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Selective inhibition by cyclohextimide of nuclear DNA synthesis in synchronous cultures of chlorella
BIOCHEMICAL
Vol. 41, No. 1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SELEWIVE INHIBI'L'ION BY CYCLOHE.&lMIDE OF NUCLEAR DNA SYNTHESIS IN SYNCHRONOUS CIJL:CURES OF CHLORELLA 2. rianka and J. 2Ioors Daboratory of Chemical Cytology, University of Nijmegen, ;'he Netherlands
Received August 2 1, 1970
In synchronous cultures of Chlorella p.yrenoidosa the synthesis of nuclear DNA was inhibited by more than 90% in the presence of 15 @I cycloheximide. Incorporation of externally applied 14C-uracil into nuclear DNA was inhibited to about the same extent, while the inhibitor had only little effect on satellite DNA synthesis. The results suggest that continuous protein synthesis is required for nuclear DNA replication.
the
Cycloheximide
(Actidione)
incorporation
of radioactive
and nucleic
acids
on protein
that
the
the
in
increase
free
of the
in
vitro
to
It
might
be due to
DNA are likely
by the
(5)
cycloheximide
in which
to respond
obtained
and reticulocytes
concentrations with
synthesis in
has been
different
85
in
evidence
(6).
to be a secondary
of DNA polymerase
interference case
ceases Direct
(3,5).
seems
resistance
effect
observation
activities
synthesis
of yeast
protein
supported enzyme
of protein
into
The inhibiting
cycloheximide
15pM
inhibit
(1,Z).
of L)NA synthesis
because
processes,
several
systems
inhibition
effect
of
inhibition
cell
The
of
has been
shown to
precursors
Chlorella
synthesis
presence
of the
of
has been
activity
up to 600pM specific
of nuclear
(7).
regulation and cytoplasmis
ways to the
inhibitor.
BIOCHEMICAL
Vol. 41, No. 1,197O
Synchronous to
study
this
satellite
cycle
p.yrenoidosa
autotrophically
by use of 2-j4
16 hours
purification,
to
nuclear
the during
in
Under
the
undergoes
and 18th
period
(IO).
suitable of two DNA,
the
entire
.
in
moieties
this
time
(8).
more
than
90% in
The labeling
This
an unambiguous of
experiment
the
strong
out
23ra
hour
after
the
beginning
this
time
label
is
incorporated
consists
density
of
of
1.689
shoulder
at
gradient
(8).
of them
(Fig.2.).
a larger g/cm3
a density The
cycloheximide
are
major
of
varied
into during
inhibited
for
light
only
into
only of the
between
(Fig.1).
affected in
the
the
1.700
86
light
the
Therefore,
from
and a smaller
The decrease
the
to be less
component
had
between
15/1&I cycloheximide
lasting
of a'bout
DNA
DNA component
be obtained
of
nuclear
the
DNA synthesis.
inhibitor
gradient
incorporated
became
cannot
nuclear
was carried
which
of
DNA seems
result
density
used,
beginning
presence
of satellite
ana
(8).
incorporation
the
CsCl
of label of
DNA was
extraction
duplications
the
amounts
(9).
after
conditions
after
High
dark
a preparative
4 successive
hour
by alternating
and,
recently
was
Gtittingen
and b hours
experimental
pyrimidine
211/8b
C-uracil
detail
usually
10th
strain
light
banded
as described
by 15~zM
presence
and synchronized
labeled
presence
contrast
are
of the
continuously
of
but
because in
periods
the
pyrenoidosa
(o).
Chlorella grown
Chlorella
possibility
DNAs which, 14 C-uracil
incorporate life
of
cultures
AND BIO~HYSICAL RESEARCH COMMUNICATIONS
19th
another until
period. the
banding
little labeling
During
satellite at
DNA
a buoyant
one appearing g/cm3
the
of the effect
as a CsCl on both
caused
0 and 40%.
This
can
VoL41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fraction
number
Inhibition by cycloheximide of labeling of nuclear DNA. A sterile solution of 20 )-IC 2-'4C-uracil and 4.5poles of cycloheximide in 2 ml dist. water was added to 300 ml of Chlorella culture 15 hours after the beginning of the light period. The cells were collected two hours later, and DNA was extracted and dissolved in 4.4 ml of 9.02 i6 2risHCl buffer pH 8. 5.5 g of CsCl were added and the solution was centrifuged for 65 hours i8 a.Spinco angle rotor type 65 at 35,000 r.p.m. and 4 . Fractions of 8 drops were collected and 2 ml of 0.02 M TrisHCl buffer were added and the optical density (o -0) and the radioactivity (oo) were measured. A. Control without cycloheximide. B. Cycloheximide present.
