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Translational control of gene expression in the human brain
Prog. Neuro-Psychophormocol. & Biol. Psychiat. Printed in Great Britain. All rights reserved
1989, Vol. 13. pp. 469-479
TRANSLATIONAL
NIKLAS
Copyright
CONTROL OF GENE EXPRESSION HUMAN BRAIN
LANGSTROM';' ANDERS
1. Department
ERIKSSON' WALLACE"
BENGT
WINBLAD:
0
027&5846/89 $0.00 + so 1989 Pergamon Press plc
IN THE
and
WILLIAM
of Psychiatry Sinai School 2. Department of Geriatric 3. Institute
and Fishberg Center for Neurobiology, Mount of Medicine, New York, N.Y. Medicine, Karolinska Institute, Huddinge, Sweden for Forensic Medicine, University of Umea, Sweden
(Final fcrm, Auqnst 1988)
Contents
1. 2. 3. 4.
Abstract Introduction: Experimental Approaches to Investigating Translational control of Gene Expression in the Human Brain Characterization of Polysomes from Human Postmortem Tissues Conclusions Acknowledgements References Abstract
Langstrom,Niklas, Anders Erikson, Bengt Winblad, and William Wallace: control of gene Translational expression in the human brain. Prog. Neuro-Psychopharmacol and Biol. Psychiat. 1989, J&469-479 1. Translational control is the regulation of protein synthesis as an efficiency of mRNA translation and is a common alteration in the mechanism by which cells regulate gene expression. 2. Alterations of total protein synthesis are often the responses of stimuli including starvation, viral infection, cells to various stress and heat shock. Numerous specific genes including ferritin heavy chain, tubulin, 3. proto-oncogene have also been shown to be under the Ick vimentin and translational control. intact organisms, Unlike cultured cells or the investigation of 4. human brain requires the measurement of control in the translational protein synthesis, especially polysomes. Therefore, we components of and characterized polysomes from human postmortem brain have purified purified tissues and compared them to polysomes from the adult rat brain. The yield (as A260 units per gram brain tissue), size (as number of 5. translational efficiency message), ribosomes per (as amount protein ability to reinitiate (as amount of synthesized per A260 unit), and all initiation inhibitors) were protein prevented by synthesis human polysomes compared with as exhibited by the significantly lower rat polysomes synthesized human and polysomes. However, the the rat similar polypeptides. differed from the rat polysomes 6. the human polysomes Thus, principally in the efficiency of mRNA translation which is likely due to
469
469 470 471 473 473 477 478
N.
470
the greatly synthesis.
reduced
Key Words: translation
human
Abbreviations:
ability
of the human
brain,
polysomes,
Translational
control
alteration
in
the
mechanism
by
which
et al.
19861.
specific
1978; Nilsen Pensiero and
protein
often
1971;
19861. of secreted
also
use
translational
of such during
of the && Protein
synthesis
in
or
protein
translational
activities efficiencies
of
is
mRNA
are (Fig
involved may
be
addition,
and
polysomes
of the protein
et al.
synthetic
et
1980;
such as the
[Walter
heavy
et al. of
mRNA
reported. chain
as a
the syntheses
19871, eIF-2,
19861,
19871,
al.
has been
tubulin,
1983; et al.
al. 1978;
regulation
genes
and vimentin
and
the expression
either
the rates
19883. by
altering
polysomal 1).
the
the sizes, determine machinery.
fraction
and therefore, To
in regulating
dynamically
In
et
processes
proteins
redistributing
that
synthesis
synthesis
experiments.
regulated
or by
mRNA
control
rate of protein chase
[Marth et al.
stimuli al.
