Vol. 84,No.
BIOCHEMICAL
1, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 45-51
September14,1978
PRIMER
DIRECTED
'C. Quagliariello, 0 Istituto di di
Bari,
POLY(A) 'R.
Gallerani,
Chimica
Via
Biologica,
Amendola
'Dipartimento
di
Calabria,
Arcavacata,
Received
SYNTHESIS
MITOCHONDRIA
'G.
and
Gadaleta
Facolta
165/A,
Biologia
IN RAT LIVER
70126
Scienze,
Saccone
Universita
Italy
Bari,
Cellulare, Cosenza,
di
'C.
Universita
della
Italy
June 6,1978
SUMMARY Rat liver mitochondria contain an endogenous factor highly specific in stimulating the homol ous poly(A) polymerase. By using an in vivo labelling with [ %! 4 orthophosphate it is possible to prepare a labelled factor and to demonstrate that it is stably incorporated in an acid insoluble molecule. This suggests that the factor probably acts as a primer in the polymerization of ATP molecules, being involved in the recognition between the mitochondrial poly(A) polymerase and the homologous RNA molecules which have to be polyadenylated. The
presence
mitochondria laboratory is
of has
(2).
dependent
and
of
the
specific
on
In
particular
an
"endogenous
purification
tion
the
The
of of
differentiates poly(A) ref.
rement
so
and
nature.
are
far
to
is
known
add
(manuscript
in
stimulating
us
that
it
the could
show
a non
its
poly(A)
separa(3).
polymerase
from
sources
cytoplasmic
poly(A)
specific
primer
to
RNAs of
its
of
requidifferent
action
of
extensive
its (5)
allows
endogenous
mechanism and
signal
mito-
different
however, of
the
confirmed which
and
tracts the
enzyme
liver
poly(A)
preparation)
synthesis contain
rat
a specific
and
liver our
dializable we
a method
from
poly(A)
rat in
RNA polymerase
on
factor; in
this
polymerase
Nuclear
about
endogenous
fication
devised
extracted
isolated
able
that
for
mitochondrial
a review).
Nothing
mitochondrial
factor
enzyme
polymerases
4 for
polymerases
this
poly(A)
this
and
partialiy
DNA dependent
of
in
(1)
Subsequently
and
homologous
dependence
molecule other
(1).
polymerase
a partial
showed
factor"
nature
poly(A)
polymerase
by Jacob Jacob
requirement
from
poly(A)
demonstrated
ribonucleotide
chondrial
(see
a specific
been
high have
(probably
the
puri-
specificity suggested
to
a specific
0006-291X/78/0841-0045$01.00/0 4.5
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in a.ry form reserved.
Vol. 84,No.
1, 1978
8lOCHEMlCALAND
nucleotide
sequence
recognized
by
This
implies
bound
to In
the
order
vitro
have
by
II
the
present for
factor
addition
may
act
demonstrate in
the
partially
the
a poly(A)
of
AMP residues.
factor
by
and
remain
the
so-called
this
paper molecules
acid
containing
we
insoluble
poly(A)
molecule
injecting
ATP
an
of
synthesized
mitochondrial
In
MATERIALS
RNA molecule)
a primer
containing
purified
of
a molecule
an
incorporation
polymerization into
as
RESEARCH COMMUNICATIONS
product.
a labelled
converted
in
the
orthophosphate.
during is
enzyme
factor
prepared
32P
originally
reaction to
endogenous in
the that
8lOPHYSlCAL
the
enzyme,
we
animals
with
demonstrate the
that
endogenous
product
which
factor behaves
as
sequences.