be mainly
ascribed
pyrimidine to
1406
nucleotides of the
cycloheximide
support content.
in
by IvIorris depends
DNA. Consequently,
synthesis
the
on the
concerning
the
inhibiting
precursor
used
about
experiments
by another 87
increase
of the
the
quantitative
was obtained
by endogenous
to
presence
conclusions
on labeling
label
were found
(1,2),
based
This
of the
which
control
As reported
the
to dilution
inhibitor
(11).
effect for
require
of
of DNA additional
of the
experiment
of
labeling
inhibition
changes
up
DNA in which
VoL41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--I
3x 102
2
E a 0 I
fraction
Fig.
2. iiesistence of satellite DNA synthesis to cycloheximide. Cycloheximide and 14C-uracil were added to 300 ml of Chlorella culture 19 hours after the beginning of the light period. The cells were collected 4 hours later. Further details as in Fig. 1.
aliquots at
of
100
different with
another
4 hours
DNA content
the
untreated
was about
95% at
period
After of the
by 5 to
the
a synchronous
replication
presence
increased
in
and
growth
for
inhibitor,
the
15%. Compared
control any time
culture during
culture
to the the
the
replication
(Fig.3). The results
apparent
cessation times
reported
failure
of protein
of
is
in
yeast
the
DNA increase
the
replication
required
seems unlikely synthesis
here
of nuclear
synthesis
during
formation
the
the
from
cycloheximide.
in
had
inhibition
during
15pUI
DNA synthesis
period
ml were withdrawn
times
supplied
It
number
that
are
in
DNA replication (14). caused period
concomitantly these
agreement
proteins
of DNA or DNA precursors, 88
with in
The almost
the
the absence
complete
by cycloheximide suggests with
that
at
all
protein
DNA synthesis.
are
enzymes
because
involved all
in
VoL41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
light
10
12 time
Fig.
dark
from
1L beginnlng
16 of the
18 Light
20
period
(h)
3. Effect
of cycloheximide on the net increase of DNA. Cells were collected at the given times and the DNA was extracted as described previously (12) and determined by the diphenylamine reaction according DNA content of the control to Burton (13). or culture, o----o DI?A found after growth for 4 hours in the presence of 15 uifi cycloheximide. For experimental details see the text.
enzyme
activities
remain
high
for
cycloheximide considered formed
that only
Mueller
(15).
regard
these
during
histone
nuclear
several (3,4,7).
certain
with
investigated
so far
hours
are
specific
DNA synthesis.
fraotions
the
Chlorella
addition
The possibility
of
has to be proteins Such
which
is
the
as shown by Gallwitz
The investigations to
after
in
regulatory
are being processes
are
case with and
continued
involved
in
DNA synthesis.
Me are much indebted to Mr. J.&A. Aelen for technical assistance and to Dr. I.K. Vasil for kindly checking the manuscript. The investigations were supported in part by the Netherlands Foundation for Chemical Research with financial aid of the Netherlands Organization for the advancement of pure Research. a9
Vol. 41,No.
1,197O
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Morris, I., Nature 211, 1190 (1960). Morris, I., J.Bxp.Bot. 18, 54 (1967). 'v;ianka, F., and Poels C.L.M., Europ.J.Biochem. 9, 478 (1969). ki'anka, F., Joppen, MIX.J., and Ku er, Ch.M.A.,-Leitschr.f. Pflanzenphysiol. 62, 146 (1970 3". Siegel, M.R. and Sisler, H., Biochim.Biophys.Acta 101, 558 (19653. ?f., and Hardesty, B., Biochem.Biophys.Res. 6. McKeehan, Corm. J& 625 (1969). Dissertation of the Faculty of Science, O.Th., 7. Schijnherr, Universi;;o;;ezij;e;e;, (1969). 8. kianka, F., and de Grip, !&.J., Arch.f. Mikrobiol., in ire&: l ' 2, 305 (1965). 9. Yianka, F., Arch.f.Mikrobiol. P.F.M., Arch.f.Mikrobiol. 58, IO. Wanka, F., and Mulders, 257 (1967). results. 11. Vereijken, P.H., un ublished Sanka, F., Planta 5,8 594 (1962). ::: Burton, J., Bi0chem.J. 62, 315 (1956). L.I., Goldring, E.S., and Marmur, J., J.Mol.Biol. 14. Grossman,
:: 2 5.
&&!
15.
Gallwitz
367 (1969).
D.,
and XIeller,
G.C.,
(1969).
