Download Isolation and Partial Characterization of Multiple

[CANCER RESEARCH 33, 1210-1216, June 1973]
Isolation and Partial Characterization of Multiple DNA
Polymerases of the Murine Myeloma, MOPC-211
Francis J. Persico,2 Diarmuid E. Nicholson,3 and A. Arthur Gottlieb
Institute of Microbiology, Rutgers University, New Brunswick, New Jersey 08903
SUMMARY
A method is presented for the isolation and initial
purification of the DNA polymerases of the myeloma line
MOPC-21. These activities are separable into two distinct
fractions by diethylaminoethyl
cellulose chromatography.
Each fraction contains a DNA polymerase that is highly active
on native calf thymus DNA that had been partially degraded
with DNase ("activated" or "nicked" DNA) and another
activity that transcribes ribopolymer strands in the presence of
a complementary primer. Some distinctive properties of these
enzymes are presented.
INTRODUCTION
An understanding of the functions of DNA-synthesizing
enzymes in a tumor cell would be helpful in developing our
concepts of the molecular basis of cancer. The availability of
homogeneous
populations
of myeloma cells provides a
well-characterized source of such enzymes in a tumor line that
maintains a high degree of differentiated function. We have
previously reported in preliminary form the 1st description of
the multiple DNA polymerases of the myeloma line MOPC-21
(10). We report herewith an efficient technique for the
fractionation of these activities and a description of some of
the properties of these enzymes.
MATERIALS
AND METHODS
Tumor Line. MOPC-21 (an IgGi producer) was originally
provided by Dr. M. Potter and was maintained by serial
transplantation
in BALB/c mice. At appropriate intervals,
tumors were excised and stored at -20° until used.
Preparation of Extract and Isolation of DNA-synthesizing
Activities. The general method used for the isolation of these
enzymes was similar to that used by Stein and Hausen (12) for
the isolation of RNA polymerase. In accordance with this
procedure,
tumor tissue was homogenized in a Servali
homogenizer at 0° with a wet weight-to-buffer (0.01 M
'Supported by Grant E-525A (NP-79B) from the American Cancer
Society, National Science Foundation Grant GB-16871, NIH Grant
GM-10395, and Damon Runyon Grant 1213. Preliminary reports of
this work were presented at the 62nd and 63rd Annual Meetings of the
American Society of Biological Chemists (June 1971, April 1972).
2Recipient of Postdoctoral Fellowship GM-46192 from the NIH.
3Fellow of the New York City Cancer Research Institute Inc.
Received July 24, 1972; accepted February 19, 1973.
1210
Tris-HCl, pH 7.8, containing 15 mM 0-mercaptoethanol) ratio
of 1:4. The extract thus obtained was treated essentially
according to the procedure of Stein and Hausen with batch
adsorption and elution from DEAE-cellulose, precipitation
with ammonium sulfate, and chromatography
on DEAEcellulose.
Two fractions (D-I and D-II) were recoverable from the
extract after stepwise elution from DEAE-cellulose with 0.1
and 0.3 M KC1, respectively. Elution with 0.3 M KC1 was not
initiated until essentially all the protein from the 0.1 M KC1
step was eliminated (as indicated by zero absorption at 280
nm). The 2 fractions obtained in this way were then
precipitated with ammonium sulfate (34 g/100 ml) and
centrifuged at 27,000 X g for 40 min, and the pellets
redissolved in 0.05 M Tris-HCl buffer, pH 7.8, containing 2
mM ß-mercaptoethanol and 50% glycerol. These fractions were
then stored at —¿20°.
Protein concentrations were determined
by the method of Lowry et al. (7). Dialysis of these fractions
prior to storage was not carried out routinely, since this
procedure appeared to result in loss of activity. The basic
procedure is outlined in Chart 1.
Assay for DNA Synthesis on DNA Templates. The assay
mixture (0.25 ml) contained 12.5 Amólesof Tris-HCl, pH 7.8;
1 fiM (3-mercaptoethanol; 50 pg of bovine serum albumin; 1
Aimole of MgCl2 ; 150 nmoles of dATP, dGTP, and dCTP; 8.7
mnoles of TTP-3H; 25 fig of template; and appropriate
dilutions of Fractions D-I or D-II. Commercial calf thymus
DNA was treated with RNase A and Pronase and then
extracted with phenol and reprecipitated from ethanol. The
DNA was used either in native form or after heating to 100°in
0.01 M Tris-HCl, pH 7.8 and 0.001 M NaCl for 10 min,
followed by rapid cooling. "Activated" (partially digested or
"nicked") DNA4 was prepared as described by Loeb (6).
