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Half-Lives
of Beta
and
Gamma
Globin
Capacity
in Cultured
By Jeffrey
The
turnover
RNAs
rates
and
human
of
than
cell
reported
increase
during
ular
hybridization
of
neonatal
are
proteins.
months
of
reference
1). After
translated
and
that
translation
the
tion
is restricted
The
ity
to the
is
blasts
appears
globin
The
genes.4’5
final
on
levels
However,
of Hbs
and
thetic
ratio
lower
in older,
F
mRNAs
begin
to be
before
gastrulation.
regulated
by
factors
a
similar
changes
Hb
induction
two
A and
is,
mature
y-$
the
(I)
(red)
burst
colonies
younger,
less mature
(pink or white)
colonies.
occurs even in cells that make predominantly
is synthesized
in bone
locytes,
HbA
while
Thus,
-y chain
emythroid
synthesis
Three
is reduced,
mechanisms
of these
13globin
could
globins:
is asynchronous,
globin
normoblasts
but
to accumulate
not
transcription.
mRNA
mRNA
account
(1)
gene
(2)
may
be
for
Transcription
ie, y globin
ct-f3 translation
mRNA.
Blood,
then
depend
solely
importance
of
in
cultured
rates,”
no
longer
produce
in normoblasts
on mRNA
mRNA
and because
with similar
and
turnover
turnover
of recent
structures
we tested
the
during
evidence
(/3 and
third
of ‘y and /3 globin
are approximately
to threefold
of /3 and
exclude
results
late-stage
erythroid
the
changes
ratio
that
b) are
hypothesis
mRNAs.
equivalent
We
in
more rapidly
‘y globins
decay
the
hypothesis
from
that
differences
cells.
in
They
because
than mRNA,
the
in parallel.
These
the
support
the
change
mRNA
the
y and
in
the
stability
in
hypothesis
/3 globin
that
genes
are
blood
from
asynchronously.
than
in
three
fused
culture
and
RNA
Blood
METHODS
isolation.
unrelated
neonates
and
patient with mild sickle
reticulocytes.
decrease
(2) HbF
AND
MATERIALS
Cell
is
cells
Umbilical
in reticu-
cytes)
per
milliliter
were
in warm
cord
peripheral
blood from
cell anemia
were used
collected
Iscove’s
one untransas sources of
in hepaninized
washed twice at room temperature
with
excess of Iscove’s medium
containing
100 U/mL
penicillin,
and 100 ,zg/mL
was resuspended
at 1 to 4 x l0 cells
to /3, in late-stage
tubes
and
a fivefold- to tenfold-volume
5% fetal calf serum (FCS),
streptomycin.
The cell pellet
(reticulocytes
plus erythro-
medium
containing
20%
FCS
fi genes
From
ceases
before
Department
efficiencies
more
There
syn-
y and
transcription
Translational
cells.
asynchronous
of the
translated
in late-stage
hypothesis.
genes
that
mRNA
the stability
turnover
rates
‘.1-fl ratio
synratio
is synchronous,
This
For
ratios
example,
is some
than
y
precedent
for
fi
and
in vitro.’#{176}”
but
mechanism
Vol 66, No 5 (November),
(3)
Transcription
‘y mRNA
turns
would
affect
1985:
over
protein
pp 1149-1154
of
faster
both
than
synthetic
the
McArdle
the
tory
for
and
supported
grant
Submitted
Address
Cancer
Laboratory
was
training
No.
Nov
reprint
Cancer
for
University
by National
McArdle
T.D.S.
