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Actinomycin inhibition of RNA synthesis in rat liver
BIOCHEMICAL
Vol. 10, No. 3, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
INFUBITION OF RNASYNTRESISIN RAT LIVER
ACTINOMYCIN
Ussx Merits Abbott Laboratories,
Received
January
14,
North Chicago, Illinois
1963
Actinomycin D hss been used successfully
as an analytical
tool in virus
research
and in studies of the synthesis of RNA. Bssed on the inhibition
cellular
RNA synthesis by actinomycin D, Reich, Franklin,
Shatkin and Tatum
(1962) suggested that the entire casplement of RRA(nuclear, transfer)
of bacterial
sensitive
and DNA-dependent reaction.
(1962) SUT~VM at in B. subtilis
~iaisr
actinomyein D blocks RNA synthesis
RNA polynerase but it does not affect
Very recently
RRA
Tsmaoki aud Mueller (1962) have studied
of all alasscs of RNAis iuhibitea
48 RNAbeing inhibited nucleotides
tot&L synthesis.
audAlexauder
of P32 orthophosphate into RRA in ReLa cells.
show that labeling
teminal
is produced in an actinomycin-
au enzyme which adds CMPen& AMP to the end of the
RNAchain.
ths inaorporation
ribommsl and
Rurwitz,Furth,Malsmy
CO~C~US~OIIS:
by inhibiting
pyrophosphorylase, transfer
and msmelieh cells
of
by actincmycin D, the
to a lesser extent than the other types. of transfer
Their data
The three
RNA (48 RNA) turn over independently of
These authors conelude that the nudleus is the reat of all
RRA synthesis in IQ&a cells. The evidence presented below indicates that aatiuomycin D inhibits incorporation
of P32 orthophosphate
in all
fozms of RXA in rat
liver.
the There
is else evidence that under these conditions the terminal triuucleotide sequmae pCpCpAin the 45 portion
of RNAcarries all the label.
Mile albino rats of the Roltsmauu strain weighing approximately were fasts& for 24 hours, then injected
intraperitoneally
254
with 1 mg.
250 g.
vol. 10. No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sctinomycin D dissolved in 0.2 ml. propylene glycol.
Control sninls3s
Three hours later
received 0.2 ml. of propylene glycol.
10 mCP32 ortho-
phosphate vas injected by the sane route and the rats were killed thereafter.
The livers
four hours
were immediately homogenized in a Waring Blendor
with 70 ml. of phenol (MsUinckrodt,
Analytical
Reagent, 884) snd 60 ml. of
a solution consisting of 0.01 M phosphate buffer 1 mMtrisodium citrate
and 0.05s sodium lauryl
ph 6.8, 1 mMVersene, sulfate to which 0.05 g.
Bentonite was added. Bentonite V&B prepared according to Fraenkel-Conrat, Singer and Tsugita (1961). lauryl
sulfate
The combined
use of phenol snd 0.05s sodium
has been shownto cause the release of over 90$ of the total
RNAkeeping its contamination with DNAat a low level (Dingmsn and Sporn, 1962).
All extraction
procedures vere carried out at 4'.
The homogenate
was shaken for one hour, the emulsion was broken by centrifugation aqueous layer extracted
and the
once more with 40 ml. phenol and 0.03 g. Bentonite.
The water solution was made 0.2 M with respect to potassium acetate buffer (pH 5.6) and the RNAwas precipitated chilled
to -20' with dry ice.
from the 0.2 M acetate buffer
by the addition of 2 volumes of ethanol
The RNAwas then reprecipitated
three times
solution with 2 volumes of ethanol.
ssntples contained less that l$ DNAand l$ protein.
The final
These
precipitate
wss dissolved in 0.15 M NsCl to yield a l$ solution of IQJA, which was quickly
frozen and stored at -20'.
Three methods were used for the fractionation In the preliminary
of the RNAprepsrations.
experiments we applied the method of Barshaw, Brown and
Graham(1962) which is based on differential with streptomycin and 1 M N&l.
The fractions
precipitation
were dissolved in 2 ml. 0.15 M
NaCl and counted In a Baird Atomic scintillation Sucrose gradient centrifugatlon swinging-bucket rotor
at 4'.
spectrometer.
was carried out in a Spinco SW-39
The sample (O.lml.)
