BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TRANSCRIPTION AND REISOLATION OF THE SIMIAN VIRUS 40 NUCLEOPROTEIN COMPLEX Mark R. Hall Department Received
March
of Cell and Molecular Georgia, Augusta,
Biology, Georgia
Medical 30902
College
of
30,1977
WY: Nucleoprotein complexes, containing cellular histones associated man Virus 40 DNA, were isolated from virus infected cells. These complexes supported RNA synthesis when incubated with E. coli DNAdependent RNA polymerase. Under the conditions of assay, transcripts synthesized by E. coli polymerase were shown to be highly specific for viral DNA by hy6rimion assay. Of particular importance is the demonstration that the viral nucleoprotein complexes isolated from the transcription mixture have not been physically altered. INTRODlJCTION Viral
nucleoprotein
complexes @PC), isolated
(SV40) or polyoma virus have been reported
virions
to contain
from Simian Virus 40
(1,2) and from virus four cellular
infected
histones
cells
(7,8).
(3-6),
These histones,
H2A, H2B, H3, and H4, are also found in the nucleosomes characteristic cellular viral
chromatin NPCs contain
to cellular
microscopic
have shown that
between viral
on transcription
NPC and cellular
chromatin
provides
(12,13).
models for studying of a defined
the effects
genome.
The present
evidence that the viral
model chromosomes (mini-chromosomes) of their
in structure
suggests the
Indeed,
have shown that polyoma and SV40 NPCs can be transcribed
and in addition
the
(10,ll).
and endogenous polymerases
ation
studies
20 nucleosomes and are similar
that NPCs might be ideal
chromosomal proteins studies
Electron
approximately
chromatin
The similarity possibility
(9).
of
report
recent
by exogenous
supports these studies
NPCs can be utilized
in a transcription
of
assay without
as alter-
structure. MATERIALSANDMETHODS
African green monkey kidney cells ((X-1) were infected with strain WI911 SV40 virus at a multiplicity of lo-20 pfu/cell. At 40 h postinfection, cultures were pulsed with [3H]TdR (15 Ci/r&i) for 120 min at 14 uCi/ml, Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
698
ISSN
0006-291X
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
Nucleoprotein complexes containing SV40 DNAwere extracted 5.0 ml/culture. as previously described (6). The crude lysates were cleared of nuclear aggregates and large cellular debris by centrifugation at 800 x g for 10 min at 4”. The resulting NPC containing supernatant was divided into three fractions for transcription studies. One fraction was deproteinized and closed circular supercoiled viral DNAisolated from propidium diiodide-CsCl gradients. The second fraction was concentrated by pressure dialysis in order to produce a “crude NPC fraction” containing viral DNA at a concentration of 2.0 ug/ml. Finally, the third fraction was fractionated by a combination of hydroxyapatite (HA) chromatography and velocity centrifugation previously described by Meinke et al. (8) to produce “purified NPCs”. Velocity centrifugation of complexes in linear 5 to 20% (w/w) sucrose gradients, and buoyant density centrifugation of glutaraldehyde fixed complexes as well as collection, processing and scintillation counting of centrifuged samples have all been previously described (5,6). In vitro transcription of SV40 NPCsand viral DNAwas carried out by a Ed-tion of Astrins (14) assay system. Except where indicated, RNAwas synthesized from NPCtemplates or purified viral DNAat 1.0 pg of DNAper assay. Each reaction also contained, in a total volume of 1.0 ml, 20 units of 5. coli DNA-dependent RNApolymerase (Miles Laboratories), 15 I&I KCl, 40 mMTRIS-HCl (pH 7.9), 0.1 n&l dithiothreitol, 5 I&I MgC12, 0.05% Triton X-100? 