From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Ribosome
Role
Biosynthesis
in
of Ribosomal
RNA
and
HE
CYTOPLASM
ribosomes
in immunoblasts.1
few
bly is
synthesis
restricted
which
in resting
accompanies
lymphocytes
an increased
to explore
regulation
lymphocyte
In
of
mammalian
subunits
units
are
of
D.
ARNOLD
The
Initiation
Growth
RUBIN
LYMPHOCYTES
the dense
clusters
has suggested
lymphocytes
and
phytohemagglutinin
that
(
contains
relatively
of these
organelles
that ribosome
assem-
the
augmented
PHA)-induced
into proliferating
immunoblasts
efficiency
of ribosome
assembly.
the association
between
ribosome
ribosome
transforma-
depends,
at least
The present
study
assembly
and
the
growth.
cells,
of unequal
distinctive
: II.
in the
of Lymphocyte
OF RESTING
when
compared
to
A previous
publication2
tion of resting
in part,
upon
will attempt
Lymphocytes
Production
Maintenance
By
T
Cultured
cytoplasmic
ribosomes
may
size.35
The
RNA
moieties
and have
been
characterized
be
dissociated
contained
by their
into
two
within
these
sedimentation
subrates
28S
( 285
455
and 18S, respectively.
Cytoplasmic
ribosomal
RNA
( rRNA)
moieties
and 185 ) originate
in the nucleolus
where
they are transcribed
as a single
rRNA
precursor
molecule
which
becomes
immediately
methylated
and
then
RNA
is cleaved
migrates
undergoes
nucleoprotein
nonconservatively
into the cytoplasm.
cytoplasm
for
ultimate
transcription
cytoplasmic
products
rRNA
45S
rRNA
processing
precursor,
were
ultimate
riboin the
with
by
kinetic
following
the
a radioactive
products
analysis
rate
for separating
time.
After
comparing
moiety.
the
28S
which
of
celiular
and
labeling
relative
simultaneously
after
initial
have
exposure
the
to PHA.
morphologic
At
this
appearance
time,
60-80
of
blasts.
the
cent
DNA
RNA
kinetics
amounts
of
displayed
per
18S
various
identifying
the newly
on
mentation
profile
of extracted
cellular
RNA.
It has been
previously
shown#{176}’7that small
lymphocytes
respond
single
cohort.
RNA
and protein
synthesis
rise to a maximum
level
culture
rRNA
and
at
precursor,
the
of
mature
Sedimentation
a means
at any given
analyzed
and
Packaged
into
separate
the 18S RNA’s
combine
to
evolved.
provided
present
precursor
intermediates
The
18S
moiety
A
an 185
nucleolus,
processing
by
moieties
gradient
moities
325
assembly.35
of
accomplished
was
synthesized
transcribed
ribosome
and
through
a sucrose
the different
rRNA
rRNA
a 325 and
still in the
further
cleavage
into
a 28S moiety.
particles
( RNP’s
), the
28S and
precursor
newly
into
While
of
rRNA
a
sedi-
to PHA
as a
at 48 hours
of the cells
replication
in the
begins
Supported
in part
by USPHS
Grant
CA
10478
from
the
Nional
Cancer
Institute
and
contract
AT(30-1)3833
from
the Atomic
Energy
Commission,
and by the Albert
A. List,
Frederick
Machim
and Anna
Ruth Lowertberg
Funds.
ARNOLD
D. RUBIN,
M.D.:
Assistant
Professor
of Medicine,
Department
of Medicine
(Hematology),
Mt.
Sinai
School
of Medicine,
New
York,
N.Y.;
Leukemia
Society
Scholar.
708
BLOOD,
VOL.
35,
No.
5
(MAY)
1970
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RIBOSOME
at
BIOSYNTHESIS
36 hours
are
about
lymphocytes
though
some
CULTURED
responding
hours,
there
protein
synthesis
may
be regarded
to
LYMPHOCYTES
709
and reaches
its maximum
at 72 hours.
recruited
into
the replicating
cycle
lymphocytes
may
undergo
Few,
after
two
if any,
72-96
or more
additional
hours.
Al-
divisions
after
is a steady
downward
trend
in the rates
of RNA,
DNA
and
as well as in the mitotic
index.
Therefore,
48-hour
cultures
as “growing.”
Resting
cultures
and cultures
which
have
been
72
allowed
IN
pass
through
“nongrowing.”
the
Cultures
morphologically
phase
at
typical
of
168
active
hours
blast
growth
are
still
might
then
comprised
cells.#{176} These
might
be
of
be
considered
per
60-80
called
cent
“nongrowing”
blasts.
The
of
kinetics
PHA
of ribosome
induced
processing
455 rRNA
changes
of
well-defined
the
earliest
assembly
lymphocyte
phases
were
growth.
precursor
in the
of the
analyzed
The
directly
rate
of
growth
during
results
the
will
relates
processing
show
different
phases
that
rate
the
to the growing
state
can
be
detected
of
and that
during
response.