Laboratory
ofPathology,
Supported
change.
efficiently
globin
mRNAs
are
translated
with different
efficiencies
in rabbit
reticulocytes,
and the concentrations
of various
reaction
components
affect
this
cells
ofeach
at different
to
in reticulocytes.9
relative
the
that
of mRNA.
cells.
thesis
globin
marrow
continues
This
HbF.8
of
indicate
quantity
production
level
embryonic
development,
human
globin
mRNAs
also
the
Inc.
late-stage
would
ratio
earlier
During
protein
synthetic
in
The
transcribed
(BFU-E)
globin
y-/3
that
determining
progenitors
the
That
more
in
especially
mRNAs.
observations
of
suggest
a role
F in erythrocytes:
erythrocytes,
decreases.8
y globin
in erythro-
observations
ratios,
globin
view
globin
& Stratton.
decays
twofold
rates of synthesis
human
of transcription
play
erythroid
possibil-
f3 and
in
production
for
matura-
cultured
meticulocytes
from an adult
patient
with mild sickle
cell anemia
and from neonatal
umbilical
cord blood.
Furthenmore,
although
neticulocyte
protein
synthetic
capacity
and
in
rate-limiting
et al. Dev
ceases
results
not
in
those
synthesis
transcription
Our
machinery.
result
with
erythroid
protein
gene
synthetic
by measuring
find that the
produc-
the
transcription.
degraded
during
protein
gene
by Grune
rate.
In
constable
In
gamma
changes
(Farquhar
during
globin
not
or
coupled
cells
that.
hemoglobin
does
rates
data.
erythroid
because
reticulocytes
in
production
of
may
of cultured
reticulocytes
is
post-
oocytes
histone
by
the
controls
differentiation
reticulocytes.
remain
investigated
and
onset
to depend
posttranscriptional
the
here
A
modulated
turnover.
becomes
quantities
stability
that
in intact
a 1985
These
suggest
).
to adult
turnover
gamma-beta
but
S-phase.”
(Hb)
cells
mRNA
beta
the
turnover.
mRNA
ensure
described
hemoglobin
emythmoid
histone
may
experiments
that
the
of
synthesis
mRNA
the
protein
gamma.
1 981
of fetal
maturation
mRNA
immature
403.
the
beta
in
with
85:
than
(reviewed
degraded
obtained
Biol
ratio
erythroid
efficiencies.
the
genes
mRNAs)
is thus
translation
and
level,
that
differences
declines
as
echinoderm
proteins
and
control
DNA
rapidly
from
by molec-
specialized
masked
to
assays.
rate
mRNAs
late
tion.
hours.
modulate
and
changing
synthetic
capacity
same
the
during
of
Synthetic
D. Sullivan
in adult
transcriptional
fertilization,
are then
hours
eight
or “masked”
of embryonic
fashion,
the
untranslated
Production
affect
to
mechanisms
(“maternal”
29
of most
at
Amphibian
a reservoir
after
expression
control
to
Thomas
synthesis
the
of Protein
Reticulocytes
Therefore.
stages
explain
protein
the
was
and
in
more
final
could
synthetic
at
controlled
goal
translation
20
of six
declines
transcriptional
for
cell-free
a half-life
messenger
As determined
and
the
the
beta-gamma
mRNAs
synthesis
some
a result
Protein
with
probably
tam
during
Such
and
synthesis
Our
RNAs
Human
Ross
is significantly
mRNA
the
reticulocytes.
LTHOUGH
mRNA
maturation.
both
decays
protein
of
in
erythroid
half-lives
cells
globin
globin
protein
measured.
globin
maturation.
ratio
gamma
globin
been
beta
gamma
erythroid
and
gamma
have
whether
stable
beta
and
reticulocytes
determine
A
of
beta
Messenger
Cancer
Research.
of Wisconsin,
Institute
Program
by a National
and
the
Madison.
grants
No.
Project
grant
Cancer
Institute
CA-071
No.
75 to
CA -23076.
predoctoral
CA -09135.
26,
1984;
requests
Research.
accepted
May
to Dr Jeffrey
450
North
16.
Ross,
Randall
1985.
McArdle
Ave.
Labora-
Madison,
WI
53706.
/3
()
1985
by Grune
& Stratton,
Inc.