Unless otherwise indicated, all incubations were carried out at
37°for 60 min, at which time 0.2 ml of a saturated solution of
sodium pyrophosphate adjusted to pH 7.8, 200 ¿/gof yeast
RNA, and 5 ml of 10% TCA previously adjusted to 5% (v/v) in
sodium pyrophosphate were added. The reaction mixtures
were filtered through B-6 membrane filters (Carl Schleicher
and Schuell), and the tubes were rinsed twice with 5%
TCA-pyrophosphate. The filters were washed 3 times with 5
ml of 5% TCA-pyrophosphate and once with 95% ethanol
4The abbreviations used are: "activated" or "nicked" DNA,
native calf thymus DNA that has been partially degraded with DNase;
TCA, trichloroacetic acid; poly rA-oligo(dT)i2—18> polyriboadenylic
acid hybridized to multiple lengths of oligodeoxythymidylate, 12 to 18
residues long.
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DNA Polymerases of Murine Myeloma
prior to counting a scintillation fluid composed of 0.02%
POPOP and 0.5% PPO in toluene. Background incorporations
achieved with reaction mixtures containing 50 Mg bovine
serum albumin in place of the enzyme-containing fractions
have been subtracted from all values reported.
Assay for DNA Synthesis on Poly rA'oligo(dT)12—is- The
basic assay conditions were as described above for DNA
templates except that MnCl2 was supplied at concentrations of
2 X 10~4 M for assays with Fraction D-I and 1 X 10 ~3 M for
assays with Fraction D-II. From 0.05 to 0.06 A26o unit of
poly rA'oligo(dT)12—is (Collaborative Research, Inc., Waltham, Mass.) was used as template. In these assays only TTP
(0.99 to 1.61 nmoles) and dATP (8.3 nmoles) were present in
the reaction mixture.
RESULTS
Each of the 2 fractions (D-I and D-II), prepared as described
above, contained 2 distinct DNA-polymerizing activities, one
of which responded very well to partially degraded DNA
("nicked" or "activated" DNA), while the other was capable
Grind myeloma tissue
i
Centiifugation (27,000 X g, 30 min) -> cell pellet
I
Batch adsorption of supernatant to DEAE-cellulose
i
Wash DEAE-cellulose
I
Elute enzymatic activities from DEAE-cellulose with 0.4 M (NH4)2SO4
I
Add 60% (NH„
)2SO4 to eluate and recover precipitate
I
DEAE-cellulose chromatography
I
0.1 M KC1cut = Fraction D-I
i
Wash until A28„¿
is 0
i
0.3 M KCl cut = Fraction D-II
Chart 1. Flow diagram for isolation of the DNA polymerases of
myeloma.
of synthesizing polydeoxythymidylate
in response to the
synthetic duplex, poly rA-oligo(dT)12_18.
In terms of specific
activity, the best purifications achieved by this procedure were
approximately 150-fold.
Table 1 demonstrates the requirements of each of these
fractions with "activated" DNA as template and demonstrates
that the product of this reaction is sensitive to digestion by
DNase, but not RNase, indicating that it is DNA. For both
fractions, the reaction was absolutely dependent upon the
addition of template. A divalent cation was required for
maximal activity of both fractions, and magnesium ion was
more effective than either manganese ion alone or an
equimolar mixture of magnesium and manganese. Activity on
this template was maximal when all 4 deoxyribonucleoside
5'-triphosphates were present. The incorporation of TTP-3 H
into acid-insoluble material, when only TTP was present in the
reaction mixture, was reduced 85% (Fraction D-I) and 92%
(Fraction D-II) relative to the incorporation attained in the
complete reaction mixtures containing all 4 deoxynucleoside
triphosphates. Table 2 demonstrates some of the requirements
of each of these fractions with regard to the utilization of the
homopolymer duplex, poly rA-oligo(dT)12_18.
Neither polyriboadenylic acid nor the 12- to 18-residue-long oligodeoxyribothymidylate alone promoted the incorporation by either
fraction of TTP-3 H or dATP-3H. The fact that dATP-3 H was
incorporated
with double-stranded
polydeoxyadenylatethymidylate of alternating sequence as template indicates that
lack of incorporation of dATP-3 H with the use of polyriboadenylic acid or 12- to 18-residue-long oligodeoxyribothymidylate was not due to a peculiarity of the dATP-3 H
used. These observations indicate that both components of the
duplex are required for optimal template activity. Further
more, the fact that neither component of the duplex by itself
was capable of promoting significant incorporation of TTP-3 H
seems to rule out the possibility that the incorporation
observed in this study was due to end addition. Moreover,
since the maximum incorporation of dATP-3 H observed with
either fraction when poly rA-oligo(dT) is used as template was
less than 4% of the amount of TTP-3 H incorporated, the
template activity of the oligodeoxyribonucleotide
component
Table 1
Requirements of the DNA polymerase activities in Fractions D-I and D-II
and nuclease sensitivity of product
The assay conditions were as described in "Materials and Methods" except for the
modifications listed. Each assay contained 50 pi of Fraction D-I or Fraction D-II containing
14.2 or 35.0 yug total protein, respectively. The specific activity of the TTP-3H was 380
cpm/pmole, and the template in all cases was 25 jugof activated DNA.