[Farrell
Jen
for ferritin
and Thomas,
et
Storti
The
as
protein
1978; Schneider,
1969;
[Hentze
proteins
[Thomas
or elongation
1982;
specific
in the
total
Calzone
metabolic
gene
of
1985,
and overall
to variousstress
et al.
control.
to iron dosages
can be
al.
a common
differences
infection
membrane
human
mammalian
mitogenesis
ribosomes
engaged
the
proto-oncogene
initiation total
normal
integral
for numerous
include
response
et
Penman,
translational
ubiquitous early
and
efficiency
examples
reversible
However,
viral
is
as an
[Moldave, either
subtle
1983;
Hacket
and
involves
of cells
19621,
1985;
and
Alterations
Etchison
[McCormick
synthesis
Such
mRNA
synthesis
expression
or more
Hershey,
McCarthy, 1982;
Lindquist,
19861
and
Lucas-Lenard,
shock
translation
control
synthesis
responses
[Howe
et al.
and
heat
protein
mRNA,
synthesis,
of protein
gene
polypeptides. the
starvation al.
mRNA
Translational
of
et
regulation
of
synthesis
Henshaw
the
regulate
total
are
is
cells
of
including
protein
to initiate
Xntroduction
efficiency
alteration
synthesis
polysomes
'I-methyl guanosine triphosphate ['l-methyl GTP]; messenger RNA [mRNA]; polyacrylamide electrophoresis [PAGE]; gel sodium lauryl sulfate [SDS] 1.
Hershey
Langstrom et al.
determine gene
levels the
of either actively whether
expression,
characterized
of
the
by pulse-
and translational distribution
and
Translational control ofgeneexpression inthehuman brain
471
TRANSLATION , Il,,-i," ,. l.-"l.,lll (// 8-w /// /. ,m.,,,, .i--..",,.i,ii ,B_"/"/ /m""1/ li___q iRIBOSOMBS i il POST-TRANSLATIONAL 1 PROCESSING il I POLYSOMAL iIU3ly f mRNA synthesized $
TRANSCRIPTION
Gene
-mRNA
polypeptid -
mature protein
re-initiation
Fig
1.
Controls
of gene expression
2. Experimental Approaches to Investigating Translational Control of Gene Expression Expression in the Human Brain However,
translational
characterized pulse-chase
experiments
amino
into
acids
synthesis
in
from such represent
studies
mRNA
the cellular prot,ein.
brain
shown
components the
[Johnson
regulation
of
total
proteins.
The purification
be
is protein
cannot
not amenable
uptake
be to
of radio-labeled
control
of protein
investigated
with
the
can be isolated
and characterized
et al.,
Because
of synthesizing
characterization
is
(Fig 2).
that most directly
efficiencies both
active
must
that mRNA
tissues
brain
human tissue
Therefore,
and polysomes
in the process
Thus,
translational
cells.
human
have
postmortem
the brain
require
which
of mRNA
in
Postmortem
individual the
characterization Earlier
control
so directly.
of
19861. proteins
reflect polysome
fundamental synthesis
of physiologically
for and
polysomes
id situ,
gene
they are
expression
sizes,
amounts
investigating that
relevant
into and the
of individual
polysomes
472
A.
lPOLYPEPTIDE$ (l**uWuPRECIPltAllOWs
MRNA (NORTHERNS)
AUTO
RADIOGRAPHI)
B.
Fig 2. Investigating VS. Tissues fBf
translational
requires
that
they
polysomal
mRNA
in the
polysomes
need
addition,
the polysomes
tissue
are
so that valid
representative
brain
to be
control:
tissue
physicslIg must
at
intact
Intact
of the
time
(A)
actively of
may be made
y
isolated
between
translated
isolation.
and translationally
be reproducibl
comparisons
the
cells
from
various
active. tissue
The In to
conditions.
473
Translational controlof gene expressionin the human brain
3. Characterization of Human Postmortem The
purification
capable been
of
previously
samples were
was
have
Campagnoni
been
and
many
of
the
age,
agonal
determine
for
investigating
for
(as
neuronal
synthesize
various
ages
in
two
variables,
Cytosolic significantly yield
per
activity
per
of
unit
and did
yield
or
et
isolated values
al.,
tissue
polysome
Polysomes
and from
translational human frontal
(A260
Yield units/g
Activities cortices
tissue)
t,he
to
human
(2.5-fold
(8-fold
of to
order
and size
ability
to
reinitiate. that
as
were
of
postmortem significant
any
activity
human
in
various
exhibit
from
fraction
tissue,
brains
underwent not
whole
a
function
of
submitted].