METHODS
Preparation
of Cell
each
normal
botomy.
From
After
allowing
( about
75-100
a
column
by
loosely
columns
were
maintaining
ready
for
blood
yielded
medium
containing
(
100
added
tritiated
certain
before
methionine-free
serum
RNA
over
\Vashed
contaminated
type
AB
), (Schwartz,
For
a frozen
slurry
of
were
immediately
Earle’s
Difco
At
)
RNA
studies,
the
cells
balanced
sodium
salt
4
solution
fifty
and
per
ml.
cent
were
platelets.
in
25
cc.
(2
mM.),
appropriate
was
of
were
and
at
resuspended
10
Ci/mM
times,
introdiuced
formate,
pCi/mi.,
1 mI/mm.
phytohemagglutinin-P
jCm./m1.
mM.
these
glutammne
Ci./mM
20
with
oven,
and
10
20
20
( Schwartz
X
with
).
19
flushing
of
95-98
to
prepared
hundred
jzCi./ml.,
methy!ated
containing
rate
erythrocytes
along
of
20
the
0.75
jCm./ml.
concentration
at
which
with
of
serum
( 100
a
drying
Three
phle-
applied
were
After
a
eluted
of
and
)
in
procedure.
cells,
by
supernatant
diameter
Leukopak.
were
the
obtained
) medium
2-cm.
sterilization
concentration
streptomycin
in
( Spinner
Fenwall
nucleated
a
human
medium
cells
NaCl
made
0.5
(0.05M
per
cent
of
60#{176}C redistilled
agitated
for
five
broken
by
minutes
once
again
) and
sodium
dodecyl
sulfate
water-saturated
at
volumes
of
ethanol
with
in
60#{176}C.After
for
phenol
the
in
( 10M
centrifugation
tracted
resuspended
),MgCL
in
volume
was
to
was
leukocyte-rich
).
per
cent
calf
Cultures
were
( 0.O1M
) buffer
washed.
Extraction
containing
was
10
the
X
suspensions
methionmne-methyl-H3
harvested
a
blood
hour,
2
achieved
grossly
harvest.
Eagle’s
and
from
having
x
cent
( H3U
intervals
long
37#{176}C throughout
cultures
uridine-5-H3
varying
No.
adjusted
) and
t/ml.
to
Eagle’s
lymphocytes
per
one
( 24-cm.
100-200
were
heparinized
for
1 : 1 with
Lymphocyte
from
of
columns
removed
at
15
cc.
to settle
and
use.
small
lymphocytes
penicillin
fiber
hour
temperature
Washed
in
These
nylon
morphologically
was
diluted
one
the
of
was
for
350
erythrocytes
fiber.
packing
water
subject,
the
nylon
distilled
ml.
human
)
cc.
of
Su.spension
at
presence
10
rapid
of
750
pH
and
briefly
phenol
was
cooling
in
a
-
for
of
cent).
shaken
g. The
jzGm.
acetate
per
in
added,
800
at
5.1
(one
precipitated
minutes
60#{176}Cand
ice-cold
bentonite
purified,
The
suspension
icewater.
and
20#{176}Cbrine
An
the
emulsion
bath,
the
was
emulsion
aqueous
supernatant
18
at
- 20#{176}Cwith
rat
liver
hours
unlabeled
equal
was
ex-
two
cytoplasmic
RNA.8
Analysis
per
cent
The
of
sucrose
precipitate
RNA
Moieties
gradient,
prepared
dissolved
in
0.3
in
ml.
buffer
of
the
without
original
MgCI0
buffer
and
was
sedimented
layered
on
at
a 5 ml.,
37,000
5-20
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
710
ARNOLD
cisTI
2puls.
1$.;----
43$
21$
D. RUBIN
#{149}
IS
4$
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11$
431
11$
113
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1SUrPIA
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ill
iii
.
IS
iu
,
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II
‘I
3.
is
1,
75
TPBE NO.
Fig.
1.-Sedimentation
patterns
of pulse-labeled
hires
exposed
to H3U (20 jzCi./ml.)
for varying
harvested.
RNA extracted
and sedimented
across
RP%1
for
fraction
RNA.
185
of
30
placed
in
hydrated
peaks
five
determined
by
same
cent
alcohol,
as
markers
specific
acid
and
precipitation.8
the
285
filter
in
75-85
left
extracted
in
in
a
three
the
on
liver
rates.
times
in
scintillation
cent
of
then
and
de-
fraction
was
vial.
lymphocyte
aqueous-phenol
aqueous
Approxiwere
TCA,
each
each
cytoplasmic
which
of
liquid
per
the
of
papers
Radioactivity
paper
yielded
peaks
filter
washed
ether.9
determinations
185
sedimentation
individual
(TCA),
finally
density
and
relative
on
RNA
RNA
Optical
the
determine
acid
appropriate
as
of
to
employed
activity
rotor.
collected
trichloracetic
the
65
positions
were
procedure
direct
SW
the
alcohol-ether,
placing
extraction
tamable
Spinco
fractions
per
by
a
located
served
12-drop
in
The
in
gradient
These
mately
the
minutes
the
RNA.
