to
confirm
the
effect
of
non-histone
proteins
upon
the
reaction.
We
found
only
two
bands
of
radioactivity
after
electrophoretic
separation
of
the
histones
(results
not
given).
The
difference
in
the
electrophoretic
profiles
for
endonuclease
digested
chromatin,
extracted
by
Method
A
and
B,
deserve
some
comment.
It
is
known
that
the
removal
of
non-histone
proteins
significantly
alters
the
susceptibility
of
chromatin
to
endonuclease
attack
(Billings
and
Bonner
1972).
However,
there
is
some
evidence
to
suggest
that
the
removal
of
non-
histone
proteins
also
involves
the
extraction
of
a
large
amount
of
salt
soluble
chromatin.
Rakowicz-
Szulczynska
and
Horst
(1981)
have
recently
described
that
extraction
of
mouse
spleen
chromatin
with
0.35
M
NaCI
removed
not
only
proteins,
but
also
50
0
of
the
chromatin.
Identically
treated
Geodia
chromatin
gave
similar
results
with
up
to
600
of
the
chromatin
being
salt
soluble.
This
then
explains
the
quantitative
dif-
ferences
in
the
banding
patterns.
Previously
the
ap-
pearance
of
base
pair
fragments
which
could
not
be
directly
attributed
to
endonuclease
digestion
of
"real"
nucleosome
multimers
indicated
a
modification
of
the
nucleosome.
Selective
removal,
or
proteolysis
of
the
histones
causes
such
a
modification
(Noll
and
Kornberg,
1977;
Smerdon
and
Lieberman,
1981
;
Klingholz
et
al.,
1981).
Under
the
experimental
con-
ditions
described
for
Method
B,
buffers
of
low
ionic
strength,
containing
a
protease
inhibitor,
were
utilised.
This
therefore
rules
out
the
possibility
of
nucleosome
modification
by
histone
proteolysis
or
extraction.
The
significance
of
the
147
base
pair
fragment
which
is
released
so
early
in
the
reaction
and
further
degraded
to
a
110
base
pair
intermediate
remains
to
be
de-
termined.
The
110
and
135
base
pair
fragments,
however,
are
two
of
the
strongest
staining
bands
seen
after
extensive
micrococcal
nuclease
digestion
of
chro-
matin
from
a
variety
of
cell
types
(Lee
Compton
et
al.,
1976).
On
the
basis
of
our
results
it
would
seem
reasonable
to
suggest
that
there
are
some
differences
in
the
specific
sites
open
to
attack
by
micrococcal
nuc-
lease
and
endogenous
endonuclease.
The
ability
of
both
DNase
I
and
endogenous
endonuclease
to
cleave
the
core
particle
at
10
base
intervals
is
further
evidence
that
Geodia
chromatin
is
structurally
similar
to
chro-
Op
ti
ca
l
de
ns
i
ty
(
5
50
nm
)
Chromatin
structure
from
the
marine
sponge
Geodia
cydonium
775
matin
from
higher
eukaryotes.
Furthermore,
exami-
nation
of
Geodia
chromatin
by
electron
microscopy
reveals
the
typical
string
of
beads
structure
associated
with
extracted
chromatin
(to
be
published).
Acknowledgement—This
work
was
supported
by
a
grant
from
the
"Stiftung
Volkswagenwerk"
(1/38
199;
W.E.G.M.).
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