from
compared
to
as
rat
[Campagnoni from
mRNA),
mRNA
1964; avoids
variations.
used
yield
each
rat
such
from
isolated
human
translational
TABLE Yields
adult
al., brain
tissues
were
of
from
et
tissue
as
polysomal
years) hours)
starting of
of from
[Langstrom
higher gram
capability
18-94
polysomes
brain
with
polysomes
A specific
the
compared
number
the
genetic
polysomes
has
the
rat
isolated
cytosol,
associated
3-14
either
were
isolated
(range: (range:
differences these
and
individual
the
within
of
human
those
of
to
polysomes
use
with
tissue assay
polysomes
[Zomzely The
control.
localized
polysome
intervals
to
translational
ribosomes
proteins
Cytosolic
and
polysomes
The of
times,
and
contrast,
inherent
polysomes
However,
brains)
19721.
suitability
the
those
19671.
number
brain
postmortem
translation
19811.
In
are
postmortem
to
enrich
al.,
brain
vitro
different
Murthy,
that
human
brains
Mahler,
et
from
human
in
investigated
1967;
complications
polysomes,
an
(three
thoroughly
state,
from
in
characterized.
brain
total
[Marotta
small
Mahler,
compared
polysomes
proteins
extensively
not
have
total
reported
analyzed
brains
We
of
synthesizing
Polysomes Tissues
Table
exhibited
cortex
frontal increase)
increase,
brain
whole
rat
and
in
I).
I of Cytosolic and adult rat
Polysomes isolated whole brain.
Translational Activity dpm (35)S-methionine ulos (per A260 Unit)
Human
1.63
(t 0.3) n=lO
695
Rat
4.22
(t 1.7) n=ll
5,770
(t212) n=lO (t650) n=S
both
translational
N.Langstrom etal.
474
In
Analysis
of
brain
largest
rat
polysomes
size
the
centrifugation whole
the
addition,
distribution that the
indicates
include
up
from the human
polysomes
ribosomes brain
than human
sucrose
by
polysomes
15-20
to
larger
were
sizes
contain
density
exhibited message,
per
polysomes. gradient by the rat whereas
5-6 ribosomes
the
per mRNA
(Fig 3).
Rat whole brain
Human FC
Fig 3. Polysomes Profiles Polysomes from Rats
The
ability
investigated vitro
of
using
translation
to differ
the a well
assay.
between
the rat
incubation
which
polysomes
synthesize
and human the
indicates
factor
under
to
characterized
throughout
decrease
translation
Tissues
rabbit
The time course
For the rat polysomes, greatly
of Rat vs Human
the
of
that there
these
synthesis
(Table 2
polypeptide
entire
60 minutes
was no exhaustion
incubation
conditions.
Larger
polypeptides
reticulocyte
of protein
polysomes
rate
Indicating
lysate
was in
was found
).
synthesis
did not
of the translation of any necessary Protein
synthesis
Translational control of geneexpression inthehuman brain
TABLE Time course of rat and absence or presence of either methyl GTP) or an inhibitor are presented as percentage JsS-methionine compared with 60 minute incubation.
30 min
73%
53%
60 min
100%
57%
by the same amount minutes synthesized support period,
(Table
rat
of
the polysomes
to
was
was capable then
of mRNA
synthesis
of only were
GTP
by minutes
specific
rat
that the initial
at
all the human
elongation
inhibitor,
anisomycin
by
initiate
60-80%. new rounds
translation
Despite rat
assay
of nascent
these and
It may
whereas
the human
polypeptide
differences human
synthesis
in yield,
polysomes
of protein 19771.
and the human These by the
synthesis
is due
On the other
hand,
both
rat
15 minutes
polysomes
The
significantly
and human
that the rat polysomal after
and
synthesis
protein
decreased
mRNA
chain
(Table 2 ).
complexes.