40 mMNaCl, 2 mMEDTA, and 1 I&I each, ATP, GTP, CI’P, and UTP ([3H]UTP 10 uC1). The reaction was terminated after 15 min incubation at 37”C, by addition of 5.0 ml of cold 10%TCA. The acid-precipitable material was collected on glass fiber filters (WhatmanGF/C), dried and counted in a liquid scintillation counter. To prepare RNAfor filter hybridization, the preceeding reaction mixtures were incubated for 3 hr at 37’C. The reaction was terminated by addition of SDSto a final concentration of 1.0%. RNAwas extracted with phenol saturated with acetate buffer pH 5.2 (13) and collected by ethanol precipitation. For DNA-RNAhybridization, SV40 DNAI, purified through propidium diiodideCsCl gradients, was heat denatured and immobilized onto 25 mmnitrocellulose filters (Schleicher 8 Schuell Co., type B6) by the methods of Aloni et al. (15). DNA-RNAhybrid analyses were performed in scintillation vials containing one blank filter and one filter containing either 2.5 or 5.0 ug of viral DNA. Incubations were for 24 hrs at 65’C with viral RNAin 2.0 ml 2 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate). The filters were then chilled, washed in 2 x SSCand exposed to 20 pg/ml pancreatic ribonuclease in 2 x SSC for one hr at room temperature. Filters were then rinsed in 2 x SSC, dried and counted in a scintillation counter. RESULTS In Vitro
Transcription
of SV40 DNA, Crude NPCs, and Purified
Prior to use in the transcription
NPCs
assay, aliquots of the purified
NPCswere analyzed to assure that they maintained the sedimentation velocity (61s) and density purified
(1.437 g/cm3) previously
NPCpreparation
coefficients 1.445 g/c+.
utilized
reported for SV40 NPCs (6).
The
in these studies had sedimentation
of between 58 and 61s and buoyant densities between 1.435 and
Vol. 76, No. 3, 1977
Table 1.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In Vitro Transcription of SV40 DNA and Nucleoprotein 5 E. coli DNA-Dependent RNA Polymerase.
RNA Synthesis *(cpm[3H]DI'P Incorporated)
Template crude
+180
NPC
Purified
Complexes
NPC
970
SV40 DNfl
4100
* RNA was synthesized from NPC templates containing 1.0 vg of DI\EAper assay or from 1.0 pg of purified Form I SV40 DNA as described in Materials and Methods. These values are corrected for approximately 85 cpm of incorporation in the absence of template. ' Corrected activity.
Purified utilized
for approximately
SV40 DNA and NPCs as well as crude NPCs were prepared and
as templates
and Methods. supported
120 cpm of endogenous polymerase
for -.E. -coli
The results
incorporation
presented
RNA catalyzed
However, it is evident
poor template
which supports
approximately
20% as much [3H]UTP as purified
that purified
viral
purified
as described
in Table 1 show that all
of [3H]UTP into
dependent RNA polymerase. is a relatively
RNA polymerase
DNA was at least
The best evidence that is accurate,
was obtained
sized from either
complex.
DNA immobilized Unfortunately, mixtures
viral
filters
we could not recover
to allow filter
hybridization
RNA synthesized
from purified
the same extent
as RNA synthesized
It is also apparent a template
of NPCs by 5 &RNA experiments.
DNA or purified
on nitrocellulose
of only
from purified 700
RNA syntheto viral
and Methods).
RNA from crude NPC reaction
experiments.
NPCs hybridized
polymerase
NPCs was hybridized
(see Materials
sufficient
as
as crude complex.
by DNA-RNA hybridization
purified
DNA
that the crude complex
4 times as efficient
transcription
three templates
by -E. coli
incorporation
complex and 20 to 22 times as effective
in Materials
As shown in Table 2,
to viral viral
DNA to approximately DNA. This suggests
BIOCHEMICAL
Vol. 76, No. 3, 1977
Hybridization of [3H]-Labeled RNASynthesized 7In Vitro from Purified SV40 Nucleoprotein Complexes or Purified Supercoiled SV40 DNA.
Table 2.