AND METHODS
Oligo-(dT) Cellulose was from Collaborative Research Inc. Waltham, Massachussetts, U.S.A., Bio-Gel P Laboratories, Richmond Cal' Ij$ATpornia and is; :SitiI i?$!f;~i~~$!{;~~~te (Sp. Act. 14 Ci/mg P. from Amersham. Insta gel was from Packard Instrument Company Inc. Downers Grove Illinois, U.S.A. Ma1 albino rats weighing about 80 gr. received each 1 mCi of PI 2P orthophosphate in Tris sterile and were killed 12 h after injection. The mitochondria were prepared by a partial modification Centrifugation at 600 xg was of a standard procedure (6). repeated twice and the last centrifugation at 12,000 xg was substituted with a second at 8,000 xg. The mitochondrial endogenous factor and the homologous poly(A) polymerase were prepared according to standard procedures (7,5). The same enzyme preparation was used throughout the experiments described in this paper. The incorporation in acid insoluble material of labelled compounds was measured as already reported (3). The specific activity of the enzyme used in the experiments described in this paper was 4,lO Units/mg of protein. One unit corresponded to a pmol of[ s l$ ATP incorporated in acid insoluble material in standard conditions (3). The protein concentration of partially purified poly(A) polymerase was determined by the Waddel method (8). RESULTS
AND DISCUSSION
The rase
specificity
for
could
an
by
the
mitochondrial (1,3,5)
present enzyme
on in
suggested
sequence
at it
poly(A)
has
nucleotide
conferring
homologous
liver
factor
a specific originally
RNA molecule
rat
endogenous
contain
nucleotide
of
the order
the to
AMP residues. 46
3'
signal start
or
that
the
it
a specific
OH termination which
polyme-
is
of recognized
addition
of
an
Vol. 84, No. 1, 1978
BKKHEMlCAL
AND BIOPHYSICAL
INCUBATION
Fig.
[I32 P
1 -
Time course of 3 [I H -ATP incorporation presence (A)
of
TIME (min)
labelled
endogenous
in
partially
RESEARCH COMMUNICATIONS
acid
factor
insoluble
purified
and
material
mitochondrial
in poly
polymerase.
The
2P labelled El column (see
the
AMP
endogenous factor purified through Bio Gel Fig. 2) (about 90,000 dpm) was mixed with p2 1.11 mg of poly(A) polymerase in an incubation mixture of standard composition (3) having a final volume of 1 ml. 80 ~1 aliquots were taken out each time (including the zero time) in 1 ml of cold 5% trichloroace radioactivity was 80 dpm, for
In primer, in
order
to
it
the
is
to
orthophosphate. paper
clearly of
unlabelled contain cubation
The in
endogenous
molecule same
conclusion
Fig.
and
[I
1 showing
3H labelling
the
is
can the
cold
incorporated drawn
time acid
by
in the
insoluble
47
to
in
an
the
or polymerase
a that
poly(A)
2P labelled $1 after in-
polymerase
acid of
analysis
insoluble poly(A).
of
incorporation material.
this
labelled
I show
sequence
dependent
in
poly(A
and
32 ca [ 32 il -
a
synthesized
factor,
ATP
linked
with
reported
Table
a
stably prepared
bound
in
adds as
labelling
purified
unlabelled
be
we
vivo
covalently
it
remains
product
partially
of
the
in
in
endogenous
presence factor
it
results
reported
containing
in
an
that
sequence
polymerase using
purpose
experimental
results
the
that
this by
poly(A)
molecule
show
For
demonstrate 32 P labelled c 3 precursor and
a poly(A)
molecule.
to factor
The
the
specific
product.
endogenous
presence
that
this
necessary
reaction
labelled
the
demonstrate
residues
data
The re
of The
constant
orted
PI2P
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 84, No. 1, 1978
TABLE
[32Pl- LABELLEB
ENUOGENOUS
FACTOR
I INCORPORATION
IN
ACID
INSOLUBLE
MATERIAL.
SYSTEM
% of
a
control
Complete
b
Poly(A)
C
Incubation
d
Poly(A)
100 polymerase mixture polymerase
omitted
22
omitted
35
and incubation
mixture
omitted
25
An aliquot of 120 yg of partially purified mitochondrial poly (A) polymerase was incubat in standard conditions in the presence of 49,700 dpm of PI P labelled endogenous factor and in the presence of a suitable incubation mixture containing The reaction was stopped by adding 5% trichlorocold ATP (3). acetic acid and the acid insoluble material formed was determined by the milli ore technique. About 0,8% (375 dpm) of the initial amount of s 2P3 labelled endogenous factor was converted into acid insoluble material. All the assays were repeated in triplicate. Counting efficiency was about 95%. The background was 10 dpm. The experiment was carried out by simultaneously incubating three different controls (b,c,d) in the mentioned conditions.
level
reached
vity
suggests
this in
time
that in
rates
far of
acid
by
32P Cl
the
[I32 P
the insoluble
to
first
the
primer
acid
insoluble
molecuies
radioacti-
taken
product
remain
stably
is
concerned,
two
PIH labelling may
the
to
the
noticed.