be concluded
of protein
period
of protein
polysome
ribosome-mRNA
of
in the presence
was
any time
15 minutes
the polysomal
and Rose,
polysomes
was a
translation
inhibitor
of the incubation
inhibited
could
polypeptide
1976; Lodish
the
itself
the 15 minute
the ability
translated
15
being
various
whether
after
were
polysomes
Therefore, under
were
a
et al.,
beyond
human
of the nascent
of existing
the synthesis
the
7-methyl [Hickey
and nearly
the
To determine
polysomes
complete
system
synthesis
synthesis
synthesis
20%
polypeptides
themselves.
protein
15
90%
by
run-off
synthesis
vitro
after
100%
translation
the
the polysomes
only slightly
82%
was essentially
translation
direct
92%
additional
protein
rat and human
suggest
rat polysomes to the
of
polypeptides
inhibited
results
no
Because
of completing
polysomes
can
2 ).
analog, cap initiation
synthesis
polysomes
that
investigated.
reinitiate,
the
of human
this cessation
conditions
human polysome-directed translation in the an inhibitor of initiation (100 mM 7of elongation (10 mM anisomycin). Results of total trichloracetic acid-precipitable uninhibited rat or human polysomes after a
polysome-directed
characteristic
2
16%
indicating
475
mRNA
in the in
can only complete
chains. size,
synthesized
and translational a
similar
size
abilities, range
of
476
N. Langstrom etal. polypeptides by
as
determined
SDS-polyacrylamide
fluorography
by
gel
separation
of total
electrophoresis
and
translation
products
visualization
by
(Fig 4).
Products of Rat (R) and Human (HI POlySOmeS SS Pig 4. Translation Comparison of free (left) vs membrane (right) separated by SDS-PAGE. Polysome products
4. Conclusions In conclusion,
intact
reproducibl. y purified When compared
and from
to polysomes
translationally various purified
human from
active
polysomes
brain
postmortem
rat whole
brains,
have been tissues. the human
477
Translational control of gene expression in the human brain
were
polysomes smaller polysomal
polypeptides
of
mRNA
However,
efficient
similar
size
range
that
the polysomes
(summarized
in Table
2
TABLE Summary
comparison
ability
the human
indicates
a
which
polysomal
less translationally
is in turn, due to the lower
to reinitiate.
mRNA
polysomes
and
smaller which
sizes,
as
due to the
of
polysomes
thoae
the human synthesized
synthesized
contain
by rat
similarly
sized
.).
3
of rat and human
polysomes
RAT POLYSOMBS
HUMAN
Whole
Frontal
Brain
Cortex
Yield:
4.2 A260
Size:
15-20 ribosomes/message
5-6 ribosomes/message 60s and monosomes prominent
Translational Efficiency:
5,770 dpm assproteins/A260 unit
695 dpm assproteins/A260
Reinitiation:
40% of proteins made by reinitiated mRNA
10% of proteins made by reinitiated mRNA
Polypeptides Synthesized:
Similar sizes SDS-PAGE)
Similar sizes SDS-PAGE)
The human control
brain
when
Polysomes
assay
individual unique
such
because
expression
during
in
synthesized of genes
gene
expression
various
brain
protein
control
translation into
tissue
in
the
the
will these
(by
conditions.
may
synthesis
proteins,
to
unit
via translational
be used
to
order
to
in
human
of polysomal
polypeptides specific
units/g
neuropathological
postmortem
translational
expression
1.7 A260
(by
overall
the in vitro
of gene
newly
regulates
from human
alterations
characterize
direct
stressed
isolated
determine
addition,
likely
units/g
POLYSOMES
brain.
mRNA
In
is the most
characterization
identify
various
the altered
of or
neuropathological
conditions.
Acknowledgements The authors in obtaining
gratefully tissues.
acknowledge In
addition,
the assistance we
express
of Christer appreciation
Gezelius to the
N. Langstrom etal.
478
Swedish
Medical
Research
Stohnes
Foundations
was
recipient
a
Medical
Research
French
Foundation
Council,
of Sweden of
a
Council
and
the
and the Mack
Visiting
Scientist
and is currently
for Alzheimer's
Gamla
Tjanarkvinnor
Foundation. Fellowship
a Fellow
William
and
Wallace
from the Swedish
of the
John Douglas
Disease.
