BIOCHEMICAL
Vol. 76, No. 3, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LOW MOLECULAR WEIGHT NUCLEAR RNA FROM SV40-TRANSFORMED WI38 CELLS; EFFECT ON TRANSCRIPTION OF WI38 CHROMATIN --IN VITRO Margarida Department Fredericton,
Received
April
0. Krause and Maurice Ringuette of Biology, University of New Brunswick, N. B., Canada. E3B 5A3
15,1977
SUMMARY: Nuclei isolated from normal and SV40-transformed WI38 cells were used as templates for RNA synthesis Comparison of template prop--in vitro. erties showed marked differences between the two chromatins with both homologous and heterologous enzymes. Addition of the 0.35 M NaCl extract from the chromatin of the SV4D-transformed cells, revealed two separate activities: one stimulating the template activity of normal WI38 chromatin and one affectSeparation of the proing the stability of the homologous RNA polymerase. tein and SnRNA fractions in the extract demonstrated that the stabilization effect is due to chromosomal protein(s). However, SnRNA alone was found to be responsible for the stimulation of the transcriptional activity of normal cells to a level undistinguishable from that of the transformed cells. Induction RNA tumor
of malignant
viruses,
transformation
has been associated
or gene copies
within
the
of some of the viral
expression
of the lar
host
genes.
cell
has been
The implication
of
by our previous
finding
phosphorylation
and turnover
(2-11).
While
there
transformation
proteins,
established. to this
the
Therefore, study
and SV40 transformed
cells
This
to test
system
was used
as an altered
viral-induced in
alteration this
histone
using
both
has been in rates
effect
of SV40-
to the metabolism changes
of addition
to be
approach
of chromatins and E. s
of
remains
on a more functional
endogenous
of
by SV40 viruses
in the
respect
properties
effect
process
transformed
to embark
template
in celluin recent
and nonhistone
of the observed
the the
with
expression
modification
of both
genes precedes
investigation
to be some consistency
we decided
by comparing
of viral
as well
of viral-induced
significance
by DNA and
Such integration
in mouse and human cells appears
integration
proteins
of mouse and human cells
chromosomal
the
in culture
(1).
of intense
of chromosomal
supported
proteins
this
the object
synthesis chromosomal
with genome genes
The mechanism
gene expression
years.
the host
of cells
from normal
RNA polymerases.
of various
components
of
chromatin. Work on chromosomal proteins in other laboratories has suggested that the 0.35 M NaCl extract from chromatin contains loosely-bound nonhistone the
proteins
presence
RNA (SnRNA) overlooked protein
Copyright AN rights
with
presumptive
in chromatin extractable
0 1977
of small at
or purification preparation.
this
has been
quantities
salt
methods It
by Academic Press, Inc. in any form reserved.
of reproduction
gene regulatory
activity of low molecular
concentration have been
claimed
(12,13).
that
(14,18)
inadequate this
However
weight
.has either to ensure
RNA is
intimately
nuclear been
an RNA-free associ-
796 ISSN
0006-291X
BIOCHEMICAL
Vol. 76, No. 3, 1977
ated
with
protein
protein
conditions. molecular rapid
(19)
fraction More weight
RNA's
--in vitro. and SnRNA component the
Marzluff
of these
effect
in order
prevent
of the
(ZO),
the
to discriminate
separation out
using
from
these improved
low methods
between
for
investigation
from chromatin
and SV40-transformed
was fractionated
the
in nondenaturing
The present
M NaCl extract
of normal extract
its
characterized
chromatin
0.35
RESEARCH COMMUNICATIONS
is carried
species.
on transcription Moreover,
two macromolecular
et al
from mouse myeloma
the
cells
chromatin
easily
purification
recovery
to test
SV40-transformed
could
protein
recently
and quantitative
was designed
and this
whenever
AND BIOPHYSICAL
into
the effects
its
of WI38
protein
of each of
components.