Replicate
lymphocyte
culintervals,
after
which
cells were
a linear
five-to-20-per
cent sucrose
RNA
interface
ob-
exhibited
phase.
RESULTS
Growing
The
must
versus
sedimentation
reflect
the
Nongrowing
profiles
moieties
Cultures
of radioactive
of newly
synthesized
RNA
extracted
RNA
from
accumulated
cultured
during
interval
of exposure
to radioactive
precursor.
If the interval
of exposure
relatively
long,
those
moieties
which
undergo
rapid
synthesis
and degradation
( i.e.,
turnover)
would
be displayed
less prominently
on the sedimentation
profile
when
compared
to more
stable
rRNA
moieties
which
steadily
cumulate.
cells
the
is
ac-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RIBOSOME
BIOSYNTHESIS
IN
1UNiPNA
CULTURED
711
LYMPHOCYTES
4tpsIss
I.
15#{149}p.ise
43$
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43$
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2.
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13
OS
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iii
OS
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ZN
10
70
30
1,
20
30
10
75
$
TUBE NO
gradient.
Resting
if);
48
two
hours
cultures
hours
(ib,
During
a
(la-ic);
(lg-ii);
168
e, h, k); four
15-minute
lated
radioactive
lower
regions
Cultures
(lj-1l).
hours
pulse
ic,
f,
gradient.
No
uridine
Fig.
la),
most
of
(
RNA,
with
of
PHA
for
pulse:
one
15
hour
d,
(la,
(id-
g,
j);
1).
i,
H3U
of
polydisperse
of the
incubated
Duration
resting
lymphocytes
which
well-defined
peaks
accumu-
sedimented
could
be
toward
discerned.
the
After
pulse
( Fig.
ib),
much
of the labeled
RNA remained
polydisperse,
but a large
discrete
peak
appeared
at 45S, together
with
an accumulation
of
radioactive
RNA
in the region
of 28 to 325. The RNA
accumulated
during
a
four-hour
pulse
( Fig.
lc)
sedimented
in discrete
peaks
at 45S and 285 with
a two-hour
the
285
creased,
constant
gested
showed
peak
predominating.
Thus,
as the duration
of the
the overall
level
of radioactive
RNA
accumulation
rate.
However,
the delayed
appearance
of radioactive
that
this moiety
was
greater
stability
than
455 rose progressively
active
cumulated
since
of the
product
more
accumulation
rate
of 28
of intracellular
of 455
lymphocytes
Preincubation
rapidly
even
cleavage,
synthesized
polydisperse
over
the
during
the
to 32S
RNA
455
namely
after
a four-hour
of resting
cultures
less rapidly,
but
once
synthesized,
RNA.
Although
the level
of radiofour-hour
interval,
28 to 325 RNA
ac-
second
RNA
two
relative
RNA,
pulse.
with
PHA
hours.
to 455
cleavage.
18S
pulse
interval
inrose
at a near455 RNA
sug-
This
RNA
was
yields
It is noteworthy
can
for
barely
be
one
hour
anticipated
an
estimate
that
detected
altered
the
other
in resting
the
pattern
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
712
ARNOLD
of RNA synthesis
( Figs.
id-f)
The overall
tion
doubled
during
a 15-minute
labeling
level of radioactive
interval
( Fig.
id),
.
appeared
in a more
discrete
peak.
During
le ) , a 30-32S
peak
became
prominent
could
be discerned
at 18S. A four-hour
accumulation
of 28-305
and some
185
caused
PHA
a prompt
a suggestion
were
more
hours
where,
during
accumulation
RNA
in
minor
the
even
rate
of
Fig.
(
resting
455
RNA
ig)
during
interval
by
synthesis
level
treated
tions
compared
when
with
PHA
for 48 hours,
to the
peaks
(
the
at 285
and
as
Fig.
well
to
and
RNA
polydisperse
the predominating
intervals,
some
45S
peak
185.
Therefore,
as
trends
for 48
of radioactive
150-fold
accumulated.
During
the two-hour
( Fig.
th ) and four-hour
there
was a marked
acceleration
in the accumulation
of 28S
Even
though
the absolute
rate of 45S RNA
synthesis
reached
in cultures
accumula45S RNA
i8S rRNA.
These
exposed
to PHA
the
level
when
compared
the brief
labeling
proportions
Furthermore,
labeling
to 28-305
and
growing
cultures
pulse
over
RNA
and
and a small
but clearly
defined
peak
labeling
interval
( Fig.
if) saw further
RNA.
Thus,
a one-hour
treatment
with
the
processing
in the
a 15-minute
increased
assumed
at 455.
increase
of more
rapid
clearly
expressed
a two-hour
D. RUBIN
( Fig.
ii ) pulses,
and
18S
its highest
assumed
peak
RNA
32S
rRNA.
level
minor
propor-
in contrast
to the
resting
culture,
growing
cultures
could
be characterized
by rapid
synthesis
of
455
RNA,
which
in turn
became
even
more
rapidly
processed
to mature
rRNA’s
sedimenting
at 28S
and
185.