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Inquires
and reprint
requests
Dr. William Wallace Department of Psychiatry Mount Sinai School of Medicine New York, NY 10029
should
be addressed
to:
of
1989, Vol. 13. pp. 469-479
TRANSLATIONAL
NIKLAS
Copyright
CONTROL OF GENE EXPRESSION HUMAN BRAIN
LANGSTROM';' ANDERS
1. Department
ERIKSSON' WALLACE"
BENGT
WINBLAD:
0
027&5846/89 $0.00 + so 1989 Pergamon Press plc
IN THE
and
WILLIAM
of Psychiatry Sinai School 2. Department of Geriatric 3. Institute
and Fishberg Center for Neurobiology, Mount of Medicine, New York, N.Y. Medicine, Karolinska Institute, Huddinge, Sweden for Forensic Medicine, University of Umea, Sweden
(Final fcrm, Auqnst 1988)
Contents
1. 2. 3. 4.
Abstract Introduction: Experimental Approaches to Investigating Translational control of Gene Expression in the Human Brain Characterization of Polysomes from Human Postmortem Tissues Conclusions Acknowledgements References Abstract
Langstrom,Niklas, Anders Erikson, Bengt Winblad, and William Wallace: control of gene Translational expression in the human brain. Prog. Neuro-Psychopharmacol and Biol. Psychiat. 1989, J&469-479 1. Translational control is the regulation of protein synthesis as an efficiency of mRNA translation and is a common alteration in the mechanism by which cells regulate gene expression. 2. Alterations of total protein synthesis are often the responses of stimuli including starvation, viral infection, cells to various stress and heat shock. Numerous specific genes including ferritin heavy chain, tubulin, 3. proto-oncogene have also been shown to be under the Ick vimentin and translational control. intact organisms, Unlike cultured cells or the investigation of 4. human brain requires the measurement of control in the translational protein synthesis, especially polysomes. Therefore, we components of and characterized polysomes from human postmortem brain have purified purified tissues and compared them to polysomes from the adult rat brain. The yield (as A260 units per gram brain tissue), size (as number of 5. translational efficiency message), ribosomes per (as amount protein ability to reinitiate (as amount of synthesized per A260 unit), and all initiation inhibitors) were protein prevented by synthesis human polysomes compared with as exhibited by the significantly lower rat polysomes synthesized human and polysomes. However, the the rat similar polypeptides. differed from the rat polysomes 6. the human polysomes Thus, principally in the efficiency of mRNA translation which is likely due to
469
469 470 471 473 473 477 478
N.
470
the greatly synthesis.
reduced
Key Words: translation
human
Abbreviations:
ability
of the human
brain,
polysomes,
Translational
control
alteration
in
the
mechanism
by
which
et al.
19861.
specific
1978; Nilsen Pensiero and
protein
often
1971;
19861. of secreted
also
use
translational
of such during
of the && Protein
synthesis
in
or
protein
translational
activities efficiencies
of
is
mRNA
are (Fig
involved may
be
addition,
and
polysomes
of the protein
et al.
synthetic
et
1980;
such as the
[Walter
heavy
et al. of
mRNA
reported. chain
as a
the syntheses
19871, eIF-2,
19861,
19871,
al.
has been
tubulin,
1983; et al.
al. 1978;
regulation
genes
and vimentin
and
the expression
either
the rates
19883. by
altering
polysomal 1).
the
the sizes, determine machinery.
fraction
and therefore, To
in regulating
dynamically
In
et
processes
proteins
redistributing
that
synthesis
synthesis
experiments.
regulated
or by
mRNA
control
rate of protein chase
[Marth et al.
stimuli al.