4.6 nip. linear gradient from 5-20s and centrifuged Fractions of 8 drops were collected
of RNAcomponents
was layered on top of a for 16 hours at 20,000 rpp.
and 3.0 ml. water added to each. 255
After
Vol. 10, No. 3, 1963
BIOCHEMICAL
measurementof the optical
AND BIOPHYSKAL
RESEARCH COMMUNICATIONS
density at 260 mu the fractions
were evaporated on
aluminum planchets and counted in a gas-flow counter (covered with Mylar film). For the chromatographic fractionation
of RNA, the methylated albumin-
Kieselguhr (MAK) columns were prepared according to a simplified
Mandell-
Hershey procedure as described by Monier, Maono, Hayes, &yes and Gras (1962). A ssmple of 0.8 ml. (8 mg. RNAin 0.3 M NaCl) was applied and eluted under a linear
concentration
gradient between 0.3 H and 1.3 M NeCl both solutions
containing 0.05 M phosphate buffer
(p3 6.7).
150 cm. gave a flow rate of 50 ml./hr.
A hydrostatic
Fractions
pressure head of
of 3.5 ml. were collected.
Base analyses were carried out by the ionophoresis method of Davidson and Smellie (1952). light
The paper strips
and in a4nradio
were then scanned under ultraviolet
chrcanatogram scanner.
Finally
on the paper strips were cut out and counted directly
the nucleotide bands in the gas-flow counter.
RESULTS
In preliminary precipitation
experiments the RNAwas fractionated
technique, both actinomycin C (Sansmycin, Farbenfarbriken
AS) and D were used as inhibitors after
by the streptcmycin
the injection
and the rats were killed
of P32 orthophosphate.
actinomycin C and D in inhibiting
one to six hours
There is no difference
FtNAsynthesis in rat liver
of labeling
In ionophoretic
2) illustrate
DNA(4s BNA) that result anslysis unfractionated
animals revealed, under ultraviolet cytidylic, activity,
adenylic,
light,
gusnylic and uridylic
of high molecular
1) and frcna chramatographic the almost caplete
of label in the ribostmlal RNApeaks and the partial of Pj2 into transfer
It
of 4s RNA is much less pronounced.
Data from sedimentation experiments (Fig. separations on a KAK column (Fig.
between
or kidney.
CSn be seen frcm Table I that actinomycin prevents the labeling Veisht RNA, while the inhibition
Bayer
disappearance
blockage of the incorporation
f+rcm actinomycin D. RNApreparations
from control
bands for the four main nucleotides: acids.
When scanned for radlo-
all of these bends showed, as expected, the presence of P32. However,
the P32 content wss greatly
reduced in the adenylic, 256
guanylic and uridylic
acid
Vol. 10, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I Incorporation
of P32 into Rat RNA
4s RN4
Time of labeling hrs.
Tissue
1
liver
CMP/g tissue
$ of control
4050 1733
42.8
5045 1500
control
cw 1
kidney
control
C 6
liver
control
C 4
liver
control
Dw
I
CMP/g tissue
$ of control
17925
456
2.5
29.8
23250 535
2.3
1745 716
40.6
64800 370
0.6
~96 1030
47.0
3~60 930
3.0
*C refers to actinomycin C treated animal. I
High molecular weight RNA
enim3J.s; D refers to actlnomycin D treated
CPM
1
OD
I
I
CPM
I
000
-
000
CONTROL
3
t
ACTINOMYCIN
D
750
750
500
500
250
250
P ‘UBE NO
I 10
Fig.
I 20
1.
I 30 Sedlmentotion
0
profiles
of rot
TUBE NO 10 liver RNA labeled
20 P32
with
30
40
‘i -
000
CONTROL
300
6-
300
6-
100
4-
ACTINOMYCIN
D
800
600
400
4s
2.4.6.6.,oo ,20 ,40 ,60 ,6j:ji$&k!^_,., ______ d 200
0
20
Fig. 2.
TUBE NO Chromotoqrophlc
fractionatmns
of rat
257
liver
RNA
40
60
labeled
80
IO0 120 TUBE NO wth P32
140
160
180 201 1
Vol. 10, No. 3, 1963
bands after activity
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the rats had been treated with actinomycin D, and most of the radio-
was present in the cytidylic
acid (Table IX).
Table II Pj2 -labeled Nucleotide Composition of Total Rat Liver RNA The values given are moles/100 moles 2'- and 3'-nuc!.eotides Cytiaylic Acid
g:,'
Control*
67.6 63.8
Actinomycin II*
*Values are duplicate
It
determinations
ia known that rat liver
similar to that of the total
specific
32.0
33.5
19.2 17.3
12.1
9.9
13.1
12.2
RNAhas a nucleotide
10.4 10.9 hydrolysates
composition quite
the fact ,that after
RNApreparations
actino-
(of which the 4s part acid content suggests some
Since the three terminal nucleotides(pCpCpA)
RNAturn over independently f+rcm the rest
may carry the label.