Polydisperse
RNA
continued
to be
synthesized
by the growing
cultures,
but these
moieties
were
masked
in the
sedimentation
After
the
profile
by the predominance
phase
of active
growth
which
PHA-stimulated
ceding
the
cultures
retransformation
treated
with
quiescence.
PHA
for
The level
entered
of
of the
reached
into
a phase
blast
cells
into
168 hours
of radioactive
(
Figs.
However,
precursor
RNA
when
slower,
and
continued
accumulated
compared
rate
of rRNA
better
precursor
studies.
In
followed
the
this
for
cultures
way
an
polydisperse
and 28S
within
from
from
resting
cultures
level
cxaccumula-
moieties
as clear
a 15-minute
of 28S RXA
The cultures
cultures
growing
of
peaks
pulse.
by
the
blasts
was
of
rapid
by a
maturation.
appreciation
to mature
A
per cent
of the nmximal
in the 168-hour
cultures,
of
be distinguished
synthesis
and
hours,
48-72
in such
a phase
of metabolic
remained
above
the resting
to 48-hour
cultures,
accumulation
185 RNA
was barely
detectable.
accumulation
nongrowing
blasts
could
rate
of rRNA
precursor
slow
to mask
at 32S
at
of metabolic
quiescence
presmall
lymphocytes.1#{176}
Cultures
lj-l)
were
accumulation
level
by about
75-fold
but was only 50-70
hibited
at 48 hours.
Unlike
resting
cultures,
tion of rRNA
of radioactive
rRNA’s.
its maximum
of
products
the
RNA
additional
with
the
differences
was achieved
synthesized
30-minute
actinomycin
D
rate
through
during
chase
and
in
of
processing
a series
a 30-minute
during
unlabeled
which
uridine
from
rRNA
pulse-chase
of
pulse
time
could
be
treatment
prevented
of
further
of labeled
RNA.
Figure
2 demonstrates
that
in a 30-minute
pulse,
cultures
of nongrowing
and
growing
blasts
accumulated
radioactive
RNA
which
sedimented
in the 30-45S
range
almost
exclusively.
Species
of rRNA
precursors
formed
discrete
peaks
at 45S and 32S. During
the chase,
nearly
all
synthesis
of
the
RNA
labeled
by
the
growing
culture
migrated
into
peaks
at
28S
and
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RIBOSOME
BIOSYNTHESIS
CULTURED
IN
LYMPHOCYTES
713
Fi No.
Fig.
2.-Fate
pairs
of pulse-labeled
lymphocyte
of
member
each
of
before
minutes
Fig.
1.
(10
typical
PHA
of
The
ment.
the
rRNA.
time
of
For
various
rRNA
on
for
Replicate
minutes.
was
for
sedimentation
hours
30
member
(0.1mM)
an
was
One
exposed
to
additional
carried
(“nongrowing
30
out
blasts”).
iii
as
Cultures
blasts”).
nongrowing
culture,
processing
the
of rRNA
sedimentation
pulse-chase
30-minute
precursor
profile
experiment
which
eliminated
in growing
cultures.
of rRNA
maturation
through
studies,
precursor
along
an
labeled
moieties.
of methyl
Methylation
of maturity,
the
The
other
cultures.
ml.)
the
and
chase
as
peaks
185 RNA
established
possibility
of
aca
de
novo
PHA
corporation
fractions.
stages
168
complete
remained
minimal.
explored
these
of RNA
for
the
jzCi.
uridine
(“growing
In
for
relationship
and 18S RNA
significance
further
PHA
nongrowing
(20
while
unlabeled
Analysis
and
to H3U
(pulse)
and
for 48 hours
32S RNA
appeared
Effects
ml.)
with
mature
precursor-product
synthesis
of 28S
Initial
/
(chase).
insufficient
of 45S and
cumulation
was
uGm.
in growing
exposed
harvested
incubated
with
provided
were
was
harvest
Cultures
incubated
18S,
pair
D
actinomvcin
RNA
cultures
In
in
analysis
PHA-induced
of
the
methylated
cultures
RNA
exposed
would
associated
bear
labeled
to
growth
of
PHA
clearly
treatidentify
to methionine-methyl-H3,
chromatin.5’
methyl
effects
served
label
into RNA
is restricted
of rRNA
immediately
follows
nucleolar
lymphocyte
initial
to ribosomal
transcription
Therefore,
groups,
and
inand
of
rRNA,
each
rRNA
transfer
the 45S
at
moiety
all
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714
ARNOLD
NORMAL
LYM?tOCYTLS
D.
RUBIN
2 HR PULSE METHIONINE-METHYL-H3
I HR PHA
RESTING
?13s
Us
400
400
200
200
aL)
lBs
5 HR PHA
20 HR PHA
600
3000
s
28
I
18s
400
2000
200
000
‘-I
a-
i:
()
30
20
to
0
0
30
Fig.