[ 3H]RNA Input wwl
Template for RNASynthesis Purified
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NPC
sv40 DNA
[ 3H]RNAHybridized (CPM) 2.5 ug DNA 5.0 1-g DNA (% of Input)
3,200
2,685
2,750
84.9
11,000
9,690
9,800
88.6
RNAwas synthesized from each template by the procedure described in Materials and Methods except that the incubation time was increased from 15 min to 3 hr. At the end of the incubation period, RNAwas extracted and purified and DNA-RNAhybridization performed as described in Materials and Methods. All values have been corrected for background counts (l-2% of input) of [3H]RNA bound to blank filters.
that viral
DNAsequences in NPCsare transcriptionally
transcribed
Recovery of Nucleoprotein
Since one of the main objectives
physical
Complex Templates
of these studies is to determine the
of NPCsfor use as model chromosomes,it was essential that the integrity
Therefore,
of the complex be retained following
purified
[3EI]-thymidine
characterize
complexes (PI-NPCs) and compare them with pretranscripNPCs. As shown in Figure 1, the sedimentation velocity
labelled F’I’-NPCsis identical
labelled pretranscriptional
to assist in determination
NPCs. A [“Cl-thymidine
sedimentation coefficients
to viral
viral
(arrow over fraction
of their
as 60 S. fixed SV40 NPCsis
Density analyses of glutaraldehyde
701
35)
for the complexes.
DNAallows calculation
The average density reported for glutaraldehyde 1.437 + .008 g/cm” (6).
labelled purified
of sedimentation coefficients
of the NPCsrelative
of
to that of [14C]-thymidine
DNAmarker (21 S) was also present in this gradient
The position
transcription.
several experiments were performed to physically
posttranscriptional tional
and are
faithfully.
Posttranscriptional
suitability
active
fixed
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IO@
9
0
IO 20 30 40 FRACTION NUM BE R
IO 20 30 FRACTION NUMBER
0
Figure 1. Velocity sedimentation, in a linear sucrose gradient, of SV40 nucleoprotein complexes. After 15 min incubation in the transcription assay mixture (see Materials and Methods), an aliquot of [3H]-thymidine labeled NPC (0.15 ml) was removed from the assay mixture mixed with 0.05 ml of [14C]-thymidine labeled marker SV40 NPC and [j4C]-thymidine labeled viral DNA (10 lambda), and centrifuged as described (5,6). An arrow marks the position of [14C]-thymidine labeled, purified viral DNA included as a marker. Figure 2. Equilibrium centrifugation of glutaraldehyde-fixed SV40 nucleoprotein complexes in linear CsCl gradients. After 15 min incubation in the transcription assay mixture (see Materials and Methods), aliquots (0.25 ml) of t3H]-thymidine labeled NPC were removed and centrifuged on 5% to 20% sucrose gradients. Peak fractions from each gradient were pooled, mixed with ['4C]-thymidinelabeled marker NPCs, and treated with glutaraldehyde (5,6). Aliquots (0.2 ml) of the glutaraldehyde fixed complexes were layered on preformed linear CsCl gradients and centrifuged to equilibrium as previously described (5,6).
pretranscriptional linear
and posttranscriptional
CsCl gradients
as previously
that these two WC preparations their
density
Although
not shown, standard
of 1.444 is within
of the DNA extracted
NPCs exhibit
similar
described
had identical
apparent
gradients
NPCs were conducted on preformed
patterns
cells
buoyant densities.
diiodide-CsCl
from pretranscriptional of banding of viral supercoiled
the same ratios
cellular
2)
In addition,
and posttranscriptional DNA with approximately
histones
90%
form.
bound to viral
as are found in eukaryotic 702
(Figure
equilibrium
DISCUSSION complexes isolated from polyoma and SV40 virus
have been shown to contain
approximately
It is evident
the normal range for SV40 NPCs.
propidium
of the DNA being in the closed circular Nucleoprotein
(5,6).
chromatin
infected DNA in (11).