In this case, after
the label should occur in cytidylic distributed
17.5 20.0
had such a high cytidylic
mechanismfor labeling.
of transfer
Uridylic Acid
RNA. Therefore,
mycin D treatment the unfractionated carried 97s of the label)
Cuanylic Acid
from the samealkaline
transfer
liver
Adenylic Acid
alkaline
of the RX?Achain, they
hydrolysis
at least 672 of
acid and the remaining P32 should be
between the nucleotides next to the pCpCpAsequence. That the
pCpCpAsequence indeed carries most, if not all,
of the label is indicated
by the data in Table II. In conclusion this investigation
showsthat
in the presence of actinomycin D
the synthesis of aJ.l forma of &A except the p~pt!pA end groups of transfer is inhibited
in rat liver.
tions of others in cell
These findings
confim
in the intact
RRA
mammaIobserva-
cultures.
The author would like to thank Mr. Werner Schulze for his excellent technical assistance. AtM.nomyuin Dwaa agift fromMerckSharp andDohrpe ReeeaFch Laboratories. 258
vol. 10, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References Davidson, J. N., EIII~R. M. S. Smellie, Blochem. J., 2, 594 (1952). Din&man, W., and M. B. @xn, BiOChim. BiOphp. ht8, 61, 164 (1962). Fraenkel-Conrat, H., Singer, B., and A. Teugita, Virology, 14, 54 (1961). Hsrshaw, J. P., Brown, R. A., and A. F. Graham, Anal. Biochem. 4, 182 (1962). Hurwitz, J., Furth, J. J., Malsmy, M., and M. Alexander, Proc. Nat. Acad. Sci. 48, 1222 (1962). Monier, R., Naono, S., Hayes, D., Hayes, F., and F. Gras, J. Mol. Biol., 2,
w. (1962).
Reich, E., Franklin, R. M., Shatkin, A. J., and E. L. Tatm, Proc. Nat. Acad. Sci., 48, 1238 (1962). Temmki, T., end 0. C. Mueller, Biochem. Biophys. Res. Coxmn.,p, 451 (1962).
259
Vol. 10, No. 3, 1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
INFUBITION OF RNASYNTRESISIN RAT LIVER
ACTINOMYCIN
Ussx Merits Abbott Laboratories,
Received
January
14,
North Chicago, Illinois
1963
Actinomycin D hss been used successfully
as an analytical
tool in virus
research
and in studies of the synthesis of RNA. Bssed on the inhibition
cellular
RNA synthesis by actinomycin D, Reich, Franklin,
Shatkin and Tatum
(1962) suggested that the entire casplement of RRA(nuclear, transfer)
of bacterial
sensitive
and DNA-dependent reaction.
(1962) SUT~VM at in B. subtilis
~iaisr
actinomyein D blocks RNA synthesis
RNA polynerase but it does not affect
Very recently
RRA
Tsmaoki aud Mueller (1962) have studied
of all alasscs of RNAis iuhibitea
48 RNAbeing inhibited nucleotides
tot&L synthesis.
audAlexauder
of P32 orthophosphate into RRA in ReLa cells.
show that labeling
teminal
is produced in an actinomycin-
au enzyme which adds CMPen& AMP to the end of the
RNAchain.
ths inaorporation
ribommsl and
Rurwitz,Furth,Malsmy
CO~C~US~OIIS:
by inhibiting
pyrophosphorylase, transfer
and msmelieh cells
of
by actincmycin D, the
to a lesser extent than the other types. of transfer
Their data
The three
RNA (48 RNA) turn over independently of
These authors conelude that the nudleus is the reat of all
RRA synthesis in IQ&a cells. The evidence presented below indicates that aatiuomycin D inhibits incorporation
of P32 orthophosphate
in all
fozms of RXA in rat
liver.
the There
is else evidence that under these conditions the terminal triuucleotide sequmae pCpCpAin the 45 portion
of RNAcarries all the label.
Mile albino rats of the Roltsmauu strain weighing approximately were fasts& for 24 hours, then injected
intraperitoneally
254
with 1 mg.
250 g.
vol. 10. No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sctinomycin D dissolved in 0.2 ml. propylene glycol.
Control sninls3s
Three hours later
received 0.2 ml. of propylene glycol.
10 mCP32 ortho-
phosphate vas injected by the sane route and the rats were killed thereafter.