3.-Sedimentation
patterns
of methylated
RNA.
Replicate
lymphocyte
tures
were
exposed
to methionine-methyl-H3
(20 ,Ci./ml.)
for two hours
harvest.
Analysis
of RNA sedimentation
was carried
out as in Fig.
1.
could
be clearly
identified
on a sedimentation
disperse
nonribosomal
components.
The
resting
45S
sedimentation
lymphocytes
with
profile
of labeled
methylated
during
a two-hour
pulse
( Fig.
a minor
peak
the purpose
of these
ignored.
Appreciable
the sedimentation
hour
prior
to the
profile
at 305
studies,
collections
profile
two-hour
and
the
prepared
pulse
a barely
RNA
showed
discernible
heavily
methylated
of methylated
285
with
3)
unobscured
the one-hour
acceleration
collection
from
cultures
treated
methionine-methyl-H3.
Accumulation
processing
of
increased.
in 45S
RNA
of 45S
accumulation
synthesis
and the five-hour
in 455 processing
labeled
455
RNA
However,
from
can
be appreciated
exceeded
the
resting
became
the
patterns
can be
less
with
hours
RNA.
peaks
with
detected
at
by about
by
at
peak
i8S.
For
)
was
on
PHA
for one
After
five hours
showed
progresOn the 20-hour
at 28S and
185.
the resting
pattern,
as an early
event.
prominent
20-hour
pattern,
as, despite
rapid
level
accumulated
a large
hetero-
transfer
RNA
( 4S
and 185 RNA
appeared
of PHA
treatment,
lymphocyte
cultures
pulsed
for two
sively
greater
accumulation
of 285 and 185 methylated
pattern,
nearly
all of the label
was contained
within
By comparing
PHA-induced
by
culbefore
as
the marked
processing,
four-fold.
the
rate
of
increase
the level
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RIBOSOME
BIOSYNTHESIS
The
near
absence
gressively
increasing
suggested
in
RNA
yields
cleavage.
groups
the
285
should
Taking
to
anticipated
exist
185:285
455
185
45S,
RNA
is not
groups
in 18S RNA
even
in small
amounts.
tamed
profile,
within
the
the amount
tativelv
for
posed
allowed
18S
pattern
and
PHA
stimulation
after
the
equimolar
rRNA’s,
amounts,
in
the
Cooper12
RNA
the
the
ratio
radioactive
to 28S
for
from
0.67
455
relatively
a
one-hour
high
of the
selected.
pulse,
complete
as
cleavage.
density
of
this
area
corncon-
RNA
on the sedimentation
could
be expressed
quanti-
RNA
optimal
resolution
technique
was
nearly
of
the rate
of 455
density
of methyl
calculated
resulting
methylated,5
required
pulse-chase
time
proalso
(
provides
a sensitive
means
of detecting
Employing
planimetry
to estimate
the
for
sufficient
resting
any variation
weights
and
of methylated
methionine-methyl-H3
to
chase
and
peaks
of
RNA relative
These
calculations
this purpose,
the
.
and,
various
of 18S
in
despite
molecular
uniformly
methyl
ponent
the
moiety
moieties
28S
ratio
in
this
an additional
dimension
of complexity
to the
RNA
processing.
Assuming
that
cleavage
of
45S
in
RNA
of
remain
unchanged
into account
the
known
Because
185
2 ) lent
and
715
LYMPHOCYTES
of labeled
1 and
accelerated
of
185 :285
CULTURED
prominence
Figs.
phenomenon
455
IN
an
455
radioactive
peaks
In cultures
ex-
additional
cleavage
one-hour
even
in resting
cultures.
Figure
4 demonstrates
lated
lymphocytes.
18S
RNA
hours.
remaining
Table
i8S:28S
predicted
Resting
from
Furthermore,
of 0.67
reach
laboratory
PHA
In
to
conservation
of RNA
Thus
maximum
of
185
resting
molecules
for
to the
RNA
appeared
still
early
and
cultures,
45S
were
cleavage,
RNA
synthesis
cleaved
nearly
one
rate
of
were
not
accompanied
by
a corresponding
introduction
of
PHA,
more
285
rapidly;
additional
products
companied
*Follo.ing
appeared
conservation
and
completion
confirming
(J.
Chem.
the
process.
report
provided
a quan-
The
data
resulting
synthesis
of
to
the
than
which
PHA,
decline
in
from
rose
maximal
while
the
rate
half
remained
at low
to 32-28S
products.
levels
and
of the
185
processed
component.
cultures
were
32-28S
more
Even
32-285
molecules
In
first
hours
455
RNA
to 32-
the
transcribed
efficiently
processed
was
processed
more
the
at the
likely
to be
byproduct.
the
earlier
slowly
lymphocyte
molecules
additional
185
of
his
Bio.