[Farrell
Jen
for ferritin
and Thomas,
et
Storti
The
as
protein
1978; Schneider,
1969;
[Hentze
proteins
[Thomas
or elongation
1982;
specific
in the
total
Calzone
metabolic
gene
of
1985,
and overall
to variousstress
et al.
control.
to iron dosages
can be
al.
a common
differences
infection
membrane
human
mammalian
mitogenesis
ribosomes
engaged
the
proto-oncogene
initiation total
normal
integral
for numerous
include
response
et
Penman,
translational
ubiquitous early
and
efficiency
examples
reversible
However,
viral
is
as an
[Moldave, either
subtle
1983;
Hacket
and
involves
of cells
19621,
1985;
and
Alterations
Etchison
[McCormick
synthesis
Such
mRNA
synthesis
expression
or more
Hershey,
McCarthy, 1982;
Lindquist,
19861
and
Lucas-Lenard,
shock
translation
control
synthesis
responses
[Howe
et al.
and
heat
protein
mRNA,
synthesis,
of protein
gene
polypeptides. the
starvation al.
mRNA
Translational
of
et
regulation
of
synthesis
Henshaw
the
regulate
total
are
is
cells
of
including
protein
to initiate
Xntroduction
efficiency
alteration
synthesis
polysomes
'I-methyl guanosine triphosphate ['l-methyl GTP]; messenger RNA [mRNA]; polyacrylamide electrophoresis [PAGE]; gel sodium lauryl sulfate [SDS] 1.
Hershey
Langstrom et al.
determine gene
levels the
of either actively whether
expression,
characterized
of
the
by pulse-
and translational distribution
and
Translational control ofgeneexpression inthehuman brain
471
TRANSLATION , Il,,-i," ,. l.-"l.,lll (// 8-w /// /. ,m.,,,, .i--..",,.i,ii ,B_"/"/ /m""1/ li___q iRIBOSOMBS i il POST-TRANSLATIONAL 1 PROCESSING il I POLYSOMAL iIU3ly f mRNA synthesized $
TRANSCRIPTION
Gene
-mRNA
polypeptid -
mature protein
re-initiation
Fig
1.
Controls
of gene expression
2. Experimental Approaches to Investigating Translational Control of Gene Expression Expression in the Human Brain However,
translational
characterized pulse-chase
experiments
amino
into
acids
synthesis
in
from such represent
studies
mRNA
the cellular prot,ein.
brain
shown
components the
[Johnson
regulation
of
total
proteins.
The purification
be
is protein
cannot
not amenable
uptake
be to
of radio-labeled
control
of protein
investigated
with
the
can be isolated
and characterized
et al.,
Because
of synthesizing
characterization
is
(Fig 2).
that most directly
efficiencies both
active
must
that mRNA
tissues
brain
human tissue
Therefore,
and polysomes
in the process
Thus,
translational
cells.
human
have
postmortem
the brain
require
which
of mRNA
in
Postmortem
individual the
characterization Earlier
control
so directly.
of
19861. proteins
reflect polysome
fundamental synthesis
of physiologically
for and
polysomes
id situ,
gene
they are
expression
sizes,
amounts
investigating that
relevant
into and the
of individual
polysomes
472
A.
lPOLYPEPTIDE$ (l**uWuPRECIPltAllOWs
MRNA (NORTHERNS)
AUTO
RADIOGRAPHI)
B.
Fig 2. Investigating VS. Tissues fBf
translational
requires
that
they
polysomal
mRNA
in the
polysomes
need
addition,
the polysomes
tissue
are
so that valid
representative
brain
to be
control:
tissue
physicslIg must
at
intact
Intact
of the
time
(A)
actively of
may be made
y
isolated
between
translated
isolation.
and translationally
be reproducibl
comparisons
the
cells
from
various
active. tissue
The In to
conditions.
473
Translational controlof gene expressionin the human brain
3. Characterization of Human Postmortem The
purification
capable been
of
previously
samples were
was
have
Campagnoni
been
and
many
of
the
age,
agonal
determine
for
investigating
for
(as
neuronal
synthesize
various
ages
in
two
variables,
Cytosolic significantly yield
per
activity
per
of
unit
and did
yield
or
et
isolated values
al.,
tissue
polysome
Polysomes
and from
translational human frontal
(A260
Yield units/g
Activities cortices
tissue)
t,he
to
human
(2.5-fold
(8-fold
of to
order
and size
ability
to
reinitiate. that
as
were
of
postmortem significant
any
activity
human
in
various
exhibit
from
fraction
tissue,
brains
underwent not
whole
a
function
of
submitted].