MATERIALS AND METHODS: All experiments utilized log-phase cultures of SV40transformed and normal WI3.8 human fibroblasts. Cell cycle analysis of both cell types revealed no significant differences in mean generation time (2Ohr.s) or length of "S"-phase (ll?l hr). Nuclei were isolated in ice-cold lysing medium containing 0.32 M Sucrose, 1% triton X-100 20 mM EDTA, 10 mM MgC12, 10 mM CaC12 and 1 mM phenylmethylsulfonyl fluoride (PMSF), washed twice with 0.01 M Tris-HCl pH 8.0, 10 mM MgC12, 10 mM CaC12 and either 0.32 M sucrose, resuspended in 10% glycerol, 1% bovine serum albumin (BSA) or chromatin was isolated by lysis in ice-cold distilled water, allowed to swell for 30 min, homogenized gently and pelleted at 20,000 g for 20 min. Isolated chromatin was resuspended in 0.35 m NaCi, 0.02 M Tris-HCl pH 7.5 at a concentration of 1 mg DNA/ml, homogenized for 20 min. in an ice bath and centrifuged at 105,000 g for 2 hrs. Fractionation of RNA and proteins from the NaCl extract was achieved by either of the following procedures: A - the extract was dialysed against 200 volumes 1% SDS-O.1 M NaCl-0.01 M trisodium acetate 1 mM EDTA (pH 5.4). The dialysate was incubated at 37OC for 15 min. and extracted with equal volumes of phenol and chloroform-isoamyl alcohol (34: 1 v/v). The aqueous phase was extracted twice more with chloroform-isoamyl alcohol, and the RNA precipitated with 3 volumes of ethanol and dried until use. B - the NaCl extract was dialysed against 200 volumes of 7 M urea 0.25 M NaCl-0.05 M Tris HCl pH 5.5 and eluted through a DEAE-cellulose column until 0.D 280 read 0. The column was washed with 0.3 M NaCl, 7 M urea, 0.05 M Tris-HCl pH 5.5 and RNA was eluted with 7 urea - 1 M NaCl in the same buffer were collected, dialysed against 0.1 M Tris HCl pH 8.0 (20) - The proteins The RNA fractions were collected, dialysed against and lyophylised until use. 5 mM EDTA and lyophlized until use. For transcription assay isolated nuclei in 10% glycerol 1% BSA were incubated in either of the following assay mixtures: A - With g. coli RNA polymerase. Assay mixtures contained in 1 ml: 50 umoles Tris-HCl pH 8.1, 3 umoles TrisHCl pH 8.1, 3 umoles MgC12, 15 nmoles 8-mercaptoethanol, 0.5 umoles each ATP, CTP and GTP, 0.2 umoles 3H-LiTP (75 uCi/ml) and 102 units E. & RNA polymerase, isolated and purified as described (21). B - For endogenous polymerase assays the reaction mixtures were similar to the above except that no polymerase of Mg++ were added, and (NH4)2S04 was added to a concentration of 200 nmoles per ml. Incubations were carried out at 2S°C and reactions stopped by addition of 10 volumes ice-cold 10% TCA-40 mu sodium pyrophosphate: precipitates were collected at 8,000 g for 10 min. 50 mM NaOH was added to disolve each pellet and nucleic acids were again precipitated by addition of equal volumes of 10% TCA and collected on GF filters. THe filters were washed with TCA and ethanol, dried and counted in a Beckman liquid scintilation spectrometer. DNA was determined either by W absorption or by the indole procedure (22) RNA by w absorption (260 nm) and protein by Lowry (23) or 0-D. 280. For assay
of
initiation
and elongation
797
of RNA transcripts,
incubation
Vol. 76, No. 3, 1977
DNA hg)
BIOCHEMICAL
AND BIOPHYSICAL
30 10 20 1NCUBATION TIME (minutes)
/ 1 unit RNA polymerase
5
10
15
INCUBATION
Fig.
1
RESEARCH COMMUNICATIONS
20
25
30
TIME (minutes)
Comparison of template activities of nuclei isolated from logphase WI38 and SVBO-transformed WI38 cells. A: with E. & RNA polymerase at increasing DNA to enzyme ratios.,(j nUdei from normal WI38 cells;Onuclei from SV40-WI38 cells. B: with 4.3 ug DNA per unit of E- coli RNA polymerase. Initiation was allowed for 40 more min. as stoped after 14 min. and elongation described under methods.0 nuclei from normal WI38 cells; 0 nuclei from SVIO-WI38 cells. C: nuclei incubated under conditions optimized for endogenous RNA polymerase II activity. 0 nuclei from normal WI38 cells; Onuclei from SV~O-WI38 cells. 798
46
Vol. 76, No. 3, 1977
BIOCHEMICAL
with E. coli polymerase at wh?ch time (NH4)2SO4 prevent further initiation added, to allow for RNA various time periods and
was carried out as above but without was added to a cont. of 0.4 M. This by RNA polymerase (24). At this chain elongation, the incubation was reaction was stopped as above.
RESULTS AND DISCUSSION: phase
The template
WI38 or SV40-transformed
RNA polymerase linearly
with
increasina
that
highest
enzyme
to cope with
additional
cells,
not
reaction
the
also
a saturation
test
whether
these
in chromatin,
rate
types allow for
level
of nuclei
indicate
but there
out
addition
is
considerably
amounts
of endogenous
present
procedure.
of nuclei apparent.
It
appears
genuine
studying
the
template
activity
therefore
seen.
WI33
there
is
In order
of initiation
to
sites both
of CTP and salt 2B.
of chain
Zero
time
cell
to
allowed values
chromatin
elongation
When nuclei
of
in
of chromatin
extent,
genous
polymerase
nuclei
than
is much higher
are
incubated
with-
in the
for
polymerase. from (26,27).
due to the low
isolated
with
transcriptional 5. coli
the
activity
WI38 cells
system
normal
are
still
polymerase
WI38 cell and as a test
Preliminary
the chromatin in
is
it
only
suggested
one affecting
799
chromatin cells,
while
experiments
showboth
to a whenever
the existence
template
cells
with
stimulated, cells
on
for
WI38 nuclei
same transformed This
for
of SV40-transformed
isolated
However, the
was utilized
and transformed
comparison
interactions.