0006-4971/85/6605-0024$03.00/0
1 149
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ROSS
1150
and the same
antibiotics
37 #{176}C
in a humidified
(growth
5% CO,
cells
were
counted
at different
sis)
was
observed.
The
staining
with
new
culture period.
To prepare
4 #{176}C
(2,400
total
culture
blue
RNA,
cells
eight
Cells
intervals,
experiment,
and
of
reticulocytes
remained
constant
were
minutes,
were cultured
In each
percentage
methylene
rpm,
medium).
atmosphere.
harvested
PRJ
at
total
no loss (hemolyassessed
by
throughout
and
the
centrifuged
centrifugation).
at
Pellets
were
ing
the
number
globin
probe
oligo
dT-cellulose.
sequences
[wt/vol]
sodium
hybridization
EDTA,
10 mmol/L
jzg/mL
of Escherichia
mixture
was
extracted
Tris-CI,
vortexed
alcohol
alone.
in RNA
10 mmol/L
24:1,
RNA
was
storage
Tris-CI,
8.0)
plus,
from
the
buffer
pH
same
Hybridization
mRNA
either
7.4).
detail.’6
number
analyses.
or
in some
y globin
mRNA.
The percentage
(0.1 mol/L
For
1 mmol/L
experiments,
then
40
cells
for each
was
hybridizations
with
resistant
and
tRNA
added.
EDTA,
time
cells
to
probes
This
been
40
was determined
measure
specific
the
were
digestion.
fi
The
H91S,’7
“P-5’-end
fi
only
discussion
In this
cpm
amount
with
one
plus
single
strand
Data
the
percentage
RNA
minus
basis.
The
the
on a per
relative
of maximum
age of time zero (Figs
regression
analysis,
1 and 2). Straight
were
drawn
than
at each
plotted
the
points,
point
plot
the
on a per
time
point
necessary
to
as a percent-
lines, determined
through
time
actually
of RNA
were
with
of RNA
number of total
rather
of mRNA
and
cpm
for each
curves
the amounts
hybridization
Cot
(5,-
v volume
the same
basis,
amounts
by comparing
solution
standard
S,-resistant
plotted
Cot
cell
all
of hybridization
harvested
Therefore,
frequently
for
of the
and
of the
maximum
Ross
and
of why hybridization
erythrocytes)
mRNA
determined
as equivalent
were
labeled
by a modification
reticulocyte
of globin
50%
probes
maximum.
case,
experiment.
microgram
to a, #{244},
or fi globin
hybridize
Because
analyzed
in
by measur-
not
y and
theoretical
for
described
does
The
divided by input cpm x 100) was
A given volume of RNA represents
in a given
give
for detailed
achieve
(reticulocytes
were
point.
nuclease
ofclone
hybridize
with
mRNA,
the theoretical
to /3 mRNA
is 50%
(see
Fig IA).
See
format.’9
times
with 5’-”P-labeled
has
to
curve
Northern
to 5,
fragment
probe
shown).
respectively.
will
Donaldson’6
in ethanol
hybridizations
method
i9 probe
and
and
not
stranded,
fails
purified by pelleting
through
a
each RNA sample was resusie, a given volume of RNA was
of total
double
The
I mmol/L
translation
This
(data
added.
two
twice
NaCI,
cell-free
of hybridization
A
and
precipitated
Solution
levels were performed
fi
NaCI,
and 0.5 vol of chloroform-
vol/vol)
then
blot analysis,
the RNA was further
CsC1 cushion.”
For each experiment,
pended in the same volume of buffer,
derived
mol/L
1 .0 vol of phenol
(CIAA,
The
pH
0.35
coli tRNA
(as carrier)
was
vigorously
at room
temperature
two times with
isoamyl
CIAA
frozen
sulfate,
resistant
bp Hinfl
SULLIVAN
labeled at +595 from the mRNA cap site, as previously
described.’6
The ‘y globin
probe
is an approximately
900 nucleotide
fragment
prepared
from the eDNA clone JWI5I.’8
After incubating
JWI5I
DNA with Sau 3A, which cuts at codons
80-81,
the mRNAcomplementary
strand was purified by affinity chromatography
on
stored at - 70 #{176}C
without
further
washing.