from
compared
to
as
rat
[Campagnoni from
mRNA),
mRNA
1964; avoids
variations.
used
yield
each
rat
such
from
isolated
human
translational
TABLE Yields
adult
al., brain
tissues
were
of
from
et
tissue
as
polysomal
years) hours)
starting of
of from
[Langstrom
higher gram
capability
18-94
polysomes
brain
with
polysomes
A specific
the
compared
number
the
genetic
polysomes
has
the
rat
isolated
cytosol,
associated
3-14
either
were
isolated
(range: (range:
differences these
and
individual
the
within
of
human
those
of
to
polysomes
use
with
tissue assay
polysomes
[Zomzely The
control.
localized
polysome
intervals
to
translational
ribosomes
proteins
Cytosolic
and
polysomes
The of
times,
and
contrast,
inherent
polysomes
However,
brains)
19721.
suitability
the
those
19671.
number
brain
postmortem
translation
19811.
In
are
postmortem
to
enrich
al.,
brain
vitro
different
Murthy,
that
human
brains
Mahler,
et
from
human
in
investigated
1967;
complications
polysomes,
an
(three
thoroughly
state,
from
in
characterized.
brain
total
[Marotta
small
Mahler,
compared
polysomes
proteins
extensively
not
have
total
reported
analyzed
brains
We
of
synthesizing
Polysomes Tissues
Table
exhibited
cortex
frontal increase)
increase,
brain
whole
rat
and
in
I).
I of Cytosolic and adult rat
Polysomes isolated whole brain.
Translational Activity dpm (35)S-methionine ulos (per A260 Unit)
Human
1.63
(t 0.3) n=lO
695
Rat
4.22
(t 1.7) n=ll
5,770
(t212) n=lO (t650) n=S
both
translational
N.Langstrom etal.
474
In
Analysis
of
brain
largest
rat
polysomes
size
the
centrifugation whole
the
addition,
distribution that the
indicates
include
up
from the human
polysomes
ribosomes brain
than human
sucrose
by
polysomes
15-20
to
larger
were
sizes
contain
density
exhibited message,
per
polysomes. gradient by the rat whereas
5-6 ribosomes
the
per mRNA
(Fig 3).
Rat whole brain
Human FC
Fig 3. Polysomes Profiles Polysomes from Rats
The
ability
investigated vitro
of
using
translation
to differ
the a well
assay.
between
the rat
incubation
which
polysomes
synthesize
and human the
indicates
factor
under
to
characterized
throughout
decrease
translation
Tissues
rabbit
The time course
For the rat polysomes, greatly
of Rat vs Human
the
of
that there
these
synthesis
(Table 2
polypeptide
entire
60 minutes
was no exhaustion
incubation
conditions.
Larger
polypeptides
reticulocyte
of protein
polysomes
rate
Indicating
lysate
was in
was found
).
synthesis
did not
of the translation of any necessary Protein
synthesis
Translational control of geneexpression inthehuman brain
TABLE Time course of rat and absence or presence of either methyl GTP) or an inhibitor are presented as percentage JsS-methionine compared with 60 minute incubation.
30 min
73%
53%
60 min
100%
57%
by the same amount minutes synthesized support period,
(Table
rat
of
the polysomes
to
was
was capable then
of mRNA
synthesis
of only were
GTP
by minutes
specific
rat
that the initial
at
all the human
elongation
inhibitor,
anisomycin
by
initiate
60-80%. new rounds
translation
Despite rat
assay
of nascent
these and
It may
whereas
the human
polypeptide
differences human
synthesis
in yield,
polysomes
of protein 19771.
and the human These by the
synthesis
is due
On the other
hand,
both
rat
15 minutes
polysomes
The
significantly
and human
that the rat polysomal after
and
synthesis
protein
decreased
mRNA
chain
(Table 2 ).
complexes.