activities,
nuclei
of SV40-transformed
from
probably
RNA
structure.
transcription
was tested
of two separate
in the
heterologous
from both
isolated
is
or SV40-transformed
template
was utilized
M NaCl extract
and endogenous
lesser
This
the heterologous
the
of components
stimulating
2C).
retained
that
experiments,
system 0.35
(Fig.
normal
RNA-polymerase-specific the
was capable
extract
rate
but
of CTP in order
in SV40-WI38
the differences
either
differences
effect
endogenous
exogenous
absence
Fig.
chromatin.
excess
we incubated
Addition (20)
the
cell
RNA polymerase from
In subsequent
ed that
in the
sites
lower
However,
isolated
possible
in normal
rate is
or both,
nuclei
still
case of normal
to number
of RNA chains
In addition,
than
incorporated
the
increase
are more initiation
is
in the
no further
initiations
cell
there
at a lower
can be attributed
E. coli -In Fig. 1A
of --E. coli RNA polymerase, under conditions where endogenous II is preferentially stimulated (25), the amount of 3H-UTP
polymerase
detecting
which
further
chromatin.
in transformed
either
log-
can be seen to increase
to proceed
E. coli RNA polymerase -to prevent elongation.
without
that
in normal
beyond
from
methods.
ratios
However,
template. appears
differences
initiation elongation
using
under product
DNA/polymerase
of elongation
with
isolated
was compared
insoluble
CTP for 15 min, is required to time CTP was also carried out for
DNA in the case of SV40-transformed
at the
only
an acid
RESEARCH COMMUNICATIONS
of nuclei
enzyme as described
of 3H-UTP into
indicating
activity
WI38 cells
or endogenous
incorporation
AND BIOPHYSICAL
structure
endo-
in this of
Vol. 76, No. 3, 1977
BIOCHEMICAL
INCUBATION
5
10 INCUBATION
Fig.
2
TIME
15
20
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(mu-wtes)
INCUBATION
TIME (muwtes)
25
TIME (minutes)
INCUBATION
TIME (minutes)
Effect of addition of nonhistone chromosomal proteins (!&NHCP) and SnRNA from the 0.35 M NaCl extract of SV40-WI38 chromatin. A- nuclei from SV40-WI38 incubated under conditions optimized for RNA polymerase II activity;. control;0 with !&NHCP in 1:l w/w proportion to DNA; 0 with SnBNA in O.l:l w/w proportion to DNA; B-nuclei from SV40-WI38 cells incubated with E. coli RNA polymerase at 4.3 ).Q DNA/unit. l control; 0 witK !kNHCP in 1:l w/w proportion to DNA; Owith SnBNA in O.l:l w/w proportion
800
BIOCHEMICAL
Vol. 76, No. 3, 1977
normal
chromatin,
the
extract
was found
proteins
(l-NHCP)
weight
nuclear
the effect
other
affecting
to contain but
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not
also
endogenous
only
small
loosely-bound
amounts
RNA (SnRNA),
non-histone
of tightly
we separated
Since
RNA polymerase. bound
the two
this
chromosomal
low molecular
fractions
in order
to test
Fig. 2A illustrates the results --in vitro. same SVBO-transformed WI38 cells using the endogenous RNA
of each on transcription
in nuclei
of the
polymerase
incubation
but
that
l-NHCP
If,
however,
the
RNA polymerase These
system.
addition
It
same experiment
no effect
experiments
formed
WI38 chromatin
specific
stimulatory
is
apparent
has a stimulatory is
carried
of either were
contain activity
that
the
effect
out with
SnRNA or l-NHCP
interpreted
heterologous could
to mean that
a protein
SnRNA has no effect
on 3H-UTP incorporation.
l-NHCP
(or proteins)
on transcription.
which
If
c. coli
be detected
these
(Fig.
2B).
from SV40-transhave a polymerase-
same l-NHCP and
SnRNA fractions are added to nuclei of normal WI38 cells incubated with either homologous (Fig. 2C) or heterologous (Fig. 2D) RNA polymerase, a similar
stimulation
cases
of 3H-UTP incorporation
by addition
of the SnRNA fraction
SnRNA purified
in a similar
from
chromatin
SV40-WI38
either
Fig.
appears able
to render
purified
the
might
responsible
RNA is
for role
indeed would
In both
likely
the active
the
while
or l-NHCP
in the conditions
stimulatory
effect
of normal
were
obtained
of
of SnRNA
nuclei
undistinguish-
contaminants molecule
SnRNA preparations
we considered
effect.
to exist
with
Since
in only
the possibility
molecule
any molecule
small first
is
prove
The use of carboxymethylcellulose-bound in vitro
RNAse prior , provided
in
that
the
the
be of a
the cell,
a serious
the If
one.
effect
is
SnRNA preparation transcription assay.
to assay
the desired
that
could suspected
quantities
in any preparation one must
which
while ensuring removal of RNAse in the otherwise affect the RNA product during the
of SnRNA on transcription
in both
chromatin,
WI38 chromatin
some contaminating
the activation is
cell
activity
activity
procedures,
contain
SV40-WI38
normal
cases
RNA can be observed
ones.