455
each
by a stable
of RNA
of
the
cleavage
An earlier
RNA
introduction
began
less
wastage.”
of 185
rate
the
slow
following
“185
after
five
rising.
transcribed
455
the
Cooper12#{176} later
increased
for
approached
Values
during
products.
conservation
hours
closely
products.
phenomenon
of
PHA
terms.
The ratio
to five hours
after
of 0.64
possibility.
an early
48
was
in quantitative
two
of 18S RNA
18S cleavage
PHA-stimu-
accumulation
with
between
cleavage
the
and
a greater
incubated
maximum
loss
this
called
contrast
a
synthesis
in
a selective
to conserve
evidence
stimulation.
gradually
the
resting
was
cultures
equimolar
and
1 provide
in
maximum
suggested
confirmation
there
phenomenon
its
assuming
between
RNA,
chase
this
of PHA.
our
Table
the
to
0.67 indicate
cultures
failed
titative
45S
after
1 expresses
value
distinction
to 285
appeared
addition
ideal
a clear
Relative
present
manuscript,
observations
244:5590,
regarding
1969).
a
publication
18S
by
wastage
Dr.
H.
and
PHA
L.
Cooper
induced
ac-
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716
ARNOLD
D.
RUBIN
TUBE NO.
Fig.
cyte
4.-Sedimentation
cultures
which
pattern
were
exposed
medium
‘as
replaced
medium
containing
10 per
additional
one
before
ned
hour
out as in Fig.
with
cent
calf
1.-Normal
Eagles
Incubation
Analysis
of
RNA
Lymphocytes-185:28S
Incubation
with
PHA
See text
serum.
Replicate
lympho-
for one
complete
harvest.
No.
hour,
2
was
after
(Spinner)
continued
for
sedimentation
car-
Ratio#{176}
Number
for experimental
of
±
1SD
Experiments
0.38±0.04
0.63 ±
0.64 ±
0.55 ±
0.48 ±
0.06
0.03
0.iO
0.07
4
4
5
5
3
Ratio
0
2
6
48
168
0
RNA.
1.
Table
Hours
of methylated
methioninemethylHa
to
details.
DISCUSSION
present
The
cyte
precursor
28S
studies
cultures.
cultures
transcription
and
of 45S
blasts,
characterized
Resting
185
rRNA
rRNA
and
rRNA.
of heterodisperse
slowed
considerably.
transcription
an
Cultures
precursor
precursor
processing,
to
PHA.
transcription
synthesis
in the
could
the
be
slower
cells
elements,
but
despite
differences
exhibited
processing
in the
appearance
of the
from
nongrowing
cultures
processed.
acceleration
of 455 RNA
detected
of
blast
a very
rapid
rate
and processing.
In cultures
of nongrowing
continued
to predominate
over the synthesis
morphologic
rate
synthesis
in three
types
of lymphorelatively
slow rate of 45S rRNA
rate of 455 processing
to mature
a
growing
nonribosomal
Therefore,
and
While
even
of
cultures
could
be distinguished
at which
rRNA
precursor
was
In PHA-stimulated
cultures,
as
rRNA
maintained
within
455
RNA
one
to two
transcription
hours
to mature
overall
rate
cells,
by
actively
the
growing
relative
transcription,
after
continued
rRNA
of RNA
rate
as well
initial
exposure
to
increase
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RIBOSOME
BIOSYNTHESIS
the
throughout
RNA
IN
entire
processing
CULTURED
period
was
a
of
active
the
function
cell growth,
initial
phases
of
tailed
analysis
of 455 RNA processing
afforded
independent
consideration.
precursor
through
32S and
finally
creased
within
one hour
after stimulation
the 455 RNA
precursor
destined
for
dependently
in pulse-chase
experiments
resting
cleavage
of 455
from
these
studies
and in PHA-stimulated
cultures
However,
reduction
within
two
of
and
hours
the
185
became
an increased
rate
of PHA
stimulation.
of
45S
Dc-
revealed
two components
which
must
be
The
rate
of cleavage
of 455
rRNA
to mature
28S RNA
became
clearly
inwith PHA.
Processing
of that portion
mature
18S RNA
was
analyzed
inwhich
allowed
sufficient
time
for
of
virtually
complete
ratios
calculated
717
LYM1’HOCYTES
RNA
even
demonstrated
cultures
deficit
in
maximal
in
resting
cells.
The
185/285
a deficit
of 185 RNA
in
at all times
after
stimulation.
PHA-stimulated
by
two
to five
cultures
hours,
appeared
before
the
overall
rate of RNA
transcription
had reached
more
than
10 per cent of its maximal
value.
Calculation
of anticipated
18S/28S
ratios
are based
on the assumptions
that
( a ) cleavage
of one molecule
of 45S RNA
ideally
yields
one molecule
of
i8S
and
tamed
one
in
selective
molecule
of 285
cytoplasmic
RNA
and
ribosomes.12
( b ) this
Therefore,
degradation
of a portion
of
products
of 45S RNA
cleavage,
32-28S
18S byproduct,
must
remain
in the
nucleus.
tion of RNA
in resting
cells indicates
degraded
if not appropriately
paired
then likely
that 185 conservation
after
the control
of 455 RNA
involved
in the initiation
the
In
scheme
becomes
rapidly
ribosomal
RNA
containing
RNP
yields
( 28S
protein
to
place
within
takes
RNA
Bound
polysome-the
cells
wastes
rate
form
to a strand
functional
( e.g.,
culture
tissue
generation.
cultures
of
an
through
growth.
of
HeLa
185
of 455
RNA.