One
initiation
of
elongation
other
elongation
be
the
contemporaneous and
thirty The
the
corresponds
molecules to
as
incorporation
probably
ly
15'
up
during
incorporated
it. As
and
after
of
(30 '-180') of
product
(up
to
30')
poly(A) already
probably molecules
different which
sequences initiated
corresponding initiated
onin
the
minutes.
existence
in
the
labelled
portions
is
clearly
obtained
by
desalting
the
reaction
product
confirmed P 2P I endogenous
48
of by
the factor
two
different
elution on
pattern Bio
Gel
BIOCHEMICAL
Vol. 84, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10
Fig.
2 -
Bio-Gel
15
20
FRACTION
NUMBER
P2 chromatography
of
25
32 P Cl
-1abeTled
endogenous
factor. An aliquot of about 5~10~ dpm of solubilized [32P 1 -1abelled endogenous factor (7) was resuspended using 1.5 ml of buffer A/lU (potassium phosphate 5 mM pH 6.8, MgC12 0.5 mM, EDTA 0.02 mM, KC1 5 mM) and applied to a (1.2x36 cm) Bio-Gel P2 column equilibra with the same buffer. Each fraction was assayed for the -1abelled endogenous factor content incubating 50 ~1 aliquots in the presence of the incubation mixture already reported (3) and 40 ug of partially purified mitochondrial poly(A) polymerase. Each fraction had a volume of 2.6 ml. Flow rate was 18 ml/h. The fractions (6-8) were pooled, lyophilized and used in the experiments described in Fig. 3.
P2 column product of
(Fig. on
the
and
by
Oligo-(dT)
acid
elling,
2)
that
characterization
cellulose
insoluble
with
the
of
(Fig.
radioactivity 32 P is in
[ 1
ot
3).
The
profile, with
reaction
corres due
agreement
the
ondence
PIH
to
our
lab-
previous
conclusions. The
experiments
product of
the
tion at
(see product
nature by
the
of
the
its
is
sensitive
to
pancreatic
affinity
made
and
[I
to
the
that
about
show eluted
part
and
enzymatic
snake
venom
ribonuclease
and
on
confirm
by molecules
column being 3 H labelled
chemical
chromatography
panels A-B)
factor
is
by
it alkali
3,
endogenous
4°C
med
by
Fig.
of
20% of
the
reac-
long
enough
to
be
retained
by
buffer.
The
the
product
LSB
phosphodiesterase, hydrolyzed
49
reaction
mechanism
only
characterization.
(9).
the
action
is
also
confirIn
fact
insensitive by
concentrated
Vol. 84, No. 1, 1978
8lOCHEMlCAL
AND 8lOPHYSlCAL
FRACTION
Fig.
3 - Oligo-(dT)
cellulose
product
made
polymerase factor
in in
[I3H
and
RESEARCH COMMUNICATIONS
NUM EIER
chromatography vitro
by
presence
tli;
of
of
the
mitochondrial
poly(A)
P3 -1abelled
[
reaction
endogenous
-ATP.
About 30,000 dpm of P* P1 labelled endogenous factor fractionated through a Bio-Gel P2 column (see Fig. 2) were absorbed on a 0.5x2.5 cm Oligo-(dT) cellulose column and left at 4'C for about 30'. The column was eluted using HSB buffer (NaCl 0.5 M, Tris 10 mM pH 7.4) (fractions l-9) and LSB buffer (Tris 10 mM pH 7.4) (fractions 10-18). Each fraction had a volume of 0.5 ml. Radioactivity was counted by mixing each f action in 10 ml of INSTA GEL (Fig. 3/A). The same amount of !!I *P labelled endogenous factor used in the previous experiment was incubated in standard conditions in the presence of 370 ug of mitochondrial poly(A) polymerase. After 30' incubation unlabelled ATP was added to a final concentration of 12 mM and NaCl to a final concentration of 0.5 M. After further 30' the column was eluted by HSB and LSB (see above). To each fraction 0.5 ml of 10% trichloroacetic acid was added and acid insoluble radioactivity determined as reported elsewhere (3) (Fig. 3/B).
The
existence
molecules by
assuming be
The remain in
led
form
that
it
of can
mainly
from
Studies
on
us
to
the the
poly(A) the
in
during
containing
molecular
our of
conclusion and
generated
of
13-17) salt
which
the
it
short
primer
can
be
results
solubilization and
too
conditions.