same results
of accidental
RNAse-sensitive, which
the
from
transcriptional
by two different
regulatory problem
2D.
of transformed
Even though preparations
manner
from
show no stimulatory
2C or Fig.
from that
into
of the effect
conditions.
to DNA. C- nuclei from normal WI38 cells incubated under conditions optimized for RNA polymerase II activity. 0 control, l with RNHCP in 1:l w/w proportion to DNA,A with SnRNA in O.l:l w/w proportion to DNA,0 with the same amount of SnRNA isolated from normal WI38 chromatin. D-nuclei from normal WI38 cells incubated with E. coli RNA polymerase at 4.3 pg DNA/unit;a control, l with R-NHCP in 1:l w/w proportion to DNA, 0 with SnRNA in O.l:l proportion to DNA, Awith the same amount of SnRNA isolated from normal WI38 chromatin.
801
Vol. 76, No. 3, 1977
BIOCHEMICAL
INCUBATION
Fig.
3
RESEARCH COMMUNICATIONS
TIME Immutes)
,og Sn RNA
added
Dose response curves for activity of SnRNA in stimulating transcription of WI38 chromatin in the presence of E. coli RNA polymerase at 4.3 )lg DNA/unit. A-SnRNA was incubated with carboxymethyl cellulose-bound RNAse for either 1 or 3 hrs. under conditions which hydrolysed 50% and 90% of the RNA respectively. 0 control, 0 with SnRNA in O.l:l w/w proportion to DNA, 0 with SnRNA pretreated for 1 hr. with RNAse,bwith SnRNA pretreated for 3 hrs. with RNAse. B- effect of addition of different amounts of SnRNA.
The results the bound
are
RNAse were
RNAse from might
AND BIOPHYSICAL
the
illustrated designed
cellulose
be responsible in the
"active"
SnRNA relative
as demonstrated
in Fig.
Different the possibility
rather
than
results.
needed
specific
These is
data
indeed
to DNA used in
of those
The presence
the
SnRNA preparation
to be in excess
3A.
to eliminate
matrix, for
molecule
in Fig.
RNA. its
even when stored
of SnRNA,
Moreover
that the
the
amounts
experiments stimulatory
"active" of
appear
activity,
3B.
in chromatin
difficult assays.
digestion
demonstrate
the transcription
to demonstrate of small
quantities
nuclear RNA has been known for some time (14,18). small quantities present and because of its close (19),it is transcription
incubation times with that release of
to isolate Moreover,
at -40°C,
and purify it
and since
is
very
most
802
in large
of low molecular However, association enough
sensitive RNAse inhibitors
weight
because of the with protein quantities
to contaminating also
interfere
for RNAse
Vol. 76, No. 3, 1977
with but
transcription, also
that
Preliminary acrylamide the
BIOCHEMICAL
gel
"active"
possible
role
transformation
we found it
could
only
that
of these
RNA's
only after
using
electrophoresis smaller
not
be stored
characterization RNA is
AND BIOPHYSICAL
tRNA.
we had to purify
very
rapidly
SnRNA separated
by autoradiography
We are
in transcription
it
lyophilization.
32P-labelled
and detected than
RESEARCH COMMUNICATIONS
currently regulation
by
indicate
investigating in general
that the
and viral
in particular.
ACKNOWLEDGEMENTS: We thank Drs. discussion, the National Research and Karen Dean for cultivation of the 1st International Congress of
E. Jay and K. Yu for advice and stimulating Council of Canada for financial support, cells. This work was presented in part cell Biology, Sept. 1976.