The
BIOCHEMICAL
Vol. 76, No. 3, 1977
similarity
of viral
be ideal
although
has led to the proposal
the effects
presented
here indicate
report
that
was deproteinized
viral
a template
DNA. Similarly,
crude NPCs were almost totally It was suggested that,
in addition
in Table 1, indicate crude extracts
that
as templates
to restriction
or ribonucleases similar
of SV40 NPCs.
RNA synthesis
inhibitory
polymerase
of transcription
(13).
as
also contained Cur results,
free of soluble
of transformed
cells
RNA polymerase.
by histones cellular presented
substances might be present
Recent work has shown that both SV40 subviral
by --E. coli
the SV40 viral
for RNA synthesis.
There is an approximately
when NFCs are purified
sequences in chromatin
that
in a study of polyoma WCs (13),
invactive
of RNA synthesis
DNA. This is in
for -E. coli
in the polyoma NPCs, the crude preparations
accurately
on gene
SV40 NPCs are transcriptionally
(12) which demonstrated
core complex was only 30% as effective
inhibitors
that NPCs might
of chromosomal proteins
at a reduced rate as compared to viral
agreement with a previous
present
RESEARCH COMMUNICATIONS
(16).
The results active
NPCs and chromatin
models for studying
expression
AND BIOPHYSICAL
five fold increase host cell
in in
proteins.
cores (12) and viral
DNA
(SV3T3) (14) can be transcribed
Significantly,
the sequences transcribed
--in vitro from SV3T3 chromatin were the same as those present in transformed cells (14). Our present work supports these studies and indicates (Table 2) that E. coli
polymerase
whether these transcripts Most important,
represent
relative
complexes for studies
intact
template
marked contrast
to recent reports
bound proteins,
(17,18).
rather
However,
true messages remains to be determined. of these viral
of gene expression,
DNA-histone
is the evidence that
from the transcription
evidence that transcription
the DNA-protein
the assay mixture
copy SV40 NFCs -__ in vitro.
to the utility
on control
complexes may be isolated is strong indirect
accurately
can
solutions,
is actually
than some deproteinized
occurring
except the polymerase,
that sarcosyl in these assays.
703
on
form and is in
in which sarcosyl was incorporated
It is likely
This
removed all
into DNA
The recovery of
Vol. 76, No. 3, 1977
unaltered transcribing
BIOCHEMICAL
NPCs after unaltered
template-complex
--in vitro
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transcription
NPCs, reisolating
(DNA or Protein),
offers
the possibility
the NPC template,
transcribing
of
modifying
the
it again and comparing
transcripts. ACKNOWLEDGEMENTS:The author thanks Ms. Brenda Kozyra and Mr. Warren Hall for skillful technical assistance. This investigation was supported by Grant Number CA 17167, awarded by the National Cancer Institute and by Grant Number NP-189 from the American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
hang, E-S., M.K. Estes, and J.S. Pagano. (1972) J. Virol. 9: 923-929. McMillen, J., and R.A. Consigli. (1974) J. Virol. 14: 1326-T536. Green, M.H., H.J. Miller, and S. Hendler. (1971) SC. Nat. Acad. Sci., U.S.A. 68: 1032-1036. White, K, and R. Eason. (1971) J. Virol. 8: 363-371. Goldstein, D., M.R. Hall, and W. Meinke. (1973) J. Virol. 12: 887-900. Hall, M.R., W. Meinke, and D.A. Goldstein. (1973) J. Virol.2: 901-908. Pett, D.M., M.K. Estes, and J.S. Pagano. (1975) J. Virol. 157379-385. Meinke, W., M.R. Hall, and D.A. Goldstein. (1975) J. VirolTl5: 439-448. Kornberg, R.D. (1974) Science 184: 868-871. Griffith, J.D. (1975) Science m: 1202-1203. Cremisi, C., P.F. Pignatti, Oxroissant, and M. Yaniv. (1976) J. Virol. 