BIOCHEMICAL
Vol. 88, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 27, 1979
Pages 1329-1336
THE MESOKARYOTE GYRODINIUM COHN11 LACKS NUCLEOSOMES Steven
Bodansky,
Lauri
B. Mints
and David
S. Holmes
Department of Biological Sciences State University of New York at Albany Albany, New York 12222
Received
May 3,1979
SUMMARY The dinoflagellate Gyrodinium cohnii has a distinct nuclear membrane but apparently lacks histones associated with its chromatin. Approximately 13% of the nuclear DNA is rapidly digested by micrococcal nuclease to acid soluble fragments and not to nucleosomal sized fragments as in the typical eukaryote. Moreover in the electron mbcroscope the chromatin of 5. cohnii appears as a thin filament of 40-60 A in width without regularly spaced nucleosomes. These observations support the view that the dinoflagellates exhibit characteristics of both prokaryotes and eukaryotes.
Gyrodinium is
cohnii --___
a unicellular
marine,
the common name for algae
in the plant
mesokaryotes organisms appears
(1).
There
are
microscopy fibrils
compared Moreover,
or morphological DNA per
cell
Abbreviations methylsulfonyl
have
Pyrophyta,
features
no normal
associated
to the
of both but
sections
(2). with reveal
prokaryotic
the nuclear remain
briefly
spindles
elements
seven
just
No histones the
never
the presence
division
is
and electron
of thin
(40-60
unicellular
with
differentiation
it
biochemical
the same amount sequences
used: EDTA, ethylenediaminetetraacetic fluoride; TCA, tricarboxylic acid.
acid;
(5).
All
rights
of
We
PMSF, pheny-
0006-291X/79/121 Copyright
1329
A)
eukaryotes
little
has approximately
DNA has reiterated
pro-
(3),
its
dis-
to mitosis
or histone-like
chromatin
nature
as
throughout
prior
and nuclear
of
and eukaryotic
condensed
of typical
as a human and this
divisions
envelope
100 i to 300 i fibrils
despite
is
have been classified
decondensing
mitotic
cohnii,
Dinoflagellate
one of the
The chromosomes
only
of nuclear
dinoflagellate.
has a nucleus
cycle,
can be detected
known as Crypthecodinium
Dinoflagellates
(1).
by microtubular
teins
(4).
they
mitosis cell
assisted
kingdom.
5. cohnii
during
also
heterotrophic
the division
in that
most of the (1).
(G. cohnii),
@ I979
329-08$01.00/O
by Academic
of reproduction
in any
Press,
Inc.
formreserved,
BIOCHEMICAL
Vol. 88, No. 4, 1979
have extended
this
nucleosome-type tron
survey
repeats
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and show that as judged
G. cohnii -___
by nuclease
has no nucleosomes
digestion
studies
or
and elec-
microscopy.
Materials
and Methods
5. cohnii strain Woods Hole was kindly provided by Dr. Beach. Cells were maiadmerile culture and nuclei were prepared essentially as described by Riszo and Nooden (3). Micrococcal nuclease digestion was carried out essentially by the method of No11 (14) as modified by Newman and Noon (15). 10 1.11 (150 units) of micrococcal nuclease (Worthington) were added to 90 ul of nuclei (130 A260 units per ml) in 10 mM Na borate-l mM CaC12, pH 7~6, and the suspensron incubated at 37'C for varying times. The reaction was quenched by placing on ice, and the nuclei sedimented at 2000 x g for 10 min at 4OC. The A260 of the supernatant was read directly or was made 7.5% tricarboxylic acid at 4OC, sedimented at 400 x g for 10 min and the A260 of the supernatant read. When nuclei were treated as described above without added en&me, 0.68% of the initial A260 absorbing material was liberated into the supernatant after 10 min of incubation at 37OC, indicating that endogenous nuclease digestion is not significant. Gel electrophoresis was carried out essentially by the method of Varshavsky Nuclei were digested for 10 min as described --et al. (17). above and the reaction quenched at 4O with the addition of an equal volume of 20 mM ethylenediaminetetraacetic acid (EDTA) and the nuclei sedimented at 2000 x g for 10 min. Nucleic acids were extracted from either the nuclei or the supernatant essentially by the method of Blin and Stafford (16) and concentrated by precipitation with ethanol. The nucleic acids were resuspended in 10 mM'Na borate - 5 mM EDTA, pH 9.2 - 20% glycerol and electrophoresed on a 5% polyacrylamide gel at 150 volts and visualized by staining with ethidium bromide (17). Chicken chromatin or chromatin isolated from G. cohnii nuclei (3) was prepared for electron microscopy by fixation with 0.1% gluteraldehyde essentially as described by Thoma and Keller (18). Unstained chromatin was supported with benzalkonium (Mobay Chemical Corp.) and picked up by the droplet procedure (18) onto carbon grids and rotary shadowed with platinum-palladium. Results
and Discussion
Rizzo tones
and Nooden
in isolated
that
yeast
least
four
similar of Rizzo
did
--G. cohnii
five
major
with
5. cohnii,
and Nooden
methylsulfonyl
chromatin.