at
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Baltimore, D. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 1187-1200. Zardi, L., Lin, J. and Baserqa, R. (1973) Nature New Biology 245, 211. Lin, J., Nicolini, C. and Baserqa, R. (1974) Biochemistry 13, 4127-4133. Cholon, J. and Studzinski, G. (1974) Cancer Research 39, 588. Krause, M.O. and Stein, G.S. (1974) Biochem. Biophys. Res. Cormn. 59, 976-803. Krause, M.O., Kleinsmith, L.J. and Stein, G.S. (1975) Exp. Cell REs. 92, 164-174. Krause, M-0. and Stein, G.S. (1975) Exp. Cell Res. 92, 175-190. Krause, M.O., Kleinsmith, L.J. and Stein, G.S. (1975) Life Sciences 16, 1047-1058. Krause, M.O., Noonan, K.D. and Stein, G.S. (1976) Cell Differentiation 5, 83-96. Pumo, D.E., Stein, G.S. and Kleinsmith, L.J. (1975) Biochem. Biophys. Acta 402, 125-130. Gonzalex, C. and Rees, K. (1975) Biochem. Biophys. Acta 393, 361-372. Nicolini, C., Nq. S. and Beserqa, R. (1975) Proc. Natl. Acad. Sci. 72, 2361-2365. Kostraba, N-C., Montaqna, R.A. and Wang, T.Y. (1975) J. Biol. Chem. 250, 1548-1555. Weinberg, R.A. and Penman, S. (1968) J. Mol. Biol. 38, 289. Moriyama, Y., Hodnett, J.L., Prestayko, A.W. and Busch, H. (1969) J. Mol. Biol. 39, 355. Monahan, J.J. and Hall, R.H. (1973) Can. J. Biochem. 51, 709. Monahan, J.J. and Hall, R.H. (1973) Can. J. Biochem. 51, 903. Holmes, D.S., Mayfield, J.E. and Bonner, J. (1974), Biochemistry 13, 849. Jacobsen, R. and Bonner, J. (1971) Arch. Biochem. Biophys. 146, 557. Marxluff, Jr., W.F., White, E-L., Benjamin, R. and Huanq, R.C.C. (1975) Biochemistry 14, 3715-3742. Burgess, R.R. and Jendrisak, J.J. (1975) Biochem. 14, 4634-4638. Ceriotti, G. (1955) J. Biol. Chem. 214, 59. Lowry, D.H., Rosenborouqh, N.J., Farr, A.L. and Randall, R.S. (1951) J. Biol. Chem. 193, 265. N. (1970) J. Mol. Biol. 50, 421-438. %-man, R.W. and Davidson, Dessureault, J. and Krause, M.O., (1975) Can. J. Biochem. 53, 149-154. Rinquette, M. and Krause, M.O. (1976) Proc. Can. Fed. Biol. Sot. 19, 167. Krause, M.O. (1976) J. Cell Biol. 70(2), A7.
803
Vol. 76, No. 3, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LOW MOLECULAR WEIGHT NUCLEAR RNA FROM SV40-TRANSFORMED WI38 CELLS; EFFECT ON TRANSCRIPTION OF WI38 CHROMATIN --IN VITRO Margarida Department Fredericton,
Received
April
0. Krause and Maurice Ringuette of Biology, University of New Brunswick, N. B., Canada. E3B 5A3
15,1977
SUMMARY: Nuclei isolated from normal and SV40-transformed WI38 cells were used as templates for RNA synthesis Comparison of template prop--in vitro. erties showed marked differences between the two chromatins with both homologous and heterologous enzymes. Addition of the 0.35 M NaCl extract from the chromatin of the SV4D-transformed cells, revealed two separate activities: one stimulating the template activity of normal WI38 chromatin and one affectSeparation of the proing the stability of the homologous RNA polymerase. tein and SnRNA fractions in the extract demonstrated that the stabilization effect is due to chromosomal protein(s). However, SnRNA alone was found to be responsible for the stimulation of the transcriptional activity of normal cells to a level undistinguishable from that of the transformed cells. Induction RNA tumor
of malignant
viruses,
transformation
has been associated
or gene copies
within
the
of some of the viral
expression
of the lar
host
genes.
cell
has been
The implication
of
by our previous
finding
phosphorylation
and turnover
(2-11).
While
there
transformation
proteins,
established. to this
the
Therefore, study
and SV40 transformed
cells
This
to test
system
was used
as an altered
viral-induced in
alteration this
histone
using
both
has been in rates
effect
of SV40-
to the metabolism changes
of addition
to be
approach
of chromatins and E. s
of
remains
on a more functional
endogenous
of
by SV40 viruses
in the
respect
properties
effect
process
transformed
to embark
template
in celluin recent
and nonhistone
of the observed
the the
with
expression
modification
of both
genes precedes
investigation
to be some consistency
we decided
by comparing
of viral
as well
of viral-induced
significance
by DNA and
Such integration
in mouse and human cells appears
integration
proteins
of mouse and human cells
chromosomal
the
in culture
(1).
of intense
of chromosomal
supported
proteins
this
the object
synthesis chromosomal
with genome genes
The mechanism
gene expression
years.
the host
of cells
from normal
RNA polymerases.
of various
components
of
chromatin. Work on chromosomal proteins in other laboratories has suggested that the 0.35 M NaCl extract from chromatin contains loosely-bound nonhistone the
proteins
presence
RNA (SnRNA) overlooked protein
Copyright AN rights
with
presumptive
in chromatin extractable
0 1977
of small at
or purification preparation.
this
has been
quantities
salt
methods It
by Academic Press, Inc. in any form reserved.
of reproduction
gene regulatory
activity of low molecular
concentration have been
claimed
(12,13).
that
(14,18)
inadequate this
However
weight
.has either to ensure
RNA is
intimately
nuclear been
an RNA-free associ-
796 ISSN
0006-291X
BIOCHEMICAL
Vol. 76, No. 3, 1977
ated
with
protein
protein
conditions. molecular rapid
(19)
fraction More weight
RNA's
--in vitro. and SnRNA component the
Marzluff
of these
effect
in order
prevent
of the
(ZO),
the
to discriminate
separation out
using
from
these improved
low methods
between
for
investigation
from chromatin
and SV40-transformed
was fractionated
the
in nondenaturing
The present
M NaCl extract
of normal extract
its
characterized
chromatin
0.35
RESEARCH COMMUNICATIONS
is carried
species.
on transcription Moreover,
two macromolecular
et al
from mouse myeloma
the
cells
chromatin
easily
purification
recovery
to test
SV40-transformed
could
protein
recently
and quantitative
was designed
and this
whenever
AND BIOPHYSICAL
into
the effects
its
of WI38
protein
of each of
components.