17: 204-211. iiing, E-S., M. Nonoyama, and J.S. Pagano. (1972) J. Virol. 9: 930-937. Shmookler, R.J., J. Buss, and M.H. Green. (1974) Virology 577122-127. Astrin, S.M. (1973) Proc. Nat. Acad, Sci., U.S.A. 70: 23047308. Aloni, Y., E. Winocour, and L. Sacks. (1968) J. Mar Biol. 31: 415-429. Germond, J-E., B. Hirt, P. Oudet, M. Gross-Bellard, and P. -bon. (1975) Proc. Nat. Acad. Sci., U.S.A. 72: 1843-1847. Gariglio, P., and S. Mousset. (1975) EBS Letters 56: 149-155. Green, M.H., and T.L. Brooks. (1976) Virology 72: X0-120. -
704
Vol. 76, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TRANSCRIPTION AND REISOLATION OF THE SIMIAN VIRUS 40 NUCLEOPROTEIN COMPLEX Mark R. Hall Department Received
March
of Cell and Molecular Georgia, Augusta,
Biology, Georgia
Medical 30902
College
of
30,1977
WY: Nucleoprotein complexes, containing cellular histones associated man Virus 40 DNA, were isolated from virus infected cells. These complexes supported RNA synthesis when incubated with E. coli DNAdependent RNA polymerase. Under the conditions of assay, transcripts synthesized by E. coli polymerase were shown to be highly specific for viral DNA by hy6rimion assay. Of particular importance is the demonstration that the viral nucleoprotein complexes isolated from the transcription mixture have not been physically altered. INTRODlJCTION Viral
nucleoprotein
complexes @PC), isolated
(SV40) or polyoma virus have been reported
virions
to contain
from Simian Virus 40
(1,2) and from virus four cellular
infected
histones
cells
(7,8).
(3-6),
These histones,
H2A, H2B, H3, and H4, are also found in the nucleosomes characteristic cellular viral
chromatin NPCs contain
to cellular
microscopic
have shown that
between viral
on transcription
NPC and cellular
chromatin
provides
(12,13).
models for studying of a defined
the effects
genome.
The present
evidence that the viral
model chromosomes (mini-chromosomes) of their
in structure
suggests the
Indeed,
have shown that polyoma and SV40 NPCs can be transcribed
and in addition
the
(10,ll).
and endogenous polymerases
ation
studies
20 nucleosomes and are similar
that NPCs might be ideal
chromosomal proteins studies
Electron
approximately
chromatin
The similarity possibility
(9).
of
report
recent
by exogenous
supports these studies
NPCs can be utilized
in a transcription
of
assay without
as alter-
structure. MATERIALSANDMETHODS
African green monkey kidney cells ((X-1) were infected with strain WI911 SV40 virus at a multiplicity of lo-20 pfu/cell. At 40 h postinfection, cultures were pulsed with [3H]TdR (15 Ci/r&i) for 120 min at 14 uCi/ml, Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
698
ISSN
0006-291X
BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
Nucleoprotein complexes containing SV40 DNAwere extracted 5.0 ml/culture. as previously described (6). The crude lysates were cleared of nuclear aggregates and large cellular debris by centrifugation at 800 x g for 10 min at 4”. The resulting NPC containing supernatant was divided into three fractions for transcription studies. One fraction was deproteinized and closed circular supercoiled viral DNAisolated from propidium diiodide-CsCl gradients. The second fraction was concentrated by pressure dialysis in order to produce a “crude NPC fraction” containing viral DNA at a concentration of 2.0 ug/ml. Finally, the third fraction was fractionated by a combination of hydroxyapatite (HA) chromatography and velocity centrifugation previously described by Meinke et al. (8) to produce “purified NPCs”. Velocity centrifugation of complexes