90
J.Biol.Chem.
244,
5947
Vol. 41, No. 1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SELEWIVE INHIBI'L'ION BY CYCLOHE.&lMIDE OF NUCLEAR DNA SYNTHESIS IN SYNCHRONOUS CIJL:CURES OF CHLORELLA 2. rianka and J. 2Ioors Daboratory of Chemical Cytology, University of Nijmegen, ;'he Netherlands
Received August 2 1, 1970
In synchronous cultures of Chlorella p.yrenoidosa the synthesis of nuclear DNA was inhibited by more than 90% in the presence of 15 @I cycloheximide. Incorporation of externally applied 14C-uracil into nuclear DNA was inhibited to about the same extent, while the inhibitor had only little effect on satellite DNA synthesis. The results suggest that continuous protein synthesis is required for nuclear DNA replication.
the
Cycloheximide
(Actidione)
incorporation
of radioactive
and nucleic
acids
on protein
that
the
the
in
increase
free
of the
in
vitro
to
It
might
be due to
DNA are likely
by the
(5)
cycloheximide
in which
to respond
obtained
and reticulocytes
concentrations with
synthesis in
has been
different
85
in
evidence
(6).
to be a secondary
of DNA polymerase
interference case
ceases Direct
(3,5).
seems
resistance
effect
observation
activities
synthesis
of yeast
protein
supported enzyme
of protein
into
The inhibiting
cycloheximide
15pM
inhibit
(1,Z).
of L)NA synthesis
because
processes,
several
systems
inhibition
effect
of
inhibition
cell
The
of
has been
shown to
precursors
Chlorella
synthesis
presence
of the
of
has been
activity
up to 600pM specific
of nuclear
(7).
regulation and cytoplasmis
ways to the
inhibitor.
BIOCHEMICAL
Vol. 41, No. 1,197O
Synchronous to
study
this
satellite
cycle
p.yrenoidosa
autotrophically
by use of 2-j4
16 hours
purification,
to
nuclear
the during
in
Under
the
undergoes
and 18th
period
(IO).
suitable of two DNA,
the
entire
.
in
moieties
this
time
(8).
more
than
90% in
The labeling
This
an unambiguous of
experiment
the
strong
out
23ra
hour
after
the
beginning
this
time
label
is
incorporated
consists
density
of
of
1.689
shoulder
at
gradient
(8).
of them
(Fig.2.).
a larger g/cm3
a density The
cycloheximide
are
major
of
varied
into during
inhibited
for
light
only
into
only of the
between
(Fig.1).
affected in
the
the
1.700
86
light
the
Therefore,
from
and a smaller
The decrease
the
to be less
component
had
between
15/1&I cycloheximide
lasting
of a'bout
DNA
DNA component
be obtained
of
nuclear
the
DNA synthesis.
inhibitor
gradient
incorporated
became
cannot
nuclear
was carried
which
of
DNA seems
result
density
used,
beginning
presence
of satellite
ana
(8).
incorporation
the
CsCl
of label of
DNA was
extraction
duplications
the
amounts
(9).
after
conditions
after
High
dark
a preparative
4 successive
hour
by alternating
and,
recently
was
Gtittingen
and b hours
experimental
pyrimidine
211/8b
C-uracil
detail
usually
10th
strain
light
banded
as described
by 15~zM
presence
and synchronized
labeled
presence
contrast
are
of the
continuously
of
but
because in
periods
the
pyrenoidosa
(o).
Chlorella grown
Chlorella
possibility
DNAs which, 14 C-uracil
incorporate life
of
cultures
AND BIO~HYSICAL RESEARCH COMMUNICATIONS
19th
another until
period. the
banding
little labeling
During
satellite at
DNA
a buoyant
one appearing g/cm3
the
of the effect
as a CsCl on both
caused
0 and 40%.
This
can
VoL41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fraction
number
Inhibition by cycloheximide of labeling of nuclear DNA. A sterile solution of 20 )-IC 2-'4C-uracil and 4.5poles of cycloheximide in 2 ml dist. water was added to 300 ml of Chlorella culture 15 hours after the beginning of the light period. The cells were collected two hours later, and DNA was extracted and dissolved in 4.4 ml of 9.02 i6 2risHCl buffer pH 8. 5.5 g of CsCl were added and the solution was centrifuged for 65 hours i8 a.Spinco angle rotor type 65 at 35,000 r.p.m. and 4 . Fractions of 8 drops were collected and 2 ml of 0.02 M TrisHCl buffer were added and the optical density (o -0) and the radioactivity (oo) were measured. A. Control without cycloheximide. B. Cycloheximide present.