The livers
four hours
were immediately homogenized in a Waring Blendor
with 70 ml. of phenol (MsUinckrodt,
Analytical
Reagent, 884) snd 60 ml. of
a solution consisting of 0.01 M phosphate buffer 1 mMtrisodium citrate
and 0.05s sodium lauryl
ph 6.8, 1 mMVersene, sulfate to which 0.05 g.
Bentonite was added. Bentonite V&B prepared according to Fraenkel-Conrat, Singer and Tsugita (1961). lauryl
sulfate
The combined
use of phenol snd 0.05s sodium
has been shownto cause the release of over 90$ of the total
RNAkeeping its contamination with DNAat a low level (Dingmsn and Sporn, 1962).
All extraction
procedures vere carried out at 4'.
The homogenate
was shaken for one hour, the emulsion was broken by centrifugation aqueous layer extracted
and the
once more with 40 ml. phenol and 0.03 g. Bentonite.
The water solution was made 0.2 M with respect to potassium acetate buffer (pH 5.6) and the RNAwas precipitated chilled
to -20' with dry ice.
from the 0.2 M acetate buffer
by the addition of 2 volumes of ethanol
The RNAwas then reprecipitated
three times
solution with 2 volumes of ethanol.
ssntples contained less that l$ DNAand l$ protein.
The final
These
precipitate
wss dissolved in 0.15 M NsCl to yield a l$ solution of IQJA, which was quickly
frozen and stored at -20'.
Three methods were used for the fractionation In the preliminary
of the RNAprepsrations.
experiments we applied the method of Barshaw, Brown and
Graham(1962) which is based on differential with streptomycin and 1 M N&l.
The fractions
precipitation
were dissolved in 2 ml. 0.15 M
NaCl and counted In a Baird Atomic scintillation Sucrose gradient centrifugatlon swinging-bucket rotor
at 4'.
spectrometer.
was carried out in a Spinco SW-39
The sample (O.lml.)
4.6 nip. linear gradient from 5-20s and centrifuged Fractions of 8 drops were collected
of RNAcomponents
was layered on top of a for 16 hours at 20,000 rpp.
and 3.0 ml. water added to each. 255
After
Vol. 10, No. 3, 1963
BIOCHEMICAL
measurementof the optical
AND BIOPHYSKAL
RESEARCH COMMUNICATIONS
density at 260 mu the fractions
were evaporated on
aluminum planchets and counted in a gas-flow counter (covered with Mylar film). For the chromatographic fractionation
of RNA, the methylated albumin-
Kieselguhr (MAK) columns were prepared according to a simplified
Mandell-
Hershey procedure as described by Monier, Maono, Hayes, &yes and Gras (1962). A ssmple of 0.8 ml. (8 mg. RNAin 0.3 M NaCl) was applied and eluted under a linear
concentration
gradient between 0.3 H and 1.3 M NeCl both solutions
containing 0.05 M phosphate buffer
(p3 6.7).
150 cm. gave a flow rate of 50 ml./hr.
A hydrostatic
Fractions
pressure head of
of 3.5 ml. were collected.
Base analyses were carried out by the ionophoresis method of Davidson and Smellie (1952). light
The paper strips
and in a4nradio
were then scanned under ultraviolet
chrcanatogram scanner.
Finally
on the paper strips were cut out and counted directly
the nucleotide bands in the gas-flow counter.
RESULTS
In preliminary precipitation
experiments the RNAwas fractionated
technique, both actinomycin C (Sansmycin, Farbenfarbriken
AS) and D were used as inhibitors after
by the streptcmycin
the injection
and the rats were killed
of P32 orthophosphate.
actinomycin C and D in inhibiting
one to six hours
There is no difference
FtNAsynthesis in rat liver
of labeling
In ionophoretic
2) illustrate
DNA(4s BNA) that result anslysis unfractionated
animals revealed, under ultraviolet cytidylic, activity,
adenylic,
light,
gusnylic and uridylic
of high molecular
1) and frcna chramatographic the almost caplete
of label in the ribostmlal RNApeaks and the partial of Pj2 into transfer
It
of 4s RNA is much less pronounced.
Data from sedimentation experiments (Fig. separations on a KAK column (Fig.
between
or kidney.
CSn be seen frcm Table I that actinomycin prevents the labeling Veisht RNA, while the inhibition
Bayer
disappearance
blockage of the incorporation
f+rcm actinomycin D. RNApreparations
from control
bands for the four main nucleotides: acids.