Label incorporated
(pmoles)
Reaction conditions
Fraction D-I
Fraction D-II
CompleteOmit
dATPOmit
dCTP, dGTP,
templateOmit
"activated" DNA
w0.5 Mg**,plus 1 jumóleMn
MnwAdditional
Mmole Mg", 0.5 jumóle
incubationwith
60-min
DNaseAdditional
incubationwith
60-min
RNaseAdditional
60-min incubation35.05.10.02.35.87.637.538.418.91.50.02.13.05.424.521.4
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1211
F. J. Persico, D. E. Nicholson, and A. A. Gottlieb
Table 2
Effect of synthetic polynucleotide templates on incorporation of TTP-3H and
Ã-Ã-A
TP-3H into an acid-insoluble form by Fractions D-I and D-II
The basic assay conditions were as described in "Materials and Methods." From 0.05 to 0.06
A260 unit of poly rA>oligo(dT),,.,,,
its 2 components alone, or double-standard polydeoxyadenylate-thymidylate of alternating sequence were used as templates. For experiments with
TTP-3H, the reaction mixture (0.25 ml) contained either 1.61 nmoles of TTP (2980
cpm/pmole) for Experiment 1 or 1.01 nmoles (2448 cpm/pmole) in Experiment 2 and 8.5
nmoles of dATP in both experiments. In some experiments, dATP was omitted as indicated. In
studies with dATP-3 H as label, the reaction mixture contained 1.39 nmoles of dATP at a
specific activity of 1727 cpm/pmole and 8.3 nmoles of TTP. In Experiment 1, 23.8 Mg of
Fraction D-I and 67.5 jig of Fraction D-II were used; whereas, for Experiment 2, Fractions D-I
and D-II were supplied at 7.9 and 25.5 MB,respectively.
Label incorporated
(pmoles)
Template
Label
Experiment
1Poly
rA-oligo(dT)Poly
rA'oligo(dT)(omit
dATP)Oligodeoxythy
Fraction D-I
Fraction D-II
HTTP-3
HTTP-3
H5.305.63<0.12.320.991<0.1
midylateTTP-3
Experiment 2
Poly rA-oligo(dT)
Poly rA-oligo(dT)
Oligodeoxythymidylate
Polyriboadenylic acid
Polydeoxyriboadenylic
acid
Double-stranded polydeoxyadenylate-thymidylate in alternating
sequence
TTP-3H
dATP-3 H
dATP-3 H
TTP-3 H
dATP-3 H
1.67
<0.06
<0.06
<0.06
<0.06
1.00
<0.06
<0.06
<0.06
<0.06
dATP-3 H
1.31
1.74
of the duplex is negligible. Thus it would seem that the
Oligodeoxythymidylate moiety serves as a primer, and incor
poration of TTP3 H appears to be directed by the ribopolymer
(riboadenylic acid) portion of the duplex.
The response of Fractions D-I and D-II to different DNA
templates is shown in Table 3. Fraction D-I appeared to be
more effective in promoting the incorporation of TTP-3 H with
all templates. "Activated" DNA was an effective template for
tograph appropriately under conditions of both stepwise and
continuous gradient elution from DEAE-cellulose. Chart 2A
illustrates the results of eluting these fractions from small
DEAE columns under conditions identical to those used in the
initial isolation. The upper 4 panels show that the polydeoxynucleotide-synthesizing activities in Fraction D-I, as measured
by the incorporation of TTP-3 H with 2 of the templates used
in this study, "activated" DNA and poly rA-oligo(dT)12—ig>
both fractions while neither fraction utilized native DNA very
well under these conditions. The DNA-synthesizing activities
in the 2 fractions appeared to be best differentiated on the
basis of their relative efficiencies in utilizing native and
denatured DNA templates. In the experiment shown in Table
3, Fraction D-I was approximately 3.5-fold more effective in
utilizing the single-stranded template, whereas Fraction D-II
was only 1.8 times more effective in promoting the
incorporation of TTP-3 H with single-stranded DNA than it was
élûtes
predominantly with 0.1 M KC1, with a smaller amount
of activity eluting with 0.3 M KC1. This is in contrast to the
DNA polymerase activity in Fraction D-II, which when
rechromatographed in the same manner élûtes
with 0.3 M KC1
(lower 2 panels). Chart 26 demonstrates the results of
rechromatography
of the fractions under conditions of
gradient elution. The upper panel in this chart depicts the
results of chromatographing an extract of the MOPC-21 tumor
prepared as described in "Materials and Methods." Elution of
with native DNA.
The kinetics of TTP-3 H incorporation
this column with a linear KC1 gradient resulted in the partial
resolution of 2 peaks. The middle panel