be mainly
ascribed
pyrimidine to
1406
nucleotides of the
cycloheximide
support content.
in
by IvIorris depends
DNA. Consequently,
synthesis
the
on the
concerning
the
inhibiting
precursor
used
about
experiments
by another 87
increase
of the
the
quantitative
was obtained
by endogenous
to
presence
conclusions
on labeling
label
were found
(1,2),
based
This
of the
which
control
As reported
the
to dilution
inhibitor
(11).
effect for
require
of
of DNA additional
of the
experiment
of
labeling
inhibition
changes
up
DNA in which
VoL41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--I
3x 102
2
E a 0 I
fraction
Fig.
2. iiesistence of satellite DNA synthesis to cycloheximide. Cycloheximide and 14C-uracil were added to 300 ml of Chlorella culture 19 hours after the beginning of the light period. The cells were collected 4 hours later. Further details as in Fig. 1.
aliquots at
of
100
different with
another
4 hours
DNA content
the
untreated
was about
95% at
period
After of the
by 5 to
the
a synchronous
replication
presence
increased
in
and
growth
for
inhibitor,
the
15%. Compared
control any time
culture during
culture
to the the
the
replication
(Fig.3). The results
apparent
cessation times
reported
failure
of protein
of
is
in
yeast
the
DNA increase
the
replication
required
seems unlikely synthesis
here
of nuclear
synthesis
during
formation
the
the
from
cycloheximide.
in
had
inhibition
during
15pUI
DNA synthesis
period
ml were withdrawn
times
supplied
It
number
that
are
in
DNA replication (14). caused period
concomitantly these
agreement
proteins
of DNA or DNA precursors, 88
with in
The almost
the
the absence
complete
by cycloheximide suggests with
that
at
all
protein
DNA synthesis.
are
enzymes
because
involved all
in
VoL41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
light
10
12 time
Fig.
dark
from
1L beginnlng
16 of the
18 Light
20
period
(h)
3. Effect
of cycloheximide on the net increase of DNA. Cells were collected at the given times and the DNA was extracted as described previously (12) and determined by the diphenylamine reaction according DNA content of the control to Burton (13). or culture, o----o DI?A found after growth for 4 hours in the presence of 15 uifi cycloheximide. For experimental details see the text.
enzyme
activities
remain
high
for
cycloheximide considered formed
that only
Mueller
(15).
regard
these
during
histone
nuclear
several (3,4,7).
certain
with
investigated
so far
hours
are
specific
DNA synthesis.
fraotions
the
Chlorella
addition
The possibility
of
has to be proteins Such
which
is
the
as shown by Gallwitz
The investigations to
after
in
regulatory
are being processes
are
case with and
continued
involved
in
DNA synthesis.
Me are much indebted to Mr. J.&A. Aelen for technical assistance and to Dr. I.K. Vasil for kindly checking the manuscript. The investigations were supported in part by the Netherlands Foundation for Chemical Research with financial aid of the Netherlands Organization for the advancement of pure Research. a9
Vol. 41,No.
1,197O
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Morris, I., Nature 211, 1190 (1960). Morris, I., J.Bxp.Bot. 18, 54 (1967). 'v;ianka, F., and Poels C.L.M., Europ.J.Biochem. 9, 478 (1969). ki'anka, F., Joppen, MIX.J., and Ku er, Ch.M.A.,-Leitschr.f. Pflanzenphysiol. 62, 146 (1970 3". Siegel, M.R. and Sisler, H., Biochim.Biophys.Acta 101, 558 (19653. ?f., and Hardesty, B., Biochem.Biophys.Res. 6. McKeehan, Corm. J& 625 (1969). Dissertation of the Faculty of Science, O.Th., 7. Schijnherr, Universi;;o;;ezij;e;e;, (1969). 8. kianka, F., and de Grip, !&.J., Arch.f. Mikrobiol., in ire&: l ' 2, 305 (1965). 9. Yianka, F., Arch.f.Mikrobiol. P.F.M., Arch.f.Mikrobiol. 58, IO. Wanka, F., and Mulders, 257 (1967). results. 11. Vereijken, P.H., un ublished Sanka, F., Planta 5,8 594 (1962). ::: Burton, J., Bi0chem.J. 62, 315 (1956). L.I., Goldring, E.S., and Marmur, J., J.Mol.Biol. 14. Grossman,
:: 2 5.
&&!
15.
Gallwitz
367 (1969).
D.,
and XIeller,
G.C.,
(1969).
90
J.Biol.Chem.
244,
5947