not have histones,
of the
ambiguity
(3) have demonstrated
using
fluoride
However,
and then
species
a well
the
later
(6, 19).
lack
of eukaryotic
it
was initially
it
was shown to have at
to repeat
characterized
protease
stages
1330
thought
Due to the possibility
we decided
(PMSF) at all.
his-
of nuclear
the
of a
experiments
inhibitor, and chromatin
phenyliso-
BIOCHEMICAL
Vol. 88, No. 4, 1979
Following
lation. the
addition
essentially
the procedure
of 0.1 mM PMSF to all
any histone-like
proteins
electrophoresis,
by extraction
tin
with
0.2
HCl,
N
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
solutions,
by sodium
the results
of Rizzo
and Nooden
acid
extractable
proteins
in either
that
have
suming
electrophoretic
that
tivated
there
organize
eukaryotes
the DNA into
discrete
is associated
number
depending
nucleosome
upon
contains
of two copies
shown). that
to eukaryotic protease
chroma-
Thus,
we con-
there
are no chromatin
histones,
that
with
for
For example, of polypeptide
is not
asinac-
unit
in the prokaryote
Thermoplasia
material
with
1 shows a time
from -~ G. cohnii
have not performed the
reaction
decrease soluble.
studied.
found
of the
with
an octomer
with
about
40 base pairs
course
of release
by
micrococcal
digestion occur
in the rate
after
utilizing
it
where
where
could
repeating other six
200 base pairs
proteins. copies
of DNA (8)
a "histone-like"
of DNA (9). of ultraviolet
absorbing
nuclease.
of G. -- cohnii
at a constant
structure
However,
in adenovirus
about
does not
An ultraviolet
of DNA, the actual
of chromosomal
but
(7).
The core
(7).
type
acidophilum,
nuclei
extensive
kingdom
which
microscopy
of DNA associated
of histones
is
are associated
Figure
being
of Hl,
nucleosomes
200 base pairs
or analogous
in the absence
is associated
termed
exception
HZa, H2b, H3 and H4 and this
VII
protein
about
the eukaryotic
a repeating
the
or by electron
the organism
a homologous
to be formed
with
units
digestion
140 base pairs
throughout
be possible
acid
or purified
or in isolated
histones,
repeating
each of histones
invariant
marked
not
nuclei
histone
the major
The nucleosome
that
nuclei
similar
is not a specific
by nuclease
or
whole
to detect
(SDS) polyacrylamide
(3) and conclude
nobilities
can be detected
unit
whole
unable
(3) with
by PMSF. In typical
is
sulfate
0.4 N H2S04 (data
firm
and Nooden
we were
dodecyl
of either
or with
of Rizzo
rate
Although
nuclei, but
it
that
we is
clear
there
is
some 13% of the DNA has been rendered
spectrum
of the released
1331
material
was
a
Vol. 88, No. 4, 1979
BIOCHEMICAL
2.0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
r
-'5cl cm
5
0.
-IO' -5 I 5
0
I I 15 20 Minutes
I IO
I 25
I 30
3 6 a-"
35
Figure 1: Time course of the release of A26O absorbing material from G. cohnii nuclei during treatment with micrococcal nuclease. Co) A260 ofthe material released from the nuclei into the supernatant. (0) A260 of the supernatant after precipitation with 1.5% TCA. typical
of purified
be noted acid,
that
chromatin
all
the liberated
indicating
that
twenty
residues
(10).
firmed
by the
fact
weight
marker
in an agarose
possibility
that,
--G. cohnii
is
it
it
agarose
gel
electrophoresis
whole
DNA (Fig.