They were subsequently
thawed but not allowed to warm up above 4 #{176}C.
An approximately
tenfold-volume
excess of urea lysis buffer’4 (7 mol/L
urea, 2%
dodecyl
of counts
is a 285
AND
taking
by linear
time
zero
to 100%.
/;r::__l
beta
30
5o
20
100?
,
2O
I0
0
a
I
0
0
a)
0
100
.
N
:r
5)
a)
0
B gamma
E
80
60
40
/
I-
-
0
,
C
-
a)
0
20
a)
aI
01
I
I
05.1
RNA(p.I
.5
per
0
5
reaction)
Fig 1 .
Solution
hybridization
analysis
of beta
and gamma
globin mRNAs
in cultured
reticulocytes.
Umbilical
cord blood was
cultured
at 10’ total cells per milliliter.
as described
in Materials
and Methods.
Aliquots
(3 ml) were harvested
at the times noted
below.
and total RNA was extracted
without
added carrier.
Each
sample was dissolved
in 300 ILl. 50 that each microliter
contained
RNA from approximately
3.3 x 10 cells (reticulocytes
plus erythrocytes).
RNA was hybridized
in solution
with 5’-P-labeled
DNAs
specific
for fi or y globin
mRNA.
each at a specific
activity
of
approximately
i07 cpm/g.
and 5,-resistant
cpm were
determined.
(A) Hybridization
with the double-stranded
P-$-globin
probe: 285 b.p. Hinfl fragment
of Hfl1 S. 5’ end-labeled
at nucleotide + 595 from the mRNA
cap site.
(B) Hybridization
with
the
single-stranded
P-y-globin
probe: 900 nt single-stranded
Sau3A
fragment
of JW151.
5’ end-labeled
at +195
from the mRNA cap
site. Closed circles.
0 hour; open circles.
61/2 hours;
closed squares.
1 6 hours; open squares.
23 hours; triangles.
41 /2 hours.
0
20
Time
Fig 2.
40
(hr.)
of beta and gamma
globin mRNAs
and of total
for fi (top panel)
and y (middle
panel)
globin mRNA decay were derived
from panels
A and B of Fig 1.
respectively.
by determining
the quantity
of RNA
required
to
protect
a given percentage
of the P-DNA
probe
(see Materials
and Methods).
Total RNA decay data (bottom
panel) were derived
from Orcinol assays#{176}of RNA recovered
at each time point. Lines
were determined
by linear regression
analysis.
including
the time
zero point as 100%.
The half-lives
of
and ‘y mRNAs
and of total
RNA are 31 . 29. and 43 hours.
respectively.
These
data are
experiment
5 of Table 1.
reticulocyte
Half-lives
RNA.
Points
$
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EOSINOPHIL
PEROXIDASE
Table
1151
1 . Summary
of Molecular
Experiment
cell
Beta
Sickle cell anemia
Neonatal
umbilical
cord
4
Neonatal
umbilical
cord
34
34
5
Neonatal
umbilical
cord
3 1
29
29 (± 6)
27 (± 8)
Fig 2. Experiments
the same
adult
are derived
the
Experiments
15
acetone,
32
34
Triton
30
22
internal
of lines
collected
3 through
such
blots
gels
mol/L
performed
in
formaldehyde,
(Biodyne
then
A,
in an
20%
samples
RNAs
Pall),
hybridized
were
rather
than
of the
agarose
gel
transferred
to nick-translated
method
FCS.
eDNA
from
2.2
transfer
a beta
was
globin
clone,
JWIO2.’8
with
20%
Grand
fetal
calf
Island,
same
ILCi/0.l
they
were
once
was
were
with
of 4 vol of water,
of protein
was
and
and
loaded
onto
with
excised,
and
scintillation
the
cultured
was
concentration
once.
saved.
previously
debris
by
was
described.”