(manuscript
mitochondrial
50
are
endogenous
preliminary
structure
insoluble be explained
experimental
that the
acid can
concentration
purification
a trinucleotide be
high
origin
partial
and
sequences
column
and
unknown.
at
poly(A)
the
Its
classes 2-6
eluted
contain by
structure
paration)
different
fractions
those
2-6) retained
two
3/B,
that
(fractions to
of
(Fig.
in
pre-
solubilized suggest procedure
RNA molecules. its
chemical
composition
BIOCHEMICAL
Vol. 84, No. 1, 1978
are
now
in
progress
in
our
laboratory
role
and
its
interaction
physiological poly(A)
AND BIOPHYSICAL
in
RESEARCH COMMUNICATIONS
order
with
to the
verify
its
homologous
polymerase.
REFEKENCES
.I 1 Jacob S.T., 2) 3) 4) 5) 6) 7) 8)
9)
Rose K.M., and Morris H.P. (1974) Biochim. Biophys. Acta 361, 312-320. Saccone C., De Giorgi C., Cantatore P., and Gallerani K. (1974) Biomembranes (Packer, L., td.), pp. 87-99, Academic Press, New York. Gallerani R., De Giorgi C., De Benedetto C., and Saccone C. (1976) Biochem. J. 157, 295-300. Edmonds M., and Winters M.A. (1976) in Proc. Nucl. Acids Res. and Mol. Biol. (Waldo E. Cohn, Ed.) Vol. 17 pp. 149179. De Benedetto C., Gallerani R., De Giorgi C., and Saccone C. (1976) Boll. Sot. It. Biol. Sper. LlI, 1343-1349. Saccone C., Gadaleta M.N., and Quagliariello E. (1967) Biochim. Biophys. Acta 138, 474-479. Gallerani R., Quagliariello C., and Saccone C. (1977) Boll. Sot. It. Biol. Sper. LIII, 1077-1083. Waddel W.J. (1956) J. Lab. Clin. Med. 48, 311-314. Quagliariello C., Saccone C., and Gallerani R. (1978) Boll. Sot. It. Biol. Sper. LTV, 2’1-26.
51
BIOCHEMICAL
1, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 45-51
September14,1978
PRIMER
DIRECTED
'C. Quagliariello, 0 Istituto di di
Bari,
POLY(A) 'R.
Gallerani,
Chimica
Via
Biologica,
Amendola
'Dipartimento
di
Calabria,
Arcavacata,
Received
SYNTHESIS
MITOCHONDRIA
'G.
and
Gadaleta
Facolta
165/A,
Biologia
IN RAT LIVER
70126
Scienze,
Saccone
Universita
Italy
Bari,
Cellulare, Cosenza,
di
'C.
Universita
della
Italy
June 6,1978
SUMMARY Rat liver mitochondria contain an endogenous factor highly specific in stimulating the homol ous poly(A) polymerase. By using an in vivo labelling with [ %! 4 orthophosphate it is possible to prepare a labelled factor and to demonstrate that it is stably incorporated in an acid insoluble molecule. This suggests that the factor probably acts as a primer in the polymerization of ATP molecules, being involved in the recognition between the mitochondrial poly(A) polymerase and the homologous RNA molecules which have to be polyadenylated. The
presence
mitochondria laboratory is
of has
(2).
dependent
and
of
the
specific
on
In
particular
an
"endogenous
purification
tion
the
The
of of
differentiates poly(A) ref.
rement
so
and
nature.
are
far
to
is
known
add
(manuscript
in
stimulating
us
that
it
the could
show
a non
its
poly(A)
separa(3).
polymerase
from
sources
cytoplasmic
poly(A)
specific
primer
to
RNAs of
its
of
requidifferent
action
of
extensive
its (5)
allows
endogenous
mechanism and
signal
mito-
different
however, of
the
confirmed which
and
tracts the
enzyme
liver
poly(A)
preparation)
synthesis contain
rat
a specific
and
liver our
dializable we
a method
from
poly(A)
rat in
RNA polymerase
on
factor; in
this
polymerase
Nuclear
about
endogenous
fication
devised
extracted
isolated
able
that
for
mitochondrial
a review).