discrete
bands
bottom
of the gel
typical
larger
than
2, lane
enzyme as judged
are
digestion
nucleases
during
digestion
sized
incubated
is
gel
rendered is
than
product
is
is
the DNA is
gradually
the nuclear 2, lane
the
membrane
B.
of
the
DNA by The majority
and co-electrophoreses
with
a smear of DNA with and extending
No significant fdr
electrophoresis acid
to the
without (Fig.
soluble)
reduced
no
digestion
10 minutes
not organized
1332
con-
DNA, we disrupted
of added
2, lane
indicating
that
Our interpretation
not significant.
of --G -cohnii
about
molecular
To eliminate
600 base pairs of DNA).
of less
the nuclear
However,
by either
(0.68%
that
C).
below
when nuclei
the chromatin
eukaryote,
600 base pairs
D) or by TCA solubility
is that
E).
should
in 5% trichloroacetic
a 70 base pair
and analyzed
(( 70 base pairs
by endogenous
2, lane
to nucleosome
is detectable
DNA is detectable
(Fig.
than
It
shown).
released
as shown in figure
is
not
soluble
of the faster
digestion
of the product purified
is
size
gel
the
(data
of polynucleotides
migrates
not permeable micrococcal
material
The small
unlike
after
--G. cohnii
consists
that
nuclei
micrococcal
for
into in size
subunits until
and it
is
Vol.
88,
No.
BIOCHEMICAL
4, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 2: Acrylamide gel electrophoresis of DNA liberated from G. cohnii after 10 min digestion with micrococcal nuclease. (A) = Hae III-restriction digest of $X174 (New England Biolabs). (B) = extracted from nuclei after micrococcal digestion. (C) = DNA extracted from nuclei without prior nuclease digestion. (D) = DNA extracted from nuclei after appropriate incubation but without added nuclease. (E) = liberated from G. cohnii nuclei after micrococcal digestion. Digestion with micrococcal nuclease was carried out for 10 min. Number of base pairs of the Hae III digest are shown on the left.
rendered
soluble.
susceptible
more
the
acid
to digestion of the chromatin
majority
giving
it
highly
condensed
a
Figure pared chicken
We do not than is
state
of the
in
10% of the chromatin It
is possible
semicrystalline
form
is that
(11)
and accounting
for
the
chromatin. micrograph
of a typical
is organized
some
from digestion
3a shows an electron
to the chromatin
some
the remainder.
of protection
measure
chromatin
know why
into
of -~G. cohnii
eukaryote
(chicken)
repeating
units
1333
which
chromatin (Fig.
3b).
measure
com-
The some
BIOCHEMICAL
Vol. 88, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 3: Electron microscope visualization (b) G. chromatin. Scale bar = 800 i. -- cohnii 115 i in diameter 60 i wide
with
whereas only
additional
thickening
shadowing,
these
either
naked
similar
due to the
dimensions
that
dimensions
The taxonomic they
occasional
are
DNA or DNA uniformly
Thus we conclude of
very
the 5. cohnii
have a nuclear
there
membrane
(a) chicken
chromatin
thickening. supporting
covered
ions
for
of the dinoflagellates
(13),
or other
about some
and to the
of nucleosomes in proteins
or
as a thread
benzalkonium
of 5
1334
appears
allowing
are no nucleosomes
and a relatively
chromatin
After
characteristic
in the chromatin position
of
(12)
and
respectively.
repeating
units
cohnii. is not enormous
clear.
amount
Since of DNA,
BIOCHEMICAL
Vol. 88, No. 4, 1979
including
reiterated
may represent
a class
intermediate survey
sequences,
between
but
involved
in the
genes.
not -~ G. cohnii determine
if
Thus
exhibit
it
appears
At least
would
another
some of these
has intervening
sequences
the mRNA of --G. cohnii
an extensive that
is
of intervening
sequences
are presumably
of heterogeneous
be interesting
they
properties
feature
the presence
and maturation it
Although
that
is
or nucleosomes,
some ancestral
and eukaryotes.
condition
in the processing
[HnRNAl to mRNA.
that
prokaryotes
of the eukaryotic
sequences
have no histones
of organisms
has not been undertaken
diagnostic
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
nuclear
to ascertain
RNA
whether
or
and HnRNA, and additionally
to
is polyadenylated.
Acknowledgments This tutes
work was supported
of Health
in part
by a grant
from the National
Insti-
[GM24295].
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