The
Stained
globin
hydrolyzed
and
counted
translation
was
(Promega-Biotec,
performed
Madison,
with
Wis)
a rabbit
containing
gel
bands
not
1 2 3 4 5 6 7 8
total
precipitated
with
in
hemoglobin
but
labeled
terminus
/3 probe
acid-urea-
marker
as an
number
the
Total
or
RNA
was
primary
other,
mRNA
extracted
structures,
of each
probe
is located
a large
anneal
with
anneal,
remains
of
culture
and
DNA
probes,
to
Although
these
the
not
percentage
40- to 48-hour
5’-”P-labeled
mRNA
levels.
example,
containing
each
because
“P-
in a region
of
segment
of
unlabeled
‘y mRNA,
probe
the
but
the
labeled
single-stranded,
and
is
by S, nuclease.
was
a given
percentage
in that
fraction.
reticulocyte
ILmol/L
of probe,
with
umbilical
cord
with
different
samples
anemia,
the lower
Summary
indicate
blood
that
B.
data
from
from
unrelated
from
the
an
the amount
five
adult
turnover
with
rates
of mRNA
experiments,
three
neonates
and
two
mild
sickle
cell
of both
mRNAs
are
23456789101112
1
.
Ay
cell
during
Re-
mRNAs.
medium
similar
might
in a liquid
9 10 11 12
globin
not
For
does
gamma
in total
shown).
possess
were
29
to determine
the volume
of RNA
required
to saturate
a given percentage
of DNA
(20% hybridization
for /3; 50% hybridization
for ‘y),
(Fig
I). The greater
the volume
of RNA
needed
to hybridize
100 g
polyacrylamide
blue.
with
The quantity
per cell of each
mRNA
decreases
as the
reticulocyte
matures
(Figs
I and 2). The relative
amount
of
mRNA
in each sample
(Fig 2) was determined
by measuring
time
lysed
The
1 2%
of
at which
Approximately
for
treated
separated
in enriched
in solution
with
/3 on ‘y globin
one,
digested
and
decrease
(data
terminus
in the
counter.
In vitro
A.
were
detects
the
(GIBCO,
saline,
acid-urea-Triton
brilliant
protein
to a final
freeze-thawed
once
resuspended
for one hour,
was
as
Coomassie
medium
were
added
washed
phosphate-buffered
supernatant
electrophoresed
stained
lysate
the
They
cultured
cold
were
essential
leucine.
‘H-leucine
Cells
washed
and
reticulocytes
in minimum
lacking
and
mL.
centrifuged,
gels
serum
NY)
medium,
200
addition
Cultured
synthesis.
were
incubation
was
was
human
ofbeta
nonhomology.’6
Protein
After
reaction
remainder
chains
was observed
mRNAs
membrane
The
protein
rates
No
annealed
quantitate
of
containing
to a nylon
to nitrocellulose.
The
containing-unlabeled
ticulocytes
times from
5 used different
by a modification
electrophoresis
the
standard.”
reticulocytes
were
After
of
acid precipitated,
protein.
globin
Turnover
newborns.
Northern
into
and
periods
Thomas.”
30 #{176}C,
a portion
RESULTS
as those
at different
at
and then trichloroacetic
17
slopes
1 and 2 used samples
patient.
unrelated
from
ILL of “S methionine.”4
1 0 /ACi/20
incorporation
3
values
plus
minutes
NaOH
Gamma
2
(± SD)
30
(h)
Sickle
half-life
anemia
hemin
mRNA
of Globin
1
Mean
from
Experiments
Half-life
Source of
Reticulocytes
No.
The
Hybridization
-
.
rS
‘-
‘
a
p.-,
_
.4
-...ai
Ay
.
-
‘-
-
-
/3a-
5
.