RNA
lines
of net
cells,
rate-limiting
steps
of
rRNA
in
accumula-
may
455
couples
Intranucleolar
cleavage
with
rRNA
specific
processing
of an
805
he
of
yields
a 605
455
RNP
of the 60
a 505 RNP
a 73S ribo-
of 735 ribosomes
forms
Continuously
propagating
a
double
their
complement
of ribosomes
calculated
from
Penman’s
data45
that
characterized
tissue
culture
line,
constantly
) must
a well
to
of such
a phenomenon
offset
the inefficient
ribosome
synthesis
precursor
as well
may
as
at
ultimate
assembly
of presomal
RNA
particles
present
data suggested
that resting
lymphocytes,
of cytoplasmic
ribosomes,
maintain
restrictions
in
any
Cooper12
The occurrence
transcription
transcription
tion
and
methylated
lack
biosynthesis,
of messenger
RNA,
a cluster
unit of protein
synthesis.
However,
mainreflects
molecule
and
by a corresponding
a mechanism,
immediately
RNP.513
that
such
ribosome
and
such
the
RNA
285 RNA
must
itself
be eventually
a corresponding
i8S moiety.
It is
stimulation
plays
a primary
role in
mammalian
805
RNP’s
45S precursor
accompanied
is rigidly
185
32S RNA
and a 40S RNP
containing
18S RNA.
Cleavage
a 505 RNP
containing
28S RNA.
Final
assembly
of
) with
a 405 RNP
( 18S RNA ) in the cytoplasm
forms
some.
every
that
with
PHA
methylated
ratio
of
However,
processing
and,
of lymphocyte
accepted
precursor
the
not
one-to-one
deficiency
the
assembly
of
455 RNA
processing
ribosomes.
may be
The
restricted
be
the
presupposes
processing.
placed
level
at
of
a high
Therefore,
the
level
of
processing
in
the
into
mature
ribosomes.
The
which
exhibit
no net synthesis
both
in the 455 RNA transcripprecise
could
mechanism
whereby
not be elucidated
by
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718
ARNOLD
the
present
could
blasts
have
data.
provide
Only
such
and
Chaudhuri
demonstrated
synthesis.
found
Perhaps
when
subject
Even
ultimate
an
isolation
answer.
of
However,
intranuclear
Ennis14
and
Lieberman15
who
a dependence
of ribosome
in lymphocytes,
Kay
and
presomal
working
studied
assembly
Korner,16
and
particles
with
L cell
fibro-
regenerating
on continued
liver
more
Cooper,12
recently
cells
protein
inhibition
of protein
synthesis
precludes
efficient
ribosorne
assembly.
the portion
of the 455 molecule
representing
precursor
to 185 RNA,
unprotected
by a protein
package,
may
be particularly
unstable
and
to early
degradation,
while
the
remainder
of the
precursor
RNP
cance
in
versed
soon
lymphocytes,
resulting
induced
after
formation
of
as
the
efficient
stimulation
presence
ribosomal
phases
of
the
transcription
inefficiency,
by ultimate
ribosome
restricted
of PHA.
805
RNP
implications
ribosome
in
the
proteases
of such
growth
adequate
for
cell
engine;
rates
of transcription
growth.
“low
The
gear”
while
may
represent
a situation
“high
gear.”
In
cultures,
clearly
preceded
is a critical
rate
these
be
would
a decline
at which
in which
the
likened
reversion
The role
sponse
to a small,
of lymphocyte
has
been
emphasized
that
intact
organism.
access
antigenic
transform
to
the
this cell has
It must
remain
all sites
recognition,
into
some
assembly
the mechanism
to
of
of
a blast
maintains
governing
appropriate
antigenic
it must
cell
active
stimulation,
its
of
of
lymphocyte
ribosome
whereby
of relative
remains
shifting
of gears
low
wastage
RNA
at
it
metabolism
may
be
immune
re-
should
requirements
so as to gain
how
remote,
yet
cytoplasmic
apparatus
Perhaps
small
assembly
and
restricted
mobile.
may
be
available
in
Perhaps
there
the delivery
of the
However,
stalls
processing
point,
cell
counterpart
synthesis.
newly
RNA
ribosomes
455
amplification
no matter
meager
in protein
the resting
restricted
delivery
unique
functional
and unencumbered
intrusion
employ
early
of
numbers
of new
45S
delivery
to the cytostudies
of nongrowing
reviews.17’18
certain
mobile
the
at relatively
precursor
growth.