MATERIALS AND METHODS: All experiments utilized log-phase cultures of SV40transformed and normal WI3.8 human fibroblasts. Cell cycle analysis of both cell types revealed no significant differences in mean generation time (2Ohr.s) or length of "S"-phase (ll?l hr). Nuclei were isolated in ice-cold lysing medium containing 0.32 M Sucrose, 1% triton X-100 20 mM EDTA, 10 mM MgC12, 10 mM CaC12 and 1 mM phenylmethylsulfonyl fluoride (PMSF), washed twice with 0.01 M Tris-HCl pH 8.0, 10 mM MgC12, 10 mM CaC12 and either 0.32 M sucrose, resuspended in 10% glycerol, 1% bovine serum albumin (BSA) or chromatin was isolated by lysis in ice-cold distilled water, allowed to swell for 30 min, homogenized gently and pelleted at 20,000 g for 20 min. Isolated chromatin was resuspended in 0.35 m NaCi, 0.02 M Tris-HCl pH 7.5 at a concentration of 1 mg DNA/ml, homogenized for 20 min. in an ice bath and centrifuged at 105,000 g for 2 hrs. Fractionation of RNA and proteins from the NaCl extract was achieved by either of the following procedures: A - the extract was dialysed against 200 volumes 1% SDS-O.1 M NaCl-0.01 M trisodium acetate 1 mM EDTA (pH 5.4). The dialysate was incubated at 37OC for 15 min. and extracted with equal volumes of phenol and chloroform-isoamyl alcohol (34: 1 v/v). The aqueous phase was extracted twice more with chloroform-isoamyl alcohol, and the RNA precipitated with 3 volumes of ethanol and dried until use. B - the NaCl extract was dialysed against 200 volumes of 7 M urea 0.25 M NaCl-0.05 M Tris HCl pH 5.5 and eluted through a DEAE-cellulose column until 0.D 280 read 0. The column was washed with 0.3 M NaCl, 7 M urea, 0.05 M Tris-HCl pH 5.5 and RNA was eluted with 7 urea - 1 M NaCl in the same buffer were collected, dialysed against 0.1 M Tris HCl pH 8.0 (20) - The proteins The RNA fractions were collected, dialysed against and lyophylised until use. 5 mM EDTA and lyophlized until use. For transcription assay isolated nuclei in 10% glycerol 1% BSA were incubated in either of the following assay mixtures: A - With g. coli RNA polymerase. Assay mixtures contained in 1 ml: 50 umoles Tris-HCl pH 8.1, 3 umoles TrisHCl pH 8.1, 3 umoles MgC12, 15 nmoles 8-mercaptoethanol, 0.5 umoles each ATP, CTP and GTP, 0.2 umoles 3H-LiTP (75 uCi/ml) and 102 units E. & RNA polymerase, isolated and purified as described (21). B - For endogenous polymerase assays the reaction mixtures were similar to the above except that no polymerase of Mg++ were added, and (NH4)2S04 was added to a concentration of 200 nmoles per ml. Incubations were carried out at 2S°C and reactions stopped by addition of 10 volumes ice-cold 10% TCA-40 mu sodium pyrophosphate: precipitates were collected at 8,000 g for 10 min. 50 mM NaOH was added to disolve each pellet and nucleic acids were again precipitated by addition of equal volumes of 10% TCA and collected on GF filters. THe filters were washed with TCA and ethanol, dried and counted in a Beckman liquid scintilation spectrometer. DNA was determined either by W absorption or by the indole procedure (22) RNA by w absorption (260 nm) and protein by Lowry (23) or 0-D. 280. For assay
of
initiation
and elongation
797
of RNA transcripts,
incubation
Vol. 76, No. 3, 1977
DNA hg)
BIOCHEMICAL
AND BIOPHYSICAL
30 10 20 1NCUBATION TIME (minutes)
/ 1 unit RNA polymerase
5
10
15
INCUBATION
Fig.
1
RESEARCH COMMUNICATIONS
20
25
30
TIME (minutes)
Comparison of template activities of nuclei isolated from logphase WI38 and SVBO-transformed WI38 cells. A: with E. & RNA polymerase at increasing DNA to enzyme ratios.,(j nUdei from normal WI38 cells;Onuclei from SV40-WI38 cells. B: with 4.3 ug DNA per unit of E- coli RNA polymerase. Initiation was allowed for 40 more min. as stoped after 14 min. and elongation described under methods.0 nuclei from normal WI38 cells; 0 nuclei from SVIO-WI38 cells. C: nuclei incubated under conditions optimized for endogenous RNA polymerase II activity. 0 nuclei from normal WI38 cells; Onuclei from SV~O-WI38 cells. 798
46
Vol. 76, No. 3, 1977
BIOCHEMICAL
with E. coli polymerase at wh?ch time (NH4)2SO4 prevent further initiation added, to allow for RNA various time periods and
was carried out as above but without was added to a cont. of 0.4 M. This by RNA polymerase (24). At this chain elongation, the incubation was reaction was stopped as above.