-
e
Pr
-
-
-
Gy
/3-
.
.
-
a-
.-f.
iV
Fig 3.
Polyacrylamide
gel electrophoresis
of reticulocyte
protein.
Reticulocyte
culture.
protein
labeling.
and cell lysis were performed
as in Table 2A legend
and Materials
and Methods.
Duplicate
samples
from different
culture
time points
were
electrophoresed
in a
polyacrylamide
gel containing
urea and Triton
X-1 00. (Panel A) Protein
stain. After electrophoresis
the gel was stained
with Coomassie
blue. destained
by diffusion.
and photographed.
(Panel
B) Fluorography.
The gel was treated
with ENHANCE
(New
England
Nuclear.
Boston)
and dried according
to the manufacturer’s
instructions.
Preflashed
film (Kodak XAR-5)
was exposed
to the gel at - 70 ‘C for 12
days. Globin proteins
are indicated
on the left. These data are experiment
3 of Table 2A. Lane 1 . 1 3 gm. 0 hour. 4.000 dpm; lane 2. 39 gsgm.
0 hour. 12.000
dpm; lanes 3 and 4. 130 gm. 0 hour. 40.000
dpm; lanes 5 and 6. 130 gm. 6/2 hours. 44.000
dpm; lanes 7 and 8. 1 30 gm. 16
hours.
20.600
dpm; lanes 9 and 1 0. 1 30 Lgm. 23 hours.
8.400 dpm; lanes 1 1 and 1 2. 1 30 gm.
41 1/2 hours. 6,250 dpm.
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ROSS
1152
similar
(Table
different
I).
between
Turnover
sis
in
rates
intact
was
reasons.
First,
ofbeta
this
Intact
cells
leucine
for one
gels
were
stained,
(see
The
results
of
similar,
approximately
protein
synthe-
this
mRNAs.
relative
Second,
it should
translational
efficien-
containing
cell lysates
in acid-urea-Triton
and
Methods)
bands
were
such
protein
seven
(Fig
3).
The
and
nine
hours
gels
that
cells
for
(Table
‘y mRNAs
depends
The structural
half-lives
molecular
hybridization
The
functional
on
decay
how
the
at similar
mRNA
are 27 to 29 hours,
with deproteinized
half-lives
are
seven
to nine
rates.
is assayed.
hours,
by
1).
as deter-
mined
by translation
in intact
cells (Table
2A). There
are at
least two possible
explanations
for this discrepancy:
(1) The
hybridization
assay
overestimates
mRNA
half-life.
This
assay
could
detect
mRNA
to anneal
to the probe
putative
neticulocytes
/3
Table
2.
Globin
of Experiments
mRNAs
in Intact
are
on the
large
Half-life
capacity
ofmRNAs.
reticulocytes
was
system,
during
to
major
B.
of Beta
of Globin
Protein
Neonate
cord
blood
time,
CsC1,
and
was
cDNA
should
hybridize
cell
electrophoresed
formaldehyde,
transfer
globin
cells
total
were
cultured
RNA
was
in cultured
for
one
hour
Lysates
9
5
6
3
8
11
(± SD)
7 (± 2)
hybridized
clone’8
to
to
/3
and
‘y globin
to
gels
sured
17
18
20
23 (± 7)
19 (± 2)
SD)
separate
radioactivity
by plotting
for
determine
decay
tNeonate
deproteinized
Methods).
supplemented,
Fig
were
chains,,
were
times
(see
and
(Materials
A single band ofapproximately
at each
time point
(Fig 4).
The
probably
because
to two
probe
hybridizes
Fig
1 . The
3),
of time
slopes
cord
blood
cells
and isolated
after
different
In vitro
translation
nuclease-treated
methionine.
Aliquots
urea-Triton
gels as
of the
in A.
44
650
support.
The
human
/3
The
probe
under
these
nucleotides
is
band
is broad,
mRNAs
and
because
discrete,
the poly A size is not uniform.’6
More important,
no
lower mol wt bands are observed.