At this
the morphologic
recent
the
rate
RNA
transcription.
assembly
prevents
resting
lymphocyte.
proliferation
in the
subject
the
to the
rate
in the rate of 455
inefficient
ribosome
growth.
during
processing
represent
the
decreasing
of sufficient
ribosomes
to support
cell
might
slacken,
a phenomenon
of which
and
by a PHAensure
the
revert
to a state
ribosome
synthesis
rapid
rates
of transcription.
In “high
gear,”
the sheer
molecules
provides
a momentum
which
ensures
ribosome
plasm
despite
the wastage.
Results
derived
from
the
blasts
re-
precursor
Alternatively,
when
may
net
efficient
gear”
signifi-
becolne
motion
would
nucleolus.
but,
may
represent
“high
into
This
of lymphocyte
most
efficient
response,
situation
would
special
wastage
of 185
set
probably
may
control
available
pools
of
a refined
control
mechanism
may
reaches
a certain
level,
assembly
and, as in the case of HeLa
cells,
in an
185
Conservation
in the regulation
assembly
becomes
lymphocyte
assumes
and
could
be
synthesis.
particles
intranucleolar
The elucidation
degradation
assembly
processing
introduction
stable
that
followed
processing
of 45S RNA
of ribosomal
protein
of certain
protein.
have
important
It appears
sensitive
RUBLX
that
particles
undergo
partial
processing
while
still in the nucleolus.
The
phenomenon
of restricted
the
D.
be
in the
ready
upon
to
ribo-
However,
exquisitely
ribosomal
pro-
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RIBOSOME
tein
BIOSYNTHESIS
may
allow
trol
CULTURED
a prompt
turn,
provides
more
protein
Cell growth
only
mune
IN
719
LYMPHOCYTES
increase
in the
rate
of 45S
RNA
processing
the cytoplasm
with
additional
ribosomes
and consequently,
more
ribosomes.
may be regulated
at several
levels.
The
on one which
may be ideally
adapted
response.
It is conceivable
that the
mechanism
may
be
critical
for
which,
the
in
synthesis
present
study
of
focuses
to the lymphocyte’s
role in the imcell environment
dictates
which
con-
in a specific
instance.
SUMMARY
Ribosomal
lated
by the
455
RNA
production
rate of 45S rRNA
processing
to
the
Cultures
of resting
both
transcription
cent
as
a
of the
RNA
result
of
to
growth
grade
five
finally
turn,
after
PHA
the
initiation
condition
may
be
ribosome
by
48
mature
ribosomes.
by maintaining
Still about
50 per
185
After
hours,
at
once
again
proliferative
despite
a brisk
of maximum.
the
in
corresponding
lymphocytes.
By
cultures
after
the
of lymphocyte
depend
on the
regulated
found
to be reguthe rate of
by
components
PHA
treat-
transcription
and
cleavage
to 28S
hour,
rose to a maximum
at 48 hours.
reached
maximal
efficiency
between
treatment.
considerably
per cent
appeared
and also
ribosome
formation
RNA at low levels.
to 285 lacked
in resting
50-75
may
components
restricted
of 45S
PHA-stimulated
At 168 hours,
slowed
lymphocytes
transcription
45S rRNA
precursor
increased
within
one
of 185 components
hours
-approximately
Thus,
growing
185
processed
degradation
selective
response,
18S RNA.
processing
and
lymphocytes
and processing
ment,
the rates
of
products,
detectably
However,
conservation
one
285
in cultured
precursor
the
began
process
rate
height
of
to selectively
had ceased,
of 455
RNA
of 45S
rRNA
de455
transcription
growth
and
the maintenance
rate of ribosome
biosynthesis
efficiency
the
precursor
of
which,
utilization
the
in
for
assembly.
REFERENCES
1. Inman,
Electron
D.
and
microscopy
stimulated
by
lymphocyte
of
1963.
A.
D. :
growth
assembly.
at
Nature
3. Perry,
220:
R. P.
Cancer
Cooper,
human
E.
Inst.
: On
H.:
lymphocytes
J. Cell.
phytohemagglutinmn.
Biol.
19:441,
2. Rubin,
Natl.
R.,
Possible
the
control
level
196,
of
of
ribosome
Blood
biogenesis.
A.
17:117,
and
of
RNA
metabolism
resting
state
lymphocytes
:
Ribosome
in the
formation
tabolism
in
cells. Nature
219:685,
1968.
6. Havemann,
K., and Rubin,
A. D. : The
animal
delayed
response
leukemia
lymphocytes
of
in vitro.
Proc.
Soc.
668,
1968.
7. Rubin,
A.
D.,
W.
: Studies
chronic
lymphocytic
to phytohemagglutinmn
Exp.
Biol.
H.
in
Nat.
in
chronic
K.,
127:
and
lympho-
L.:
dur-
to
active
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Ribosome Biosynthesis in Cultured Lymphocytes: II. The Role of
Ribosomal RNA Production in the Initiation and Maintenance of
Lymphocyte Growth
ARNOLD D. RUBIN
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