in linear 5 to 20% (w/w) sucrose gradients, and buoyant density centrifugation of glutaraldehyde fixed complexes as well as collection, processing and scintillation counting of centrifuged samples have all been previously described (5,6). In vitro transcription of SV40 NPCsand viral DNAwas carried out by a Ed-tion of Astrins (14) assay system. Except where indicated, RNAwas synthesized from NPCtemplates or purified viral DNAat 1.0 pg of DNAper assay. Each reaction also contained, in a total volume of 1.0 ml, 20 units of 5. coli DNA-dependent RNApolymerase (Miles Laboratories), 15 I&I KCl, 40 mMTRIS-HCl (pH 7.9), 0.1 n&l dithiothreitol, 5 I&I MgC12, 0.05% Triton X-100? 40 mMNaCl, 2 mMEDTA, and 1 I&I each, ATP, GTP, CI’P, and UTP ([3H]UTP 10 uC1). The reaction was terminated after 15 min incubation at 37”C, by addition of 5.0 ml of cold 10%TCA. The acid-precipitable material was collected on glass fiber filters (WhatmanGF/C), dried and counted in a liquid scintillation counter. To prepare RNAfor filter hybridization, the preceeding reaction mixtures were incubated for 3 hr at 37’C. The reaction was terminated by addition of SDSto a final concentration of 1.0%. RNAwas extracted with phenol saturated with acetate buffer pH 5.2 (13) and collected by ethanol precipitation. For DNA-RNAhybridization, SV40 DNAI, purified through propidium diiodideCsCl gradients, was heat denatured and immobilized onto 25 mmnitrocellulose filters (Schleicher 8 Schuell Co., type B6) by the methods of Aloni et al. (15). DNA-RNAhybrid analyses were performed in scintillation vials containing one blank filter and one filter containing either 2.5 or 5.0 ug of viral DNA. Incubations were for 24 hrs at 65’C with viral RNAin 2.0 ml 2 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate). The filters were then chilled, washed in 2 x SSCand exposed to 20 pg/ml pancreatic ribonuclease in 2 x SSC for one hr at room temperature. Filters were then rinsed in 2 x SSC, dried and counted in a scintillation counter. RESULTS In Vitro
Transcription
of SV40 DNA, Crude NPCs, and Purified
Prior to use in the transcription
NPCs
assay, aliquots of the purified
NPCswere analyzed to assure that they maintained the sedimentation velocity (61s) and density purified
(1.437 g/cm3) previously
NPCpreparation
coefficients 1.445 g/c+.
utilized
reported for SV40 NPCs (6).
The
in these studies had sedimentation
of between 58 and 61s and buoyant densities between 1.435 and
Vol. 76, No. 3, 1977
Table 1.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In Vitro Transcription of SV40 DNA and Nucleoprotein 5 E. coli DNA-Dependent RNA Polymerase.
RNA Synthesis *(cpm[3H]DI'P Incorporated)
Template crude
+180
NPC
Purified
Complexes
NPC
970
SV40 DNfl
4100
* RNA was synthesized from NPC templates containing 1.0 vg of DI\EAper assay or from 1.0 pg of purified Form I SV40 DNA as described in Materials and Methods. These values are corrected for approximately 85 cpm of incorporation in the absence of template. ' Corrected activity.
Purified utilized
for approximately
SV40 DNA and NPCs as well as crude NPCs were prepared and
as templates
and Methods. supported
120 cpm of endogenous polymerase
for -.E. -coli
The results
incorporation
presented
RNA catalyzed
However, it is evident
poor template
which supports
approximately
20% as much [3H]UTP as purified
that purified
viral
purified
as described
in Table 1 show that all
of [3H]UTP into
dependent RNA polymerase. is a relatively
RNA polymerase
DNA was at least
The best evidence that is accurate,
was obtained
sized from either
complex.