Bands containing
20%
or less of the input
can easily
be detected
(lane
1).
Therefore,
there
is no evidence
for the accumulation
of
234
622
-
and
and
Methods).
the
Fig
amount
Half-lives
were
v culture
zero)
of the
of
the
rabbit
translation
were
cultured,
culture
lines
RNA
was
reticulocyte
mixture
and
times
of
mea-
time,
as
used
to
were
total
RNA
(Materials
performed
lysates
were
439
-
370
-
227
-
181
-
83
pulse-labeled
rates.
umbilical
or
and
in polyacrylamide
determined.
percentage
Table
cultured
electrophoresed
was
band
(as the
2 and
cells
at different
globin
cpm
described
blood
samples
globin
in each
mRNAs
conditions.
observed
the
20,
by pelleting
gel containing
nylon
with
a nick-translated
and
autoradiographed.
9 (± 3)
28
3H leucine
a
0,
1875
5
prepared,
for
purified
(h)
4
with
were
functional
reticulocytes
2
cord
of
in an agarose
transferred
Gamma
9
umbilical
levels
from
protein
Extracts
Synthesis
1
(±
isolated
in a cell-free
the
perwas
breakdown
RNA
translated
In vitro translationt
Mean
were
analysis
mRNA
Second,
determine
To address
culture.
At each
through
time.
Is.
Mean
translation
enough
v Cell-free
Beta
synthesis
a limiting
For example,
Translation
Reticulocytes
.
Globin
of
degradation
products
in rabbit
by hybridization.’5
(2) The func-
Experiment
No.
A.
that
be translated.
cultured
synthesis
with
1
globin
mRNA
are detectable
Summary
Gamma
fragments
but cannot
to determine
accumulate
Umbilical
is
as measured
RNA
(Table
translational
products
hours.
the
in intact
each
and
mRNA
counted.
indicate
synthesis
to
‘Hwere
polyacryl-
excised
experiments
‘y globin
rate
half-life
whether
/3 and
the
the
mRNA
That
is,
not mRNA.
Size
as opposed
culture.
globin
To summarize,
measures
exploited,
the functional,
2A).
However,
assay
SULLIVAN
underestimates
limits
translation.
additional
experiments
blot
hybridization
in the
three
of /3 and
tional
assay
in whole
meticulocytes
half-life
because
something
else
were
these
two possibilities,
formed.
First,
Northern
Materials
half-life
globin
incubated
in medium
Globin
chains
in crude
and
values
measures
by electrophoresis
amide
the
factor(s),
during
were
hour.
gamma
of the
if any,
why
2.
Synthesis
of y and /3 globin
in cultured
reticulocytes
for several
assay
mRNAs
separated
know
1 and
half-lives
changes,
of the
not
and
monitored
to the structural,
cies
do
reticulocytes.
proteins
detect
We
experiments
AND
was
and
in hemincontaining
analyzed
35
in
acid-
4.
Blot
transfer
analysis
of
reticulocyte
globin
RNAs.
Umbilical
cord blood cells were
cultured
for 0. 20. or 44 hours.
Total
RNA was prepared
by phenol
extraction
without
added
carrier
and was purified
by pelleting
through
a CsCI cushion
(see
Materials
and Methods).
RNA samples
were electrophoresed
in a
1 .5% agarose
gel containing
2.2 mol/L
formaldehyde#{176} and were
transferred
to a nylon
support
(Biodyne
A paper.
Pall).
The
transfer
was hybridized
with nick-translated
globin cDNA clone
JW1O2.
and the autoradiogram
was exposed
for six days at
- 70
‘C with Kodak
XAR film and a Lightning
Plus intensifying
screen.
Size
marker
fragments
in nucleotides
are noted on the right side of the
figure (pBR322
DNA treated
with Haell and kinased).
Lane 1 : t,,,. 0.2
ILg; lane 2:
t0.