Nothing
mitochondrial
factor
enzyme
polymerases
4 for
polymerases
this
poly(A)
this
and
partialiy
DNA dependent
of
in
(1)
Subsequently
and
homologous
dependence
molecule other
(1).
polymerase
a partial
showed
factor"
nature
poly(A)
polymerase
by Jacob Jacob
requirement
from
poly(A)
demonstrated
ribonucleotide
chondrial
(see
a specific
been
high have
(probably
the
puri-
specificity suggested
to
a specific
0006-291X/78/0841-0045$01.00/0 4.5
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in a.ry form reserved.
Vol. 84,No.
1, 1978
8lOCHEMlCALAND
nucleotide
sequence
recognized
by
This
implies
bound
to In
the
order
vitro
have
by
II
the
present for
factor
addition
may
act
demonstrate in
the
partially
the
a poly(A)
of
AMP residues.
factor
by
and
remain
the
so-called
this
paper molecules
acid
containing
we
insoluble
poly(A)
molecule
injecting
ATP
an
of
synthesized
mitochondrial
In
MATERIALS
RNA molecule)
a primer
containing
purified
of
a molecule
an
incorporation
polymerization into
as
RESEARCH COMMUNICATIONS
product.
a labelled
converted
in
the
orthophosphate.
during is
enzyme
factor
prepared
32P
originally
reaction to
endogenous in
the that
8lOPHYSlCAL
the
enzyme,
we
animals
with
demonstrate the
that
endogenous
product
which
factor behaves
as
sequences.
AND METHODS
Oligo-(dT) Cellulose was from Collaborative Research Inc. Waltham, Massachussetts, U.S.A., Bio-Gel P Laboratories, Richmond Cal' Ij$ATpornia and is; :SitiI i?$!f;~i~~$!{;~~~te (Sp. Act. 14 Ci/mg P. from Amersham. Insta gel was from Packard Instrument Company Inc. Downers Grove Illinois, U.S.A. Ma1 albino rats weighing about 80 gr. received each 1 mCi of PI 2P orthophosphate in Tris sterile and were killed 12 h after injection. The mitochondria were prepared by a partial modification Centrifugation at 600 xg was of a standard procedure (6). repeated twice and the last centrifugation at 12,000 xg was substituted with a second at 8,000 xg. The mitochondrial endogenous factor and the homologous poly(A) polymerase were prepared according to standard procedures (7,5). The same enzyme preparation was used throughout the experiments described in this paper. The incorporation in acid insoluble material of labelled compounds was measured as already reported (3). The specific activity of the enzyme used in the experiments described in this paper was 4,lO Units/mg of protein. One unit corresponded to a pmol of[ s l$ ATP incorporated in acid insoluble material in standard conditions (3). The protein concentration of partially purified poly(A) polymerase was determined by the Waddel method (8). RESULTS
AND DISCUSSION
The rase
specificity
for
could
an
by
the
mitochondrial (1,3,5)
present enzyme
on in
suggested
sequence
at it
poly(A)
has
nucleotide
conferring
homologous
liver
factor
a specific originally
RNA molecule
rat
endogenous
contain
nucleotide
of
the order
the to
AMP residues. 46
3'
signal start
or
that
the
it
a specific
OH termination which
polyme-
is
of recognized
addition
of
an
Vol. 84, No. 1, 1978
BKKHEMlCAL
AND BIOPHYSICAL
INCUBATION
Fig.
[I32 P
1 -
Time course of 3 [I H -ATP incorporation presence (A)
of
TIME (min)
labelled
endogenous
in
partially
RESEARCH COMMUNICATIONS
acid
factor
insoluble
purified
and
material
mitochondrial
in poly
polymerase.
The
2P labelled El column (see
the
AMP
endogenous factor purified through Bio Gel Fig. 2) (about 90,000 dpm) was mixed with p2 1.11 mg of poly(A) polymerase in an incubation mixture of standard composition (3) having a final volume of 1 ml. 80 ~1 aliquots were taken out each time (including the zero time) in 1 ml of cold 5% trichloroace radioactivity was 80 dpm, for
In primer, in
order
to
it
the
is
to
orthophosphate. paper
clearly of
unlabelled contain cubation
The in
endogenous
molecule same
conclusion
Fig.
and
[I
1 showing
3H labelling
the
is
can the
cold
incorporated drawn
time acid
by
in the
insoluble
47
to
in
an
the
or polymerase
a that
poly(A)
2P labelled $1 after in-
polymerase
acid of
analysis
insoluble poly(A).