be concluded
of protein
period
of protein
polysome
ribosome-mRNA
of
in the presence
was
any time
15 minutes
the polysomal
and Rose,
polysomes
was a
translation
inhibitor
of the incubation
inhibited
could
polypeptide
1976; Lodish
the
itself
the 15 minute
the ability
translated
15
being
various
whether
after
were
polysomes
Therefore, under
were
a
et al.,
beyond
human
of the nascent
of existing
the synthesis
the
7-methyl [Hickey
and nearly
the
To determine
polysomes
complete
system
synthesis
synthesis
synthesis
20%
polypeptides
themselves.
protein
15
90%
by
run-off
synthesis
vitro
after
100%
translation
the
the polysomes
only slightly
82%
was essentially
translation
direct
92%
additional
protein
rat and human
suggest
rat polysomes to the
of
polypeptides
inhibited
results
no
Because
of completing
polysomes
can
2 ).
analog, cap initiation
synthesis
polysomes
that
investigated.
reinitiate,
the
of human
this cessation
conditions
human polysome-directed translation in the an inhibitor of initiation (100 mM 7of elongation (10 mM anisomycin). Results of total trichloracetic acid-precipitable uninhibited rat or human polysomes after a
polysome-directed
characteristic
2
16%
indicating
475
mRNA
in the in
can only complete
chains. size,
synthesized
and translational a
similar
size
abilities, range
of
476
N. Langstrom etal. polypeptides by
as
determined
SDS-polyacrylamide
fluorography
by
gel
separation
of total
electrophoresis
and
translation
products
visualization
by
(Fig 4).
Products of Rat (R) and Human (HI POlySOmeS SS Pig 4. Translation Comparison of free (left) vs membrane (right) separated by SDS-PAGE. Polysome products
4. Conclusions In conclusion,
intact
reproducibl. y purified When compared
and from
to polysomes
translationally various purified
human from
active
polysomes
brain
postmortem
rat whole
brains,
have been tissues. the human
477
Translational control of gene expression in the human brain
were
polysomes smaller polysomal
polypeptides
of
mRNA
However,
efficient
similar
size
range
that
the polysomes
(summarized
in Table
2
TABLE Summary
comparison
ability
the human
indicates
a
which
polysomal
less translationally
is in turn, due to the lower
to reinitiate.
mRNA
polysomes
and
smaller which
sizes,
as
due to the
of
polysomes
thoae
the human synthesized
synthesized
contain
by rat
similarly
sized
.).
3
of rat and human
polysomes
RAT POLYSOMBS
HUMAN
Whole
Frontal
Brain
Cortex
Yield:
4.2 A260
Size:
15-20 ribosomes/message
5-6 ribosomes/message 60s and monosomes prominent
Translational Efficiency:
5,770 dpm assproteins/A260 unit
695 dpm assproteins/A260
Reinitiation:
40% of proteins made by reinitiated mRNA
10% of proteins made by reinitiated mRNA
Polypeptides Synthesized:
Similar sizes SDS-PAGE)
Similar sizes SDS-PAGE)
The human control
brain
when
Polysomes
assay
individual unique
such
because
expression
during
in
synthesized of genes
gene
expression
various
brain
protein
control
translation into
tissue
in
the
the
will these
(by
conditions.
may
synthesis
proteins,
to
unit
via translational
be used
to
order
to
in
human
of polysomal
polypeptides specific
units/g
neuropathological
postmortem
translational
expression
1.7 A260
(by
overall
the in vitro
of gene
newly
regulates
from human
alterations
characterize
direct
stressed
isolated
determine
addition,
likely
units/g
POLYSOMES
brain.
mRNA
In
is the most
characterization
identify
various
the altered
of or
neuropathological
conditions.
Acknowledgements The authors in obtaining
gratefully tissues.
acknowledge In
addition,
the assistance we
express
of Christer appreciation
Gezelius to the
N. Langstrom etal.
478
Swedish
Medical
Research
Stohnes
Foundations
was
recipient
a
Medical
Research
French
Foundation
Council,
of Sweden of
a
Council
and
the
and the Mack
Visiting
Scientist
and is currently
for Alzheimer's
Gamla
Tjanarkvinnor
Foundation. Fellowship
a Fellow
William
and
Wallace
from the Swedish
of the
John Douglas
Disease.
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