1 .0 Lg; lane 3: t20. 1 .0 Mg; lane
4: t44.
1 .0 g.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
EOSINOPHIL
discrete,
stable
small
RNA
mRNA
Low
levels
long
result
that
of shortened
stable
accumulate
time
below
For this
fragments
not shown).
degradation
with
Of
poorly
Work
course,
that
and
the intact
reason
we
experiments
with a DNA
third of /3 globin
mRNA.’5
protected
(data
products.
or anneal
immediately
in this assay.
5, mapping
to the 3’ one
exposures
from
transfer
that migrate
go undetected
also performed
that anneals
with
degradation
fragments
large
fragments
mRNA
might
have
probe
1 153
PEROXIDASE
were
These
products,
in culture
and
observed
fragments
but
were
not
did
not
The
functional
state
translation
lysates.”
with the
Subtle
Northern
of
reticulocyte
mRNAs
(Table
/3 and
capacity
the globin
analyzed
by
reticulocyte
that
escape
assays
but
mRNAs
and in vitro
the observation
of a reticulocyte
are
similar,
disappears
as
We
draw
two
mRNAs
cytes.
(2)
disappears
BFU-E
and
late
demonstrated
with
globin
erythroblast
chain
stages
of
conclusion
genes
makes
is repressed
it probable
that
asynchronously.
transcription
Thus,
of these
expression
of these
erythroid
cell maturation
is determined
affect
both the rate of transcription
and
time period
during
which
The second
conclusion,
each gene is transcribed.
that protein
synthesis
decays
by
the
faster
extends
the original
that translation
in rabbit
limited
by the amount
results
have
been
amphibian
oocytes’8
and in the lens of the
eye.’9 Additional
experiments
are required
which
protein
synthesis
factor(s)
become
of endogeobserved
in
developing
chick
to characterize
rate limiting
in
reticulocytes.
than
ACKNOWLEDGMENT
mRNA.
DISCUSSION
globin
the
already
in parallel
development.’7
These
data,
along
with those
reported
here,
indicate
that the shift in ‘y-f3 synthetic
ratios or in HbF-HbA
accumulation
ratios
in late-stage
erythroid
cells does not
result
from differential
stability
of ‘y and /3 globin
mRNAs.
maturing
assays.
protein
faster
between
had
rise
reticulocyte
lysates
is not
nous
mRNA.”
Analogous
in the
rates
functional
that the
laboratories
levels
than
mRNA
levels
in reticulocytes,
observation
of Temple
and Lodish
detection
that alter
decay
with half-lives
2B). Thus,
the turnover
y globin
measured
by both structural
This conclusion
highlights
does
was
rabbit
be detected
by in vitro translation.
parallel
the molecular
hybridization
results:
/3 and ‘y globin
range of 20 to 30 hours
synthetic
mRNA
structural
changes
blot or S mapping
mRNA
function
might
The results
of this assay
of
each
in nuclease-treated
other
mRNA
genes
during
controls
that
investigated
further.
cell-free
synthesis
This
could
they
from
globin
conclusions
decay
The
protein
faster
than
from
at the
this
same
rate
synthetic
does
work:
capacity
either
( I ) /3
in cultured
globin
of
and
‘y
reticulo-
We are grateful to Drs B.G. Forget and SM. Weissman
for globin
eDNA clones, to Dr T. Maniatis
for fi globin genomic
clones, to
Debra
Donaldson
for expert
technical
assistance,
and to M.J.
Markham
for excellent
typing. These investigations
were performed
with
reticulocytes
the
approval
Committee.
formed
mRNA.
of the
University
Recombinant
using
of Wisconsin
DNA
cloning
Human
and
Subjects
handling
was
per-
P1 restriction.
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1985 66: 1149-1154
Half-lives of beta and gamma globin messenger RNAs and of protein
synthetic capacity in cultured human reticulocytes
J Ross and TD Sullivan
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