of
incorporation material.
this
labelled
I show
sequence
dependent
in
poly(A
and
32 ca [ 32 il -
a
synthesized
factor,
ATP
linked
with
reported
Table
a
stably prepared
bound
in
adds as
labelling
purified
unlabelled
be
we
vivo
covalently
it
remains
product
partially
of
the
in
in
endogenous
presence factor
it
results
reported
containing
in
an
that
sequence
polymerase using
purpose
experimental
results
the
that
this by
poly(A)
molecule
show
For
demonstrate 32 P labelled c 3 precursor and
a poly(A)
molecule.
to factor
The
the
specific
product.
endogenous
presence
that
this
necessary
reaction
labelled
the
demonstrate
residues
data
The re
of The
constant
orted
PI2P
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 84, No. 1, 1978
TABLE
[32Pl- LABELLEB
ENUOGENOUS
FACTOR
I INCORPORATION
IN
ACID
INSOLUBLE
MATERIAL.
SYSTEM
% of
a
control
Complete
b
Poly(A)
C
Incubation
d
Poly(A)
100 polymerase mixture polymerase
omitted
22
omitted
35
and incubation
mixture
omitted
25
An aliquot of 120 yg of partially purified mitochondrial poly (A) polymerase was incubat in standard conditions in the presence of 49,700 dpm of PI P labelled endogenous factor and in the presence of a suitable incubation mixture containing The reaction was stopped by adding 5% trichlorocold ATP (3). acetic acid and the acid insoluble material formed was determined by the milli ore technique. About 0,8% (375 dpm) of the initial amount of s 2P3 labelled endogenous factor was converted into acid insoluble material. All the assays were repeated in triplicate. Counting efficiency was about 95%. The background was 10 dpm. The experiment was carried out by simultaneously incubating three different controls (b,c,d) in the mentioned conditions.
level
reached
vity
suggests
this in
time
that in
rates
far of
acid
by
32P Cl
the
[I32 P
the insoluble
to
first
the
primer
acid
insoluble
molecuies
radioacti-
taken
product
remain
stably
is
concerned,
two
PIH labelling may
the
to
the
noticed.
One
initiation
of
elongation
other
elongation
be
the
contemporaneous and
thirty The
the
corresponds
molecules to
as
incorporation
probably
ly
15'
up
during
incorporated
it. As
and
after
of
(30 '-180') of
product
(up
to
30')
poly(A) already
probably molecules
different which
sequences initiated
corresponding initiated
onin
the
minutes.
existence
in
the
labelled
portions
is
clearly
obtained
by
desalting
the
reaction
product
confirmed P 2P I endogenous
48
of by
the factor
two
different
elution on
pattern Bio
Gel
BIOCHEMICAL
Vol. 84, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10
Fig.
2 -
Bio-Gel
15
20
FRACTION
NUMBER
P2 chromatography
of
25
32 P Cl
-1abeTled
endogenous
factor. An aliquot of about 5~10~ dpm of solubilized [32P 1 -1abelled endogenous factor (7) was resuspended using 1.5 ml of buffer A/lU (potassium phosphate 5 mM pH 6.8, MgC12 0.5 mM, EDTA 0.02 mM, KC1 5 mM) and applied to a (1.2x36 cm) Bio-Gel P2 column equilibra with the same buffer. Each fraction was assayed for the -1abelled endogenous factor content incubating 50 ~1 aliquots in the presence of the incubation mixture already reported (3) and 40 ug of partially purified mitochondrial poly(A) polymerase. Each fraction had a volume of 2.6 ml. Flow rate was 18 ml/h. The fractions (6-8) were pooled, lyophilized and used in the experiments described in Fig. 3.
P2 column product of
(Fig. on
the
and
by
Oligo-(dT)
acid
elling,
2)
that
characterization
cellulose
insoluble
with
the
of
(Fig.
radioactivity 32 P is in
[ 1
ot
3).
The
profile, with
reaction
corres due
agreement
the
ondence
PIH
to
our
lab-
previous
conclusions. The
experiments
product of
the
tion at
(see product
nature by
the
of
the
its
is
sensitive
to
pancreatic
affinity
made
and
[I
to
the
that
about
show eluted
part
and
enzymatic
snake
venom
ribonuclease
and
on
confirm
by molecules
column being 3 H labelled
chemical
chromatography
panels A-B)
factor
is
by
it alkali
3,
endogenous
4°C
med
by
Fig.
of
20% of
the
reac-
long
enough
to
be
retained
by
buffer.