DNA immobilized Unfortunately, mixtures
viral
filters
we could not recover
to allow filter
hybridization
RNA synthesized
from purified
the same extent
as RNA synthesized
It is also apparent a template
of NPCs by 5 &RNA experiments.
DNA or purified
on nitrocellulose
of only
from purified 700
RNA syntheto viral
and Methods).
RNA from crude NPC reaction
experiments.
NPCs hybridized
polymerase
NPCs was hybridized
(see Materials
sufficient
as
as crude complex.
by DNA-RNA hybridization
purified
DNA
that the crude complex
4 times as efficient
transcription
three templates
by -E. coli
incorporation
complex and 20 to 22 times as effective
in Materials
As shown in Table 2,
to viral viral
DNA to approximately DNA. This suggests
BIOCHEMICAL
Vol. 76, No. 3, 1977
Hybridization of [3H]-Labeled RNASynthesized 7In Vitro from Purified SV40 Nucleoprotein Complexes or Purified Supercoiled SV40 DNA.
Table 2.
[ 3H]RNA Input wwl
Template for RNASynthesis Purified
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NPC
sv40 DNA
[ 3H]RNAHybridized (CPM) 2.5 ug DNA 5.0 1-g DNA (% of Input)
3,200
2,685
2,750
84.9
11,000
9,690
9,800
88.6
RNAwas synthesized from each template by the procedure described in Materials and Methods except that the incubation time was increased from 15 min to 3 hr. At the end of the incubation period, RNAwas extracted and purified and DNA-RNAhybridization performed as described in Materials and Methods. All values have been corrected for background counts (l-2% of input) of [3H]RNA bound to blank filters.
that viral
DNAsequences in NPCsare transcriptionally
transcribed
Recovery of Nucleoprotein
Since one of the main objectives
physical
Complex Templates
of these studies is to determine the
of NPCsfor use as model chromosomes,it was essential that the integrity
Therefore,
of the complex be retained following
purified
[3EI]-thymidine
characterize
complexes (PI-NPCs) and compare them with pretranscripNPCs. As shown in Figure 1, the sedimentation velocity
labelled F’I’-NPCsis identical
labelled pretranscriptional
to assist in determination
NPCs. A [“Cl-thymidine
sedimentation coefficients
to viral
viral
(arrow over fraction
of their
as 60 S. fixed SV40 NPCsis
Density analyses of glutaraldehyde
701
35)
for the complexes.
DNAallows calculation
The average density reported for glutaraldehyde 1.437 + .008 g/cm” (6).
labelled purified
of sedimentation coefficients
of the NPCsrelative
of
to that of [14C]-thymidine
DNAmarker (21 S) was also present in this gradient
The position
transcription.
several experiments were performed to physically
posttranscriptional tional
and are
faithfully.
Posttranscriptional
suitability
active
fixed
Vol. 76, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IO@
9
0
IO 20 30 40 FRACTION NUM BE R
IO 20 30 FRACTION NUMBER
0
Figure 1. Velocity sedimentation, in a linear sucrose gradient, of SV40 nucleoprotein complexes. After 15 min incubation in the transcription assay mixture (see Materials and Methods), an aliquot of [3H]-thymidine labeled NPC (0.15 ml) was removed from the assay mixture mixed with 0.05 ml of [14C]-thymidine labeled marker SV40 NPC and [j4C]-thymidine labeled viral DNA (10 lambda), and centrifuged as described (5,6). An arrow marks the position of [14C]-thymidine labeled, purified viral DNA included as a marker. Figure 2. Equilibrium centrifugation of glutaraldehyde-fixed SV40 nucleoprotein complexes in linear CsCl gradients. After 15 min incubation in the transcription assay mixture (see Materials and Methods), aliquots (0.25 ml) of t3H]-thymidine labeled NPC were removed and centrifuged on 5% to 20% sucrose gradients. Peak fractions from each gradient were pooled, mixed with ['4C]-thymidinelabeled marker NPCs, and treated with glutaraldehyde (5,6). Aliquots (0.2 ml) of the glutaraldehyde fixed complexes were layered on preformed linear CsCl gradients and centrifuged to equilibrium as previously described (5,6).