When scanned for radlo-
all of these bends showed, as expected, the presence of P32. However,
the P32 content wss greatly
reduced in the adenylic, 256
guanylic and uridylic
acid
Vol. 10, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I Incorporation
of P32 into Rat RNA
4s RN4
Time of labeling hrs.
Tissue
1
liver
CMP/g tissue
$ of control
4050 1733
42.8
5045 1500
control
cw 1
kidney
control
C 6
liver
control
C 4
liver
control
Dw
I
CMP/g tissue
$ of control
17925
456
2.5
29.8
23250 535
2.3
1745 716
40.6
64800 370
0.6
~96 1030
47.0
3~60 930
3.0
*C refers to actinomycin C treated animal. I
High molecular weight RNA
enim3J.s; D refers to actlnomycin D treated
CPM
1
OD
I
I
CPM
I
000
-
000
CONTROL
3
t
ACTINOMYCIN
D
750
750
500
500
250
250
P ‘UBE NO
I 10
Fig.
I 20
1.
I 30 Sedlmentotion
0
profiles
of rot
TUBE NO 10 liver RNA labeled
20 P32
with
30
40
‘i -
000
CONTROL
300
6-
300
6-
100
4-
ACTINOMYCIN
D
800
600
400
4s
2.4.6.6.,oo ,20 ,40 ,60 ,6j:ji$&k!^_,., ______ d 200
0
20
Fig. 2.
TUBE NO Chromotoqrophlc
fractionatmns
of rat
257
liver
RNA
40
60
labeled
80
IO0 120 TUBE NO wth P32
140
160
180 201 1
Vol. 10, No. 3, 1963
bands after activity
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the rats had been treated with actinomycin D, and most of the radio-
was present in the cytidylic
acid (Table IX).
Table II Pj2 -labeled Nucleotide Composition of Total Rat Liver RNA The values given are moles/100 moles 2'- and 3'-nuc!.eotides Cytiaylic Acid
g:,'
Control*
67.6 63.8
Actinomycin II*
*Values are duplicate
It
determinations
ia known that rat liver
similar to that of the total
specific
32.0
33.5
19.2 17.3
12.1
9.9
13.1
12.2
RNAhas a nucleotide
10.4 10.9 hydrolysates
composition quite
the fact ,that after
RNApreparations
actino-
(of which the 4s part acid content suggests some
Since the three terminal nucleotides(pCpCpA)
RNAturn over independently f+rcm the rest
may carry the label.
In this case, after
the label should occur in cytidylic distributed
17.5 20.0
had such a high cytidylic
mechanismfor labeling.
of transfer
Uridylic Acid
RNA. Therefore,
mycin D treatment the unfractionated carried 97s of the label)
Cuanylic Acid
from the samealkaline
transfer
liver
Adenylic Acid
alkaline
of the RX?Achain, they
hydrolysis
at least 672 of
acid and the remaining P32 should be
between the nucleotides next to the pCpCpAsequence. That the
pCpCpAsequence indeed carries most, if not all,
of the label is indicated
by the data in Table II. In conclusion this investigation
showsthat
in the presence of actinomycin D
the synthesis of aJ.l forma of &A except the p~pt!pA end groups of transfer is inhibited
in rat liver.
tions of others in cell
These findings
confim
in the intact
RRA
mammaIobserva-
cultures.
The author would like to thank Mr. Werner Schulze for his excellent technical assistance. AtM.nomyuin Dwaa agift fromMerckSharp andDohrpe ReeeaFch Laboratories. 258
vol. 10, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References Davidson, J. N., EIII~R. M. S. Smellie, Blochem. J., 2, 594 (1952). Din&man, W., and M. B. @xn, BiOChim. BiOphp. ht8, 61, 164 (1962). Fraenkel-Conrat, H., Singer, B., and A. Teugita, Virology, 14, 54 (1961). Hsrshaw, J. P., Brown, R. A., and A. F. Graham, Anal. Biochem. 4, 182 (1962). Hurwitz, J., Furth, J. J., Malsmy, M., and M. Alexander, Proc. Nat. Acad. Sci. 48, 1222 (1962). Monier, R., Naono, S., Hayes, D., Hayes, F., and F. Gras, J. Mol. Biol., 2,
w. (1962).
Reich, E., Franklin, R. M., Shatkin, A. J., and E. L. Tatm, Proc. Nat. Acad. Sci., 48, 1238 (1962). Temmki, T., end 0. C. Mueller, Biochem. Biophys. Res. Coxmn.,p, 451 (1962).
259