The
the
product
LSB
phosphodiesterase, hydrolyzed
49
reaction
mechanism
only
characterization.
(9).
the
action
is
also
confirIn
fact
insensitive by
concentrated
Vol. 84, No. 1, 1978
8lOCHEMlCAL
AND 8lOPHYSlCAL
FRACTION
Fig.
3 - Oligo-(dT)
cellulose
product
made
polymerase factor
in in
[I3H
and
RESEARCH COMMUNICATIONS
NUM EIER
chromatography vitro
by
presence
tli;
of
of
the
mitochondrial
poly(A)
P3 -1abelled
[
reaction
endogenous
-ATP.
About 30,000 dpm of P* P1 labelled endogenous factor fractionated through a Bio-Gel P2 column (see Fig. 2) were absorbed on a 0.5x2.5 cm Oligo-(dT) cellulose column and left at 4'C for about 30'. The column was eluted using HSB buffer (NaCl 0.5 M, Tris 10 mM pH 7.4) (fractions l-9) and LSB buffer (Tris 10 mM pH 7.4) (fractions 10-18). Each fraction had a volume of 0.5 ml. Radioactivity was counted by mixing each f action in 10 ml of INSTA GEL (Fig. 3/A). The same amount of !!I *P labelled endogenous factor used in the previous experiment was incubated in standard conditions in the presence of 370 ug of mitochondrial poly(A) polymerase. After 30' incubation unlabelled ATP was added to a final concentration of 12 mM and NaCl to a final concentration of 0.5 M. After further 30' the column was eluted by HSB and LSB (see above). To each fraction 0.5 ml of 10% trichloroacetic acid was added and acid insoluble radioactivity determined as reported elsewhere (3) (Fig. 3/B).
The
existence
molecules by
assuming be
The remain in
led
form
that
it
of can
mainly
from
Studies
on
us
to
the the
poly(A) the
in
during
containing
molecular
our of
conclusion and
generated
of
13-17) salt
which
the
it
short
primer
can
be
results
solubilization and
too
conditions.
(manuscript
mitochondrial
50
are
endogenous
preliminary
structure
insoluble be explained
experimental
that the
acid can
concentration
purification
a trinucleotide be
high
origin
partial
and
sequences
column
and
unknown.
at
poly(A)
the
Its
classes 2-6
eluted
contain by
structure
paration)
different
fractions
those
2-6) retained
two
3/B,
that
(fractions to
of
(Fig.
in
pre-
solubilized suggest procedure
RNA molecules. its
chemical
composition
BIOCHEMICAL
Vol. 84, No. 1, 1978
are
now
in
progress
in
our
laboratory
role
and
its
interaction
physiological poly(A)
AND BIOPHYSICAL
in
RESEARCH COMMUNICATIONS
order
with
to the
verify
its
homologous
polymerase.
REFEKENCES
.I 1 Jacob S.T., 2) 3) 4) 5) 6) 7) 8)
9)
Rose K.M., and Morris H.P. (1974) Biochim. Biophys. Acta 361, 312-320. Saccone C., De Giorgi C., Cantatore P., and Gallerani K. (1974) Biomembranes (Packer, L., td.), pp. 87-99, Academic Press, New York. Gallerani R., De Giorgi C., De Benedetto C., and Saccone C. (1976) Biochem. J. 157, 295-300. Edmonds M., and Winters M.A. (1976) in Proc. Nucl. Acids Res. and Mol. Biol. (Waldo E. Cohn, Ed.) Vol. 17 pp. 149179. De Benedetto C., Gallerani R., De Giorgi C., and Saccone C. (1976) Boll. Sot. It. Biol. Sper. LlI, 1343-1349. Saccone C., Gadaleta M.N., and Quagliariello E. (1967) Biochim. Biophys. Acta 138, 474-479. Gallerani R., Quagliariello C., and Saccone C. (1977) Boll. Sot. It. Biol. Sper. LIII, 1077-1083. Waddel W.J. (1956) J. Lab. Clin. Med. 48, 311-314. Quagliariello C., Saccone C., and Gallerani R. (1978) Boll. Sot. It. Biol. Sper. LTV, 2’1-26.
51