pretranscriptional linear
and posttranscriptional
CsCl gradients
as previously
that these two WC preparations their
density
Although
not shown, standard
of 1.444 is within
of the DNA extracted
NPCs exhibit
similar
described
had identical
apparent
gradients
NPCs were conducted on preformed
patterns
cells
buoyant densities.
diiodide-CsCl
from pretranscriptional of banding of viral supercoiled
the same ratios
cellular
2)
In addition,
and posttranscriptional DNA with approximately
histones
90%
form.
bound to viral
as are found in eukaryotic 702
(Figure
equilibrium
DISCUSSION complexes isolated from polyoma and SV40 virus
have been shown to contain
approximately
It is evident
the normal range for SV40 NPCs.
propidium
of the DNA being in the closed circular Nucleoprotein
(5,6).
chromatin
infected DNA in (11).
The
BIOCHEMICAL
Vol. 76, No. 3, 1977
similarity
of viral
be ideal
although
has led to the proposal
the effects
presented
here indicate
report
that
was deproteinized
viral
a template
DNA. Similarly,
crude NPCs were almost totally It was suggested that,
in addition
in Table 1, indicate crude extracts
that
as templates
to restriction
or ribonucleases similar
of SV40 NPCs.
RNA synthesis
inhibitory
polymerase
of transcription
(13).
as
also contained Cur results,
free of soluble
of transformed
cells
RNA polymerase.
by histones cellular presented
substances might be present
Recent work has shown that both SV40 subviral
by --E. coli
the SV40 viral
for RNA synthesis.
There is an approximately
when NFCs are purified
sequences in chromatin
that
in a study of polyoma WCs (13),
invactive
of RNA synthesis
DNA. This is in
for -E. coli
in the polyoma NPCs, the crude preparations
accurately
on gene
SV40 NPCs are transcriptionally
(12) which demonstrated
core complex was only 30% as effective
inhibitors
that NPCs might
of chromosomal proteins
at a reduced rate as compared to viral
agreement with a previous
present
RESEARCH COMMUNICATIONS
(16).
The results active
NPCs and chromatin
models for studying
expression
AND BIOPHYSICAL
five fold increase host cell
in in
proteins.
cores (12) and viral
DNA
(SV3T3) (14) can be transcribed
Significantly,
the sequences transcribed
--in vitro from SV3T3 chromatin were the same as those present in transformed cells (14). Our present work supports these studies and indicates (Table 2) that E. coli
polymerase
whether these transcripts Most important,
represent
relative
complexes for studies
intact
template
marked contrast
to recent reports
bound proteins,
(17,18).
rather
However,
true messages remains to be determined. of these viral
of gene expression,
DNA-histone
is the evidence that
from the transcription
evidence that transcription
the DNA-protein
the assay mixture
copy SV40 NFCs -__ in vitro.
to the utility
on control
complexes may be isolated is strong indirect
accurately
can
solutions,
is actually
than some deproteinized
occurring
except the polymerase,
that sarcosyl in these assays.
703
on
form and is in
in which sarcosyl was incorporated
It is likely
This
removed all
into DNA
The recovery of
Vol. 76, No. 3, 1977
unaltered transcribing
BIOCHEMICAL
NPCs after unaltered
template-complex
--in vitro
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transcription
NPCs, reisolating
(DNA or Protein),
offers
the possibility
the NPC template,
transcribing
of
modifying
the
it again and comparing
transcripts. ACKNOWLEDGEMENTS:The author thanks Ms. Brenda Kozyra and Mr. Warren Hall for skillful technical assistance. This investigation was supported by Grant Number CA 17167, awarded by the National Cancer Institute and by Grant Number NP-189 from the American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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