RESULTS AND DISCUSSION: phase
The template
WI38 or SV40-transformed
RNA polymerase linearly
with
increasina
that
highest
enzyme
to cope with
additional
cells,
not
reaction
the
also
a saturation
test
whether
these
in chromatin,
rate
types allow for
level
of nuclei
indicate
but there
out
addition
is
considerably
amounts
of endogenous
present
procedure.
of nuclei apparent.
It
appears
genuine
studying
the
template
activity
therefore
seen.
WI33
there
is
In order
of initiation
to
sites both
of CTP and salt 2B.
of chain
Zero
time
cell
to
allowed values
chromatin
elongation
When nuclei
of
in
of chromatin
extent,
genous
polymerase
nuclei
than
is much higher
are
incubated
with-
in the
for
polymerase. from (26,27).
due to the low
isolated
with
transcriptional 5. coli
the
activity
WI38 cells
system
normal
are
still
polymerase
WI38 cell and as a test
Preliminary
the chromatin in
is
it
only
suggested
one affecting
799
chromatin cells,
while
experiments
showboth
to a whenever
the existence
template
cells
with
stimulated, cells
on
for
WI38 nuclei
same transformed This
for
of SV40-transformed
isolated
However, the
was utilized
and transformed
comparison
interactions.
activities,
nuclei
of SV40-transformed
from
probably
RNA
structure.
transcription
was tested
of two separate
in the
heterologous
from both
isolated
is
or SV40-transformed
template
was utilized
M NaCl extract
and endogenous
lesser
This
the heterologous
the
of components
stimulating
2C).
retained
that
experiments,
system 0.35
(Fig.
normal
RNA-polymerase-specific the
was capable
extract
rate
but
of CTP in order
in SV40-WI38
the differences
either
differences
effect
endogenous
exogenous
absence
Fig.
chromatin.
excess
we incubated
Addition (20)
the
cell
RNA polymerase from
In subsequent
ed that
in the
sites
lower
However,
isolated
possible
in normal
rate is
or both,
nuclei
still
case of normal
to number
of RNA chains
In addition,
than
incorporated
the
increase
are more initiation
is
in the
no further
initiations
cell
there
at a lower
can be attributed
E. coli -In Fig. 1A
of --E. coli RNA polymerase, under conditions where endogenous II is preferentially stimulated (25), the amount of 3H-UTP
polymerase
detecting
which
further
chromatin.
in transformed
either
log-
can be seen to increase
to proceed
E. coli RNA polymerase -to prevent elongation.
without
that
in normal
beyond
from
methods.
ratios
However,
template. appears
differences
initiation elongation
using
under product
DNA/polymerase
of elongation
with
isolated
was compared
insoluble
CTP for 15 min, is required to time CTP was also carried out for
DNA in the case of SV40-transformed
at the
only
an acid
RESEARCH COMMUNICATIONS
of nuclei
enzyme as described
of 3H-UTP into
indicating
activity
WI38 cells
or endogenous
incorporation
AND BIOPHYSICAL
structure
endo-
in this of
Vol. 76, No. 3, 1977
BIOCHEMICAL
INCUBATION
5
10 INCUBATION
Fig.
2
TIME
15
20
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(mu-wtes)
INCUBATION
TIME (muwtes)
25
TIME (minutes)
INCUBATION
TIME (minutes)
Effect of addition of nonhistone chromosomal proteins (!&NHCP) and SnRNA from the 0.35 M NaCl extract of SV40-WI38 chromatin. A- nuclei from SV40-WI38 incubated under conditions optimized for RNA polymerase II activity;. control;0 with !&NHCP in 1:l w/w proportion to DNA; 0 with SnBNA in O.l:l w/w proportion to DNA; B-nuclei from SV40-WI38 cells incubated with E. coli RNA polymerase at 4.3 ).Q DNA/unit. l control; 0 witK !kNHCP in 1:l w/w proportion to DNA; Owith SnBNA in O.l:l w/w proportion
800
BIOCHEMICAL
Vol. 76, No. 3, 1977
normal
chromatin,
the
extract
was found
proteins
(l-NHCP)
weight
nuclear
the effect
other
affecting
to contain but
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
not
also
endogenous
only
small
loosely-bound
amounts
RNA (SnRNA),
non-histone
of tightly
we separated
Since
RNA polymerase. bound
the two
this
chromosomal
low molecular
fractions
in order
to test
Fig. 2A illustrates the results --in vitro. same SVBO-transformed WI38 cells using the endogenous RNA
of each on transcription
in nuclei
of the
polymerase
incubation
but
that
l-NHCP
If,
however,
the
RNA polymerase These
system.
addition
It
same experiment
no effect
experiments
formed
WI38 chromatin
specific
stimulatory
is
apparent
has a stimulatory is
carried
of either were
contain activity
that
the
effect
out with
SnRNA or l-NHCP
interpreted
heterologous could
to mean that
a protein
SnRNA has no effect
on 3H-UTP incorporation.
l-NHCP
(or proteins)
on transcription.
which
If
c. coli
be detected
these
(Fig.
2B).
from SV40-transhave a polymerase-
same l-NHCP and
SnRNA fractions are added to nuclei of normal WI38 cells incubated with either homologous (Fig. 2C) or heterologous (Fig. 2D) RNA polymerase, a similar
stimulation
cases
of 3H-UTP incorporation
by addition
of the SnRNA fraction
SnRNA purified
in a similar
from
chromatin
SV40-WI38
either
Fig.
appears able
to render
purified
the
might
responsible
RNA is
for role
indeed would
In both
likely
the active
the
while
or l-NHCP
in the conditions
stimulatory
effect
of normal
were
obtained
of
of SnRNA
nuclei
undistinguish-
contaminants molecule
SnRNA preparations
we considered
effect.
to exist
with
Since
in only
the possibility
molecule
any molecule
small first
is
prove
The use of carboxymethylcellulose-bound in vitro
RNAse prior , provided
in
that
the
the
be of a
the cell,
a serious
the If
one.
effect
is
SnRNA preparation transcription assay.
to assay
the desired
that
could suspected
quantities
in any preparation one must
which
while ensuring removal of RNAse in the otherwise affect the RNA product during the
of SnRNA on transcription
in both
chromatin,
WI38 chromatin
some contaminating
the activation is
cell
activity
activity
procedures,
contain
SV40-WI38
normal
cases
RNA can be observed
ones.
same results
of accidental
RNAse-sensitive, which
the
from
transcriptional
by two different
regulatory problem
2D.
of transformed
Even though preparations
manner
from
show no stimulatory
2C or Fig.
from that
into
of the effect
conditions.
to DNA. C- nuclei from normal WI38 cells incubated under conditions optimized for RNA polymerase II activity. 0 control, l with RNHCP in 1:l w/w proportion to DNA,A with SnRNA in O.l:l w/w proportion to DNA,0 with the same amount of SnRNA isolated from normal WI38 chromatin. D-nuclei from normal WI38 cells incubated with E. coli RNA polymerase at 4.3 pg DNA/unit;a control, l with R-NHCP in 1:l w/w proportion to DNA, 0 with SnRNA in O.l:l proportion to DNA, Awith the same amount of SnRNA isolated from normal WI38 chromatin.
801
Vol. 76, No. 3, 1977
BIOCHEMICAL
INCUBATION
Fig.
3
RESEARCH COMMUNICATIONS
TIME Immutes)
,og Sn RNA
added
Dose response curves for activity of SnRNA in stimulating transcription of WI38 chromatin in the presence of E. coli RNA polymerase at 4.3 )lg DNA/unit. A-SnRNA was incubated with carboxymethyl cellulose-bound RNAse for either 1 or 3 hrs. under conditions which hydrolysed 50% and 90% of the RNA respectively. 0 control, 0 with SnRNA in O.l:l w/w proportion to DNA, 0 with SnRNA pretreated for 1 hr. with RNAse,bwith SnRNA pretreated for 3 hrs. with RNAse. B- effect of addition of different amounts of SnRNA.
The results the bound
are
RNAse were
RNAse from might
AND BIOPHYSICAL
the
illustrated designed
cellulose
be responsible in the
"active"
SnRNA relative
as demonstrated
in Fig.
Different the possibility
rather
than
results.
needed
specific
These is
data
indeed
to DNA used in
of those
The presence
the
SnRNA preparation
to be in excess
3A.
to eliminate
matrix, for
molecule
in Fig.
RNA. its
even when stored
of SnRNA,
Moreover
that the
the
amounts
experiments stimulatory
"active" of
appear
activity,
3B.
in chromatin
difficult assays.
digestion
demonstrate
the transcription
to demonstrate of small
quantities
nuclear RNA has been known for some time (14,18). small quantities present and because of its close (19),it is transcription
incubation times with that release of
to isolate Moreover,
at -40°C,
and purify it
and since
is
very
most
802
in large
of low molecular However, association enough
sensitive RNAse inhibitors
weight
because of the with protein quantities
to contaminating also
interfere
for RNAse
Vol. 76, No. 3, 1977
with but
transcription, also
that
Preliminary acrylamide the
BIOCHEMICAL
gel
"active"
possible
role
transformation
we found it
could
only
that
of these
RNA's
only after
using
electrophoresis smaller
not
be stored
characterization RNA is
AND BIOPHYSICAL
tRNA.
we had to purify
very
rapidly
SnRNA separated
by autoradiography
We are
in transcription
it
lyophilization.
32P-labelled
and detected than
RESEARCH COMMUNICATIONS
currently regulation
by
indicate
investigating in general
that the
and viral
in particular.
ACKNOWLEDGEMENTS: We thank Drs. discussion, the National Research and Karen Dean for cultivation of the 1st International Congress of
E. Jay and K. Yu for advice and stimulating Council of Canada for financial support, cells. This work was presented in part cell Biology, Sept. 1976.
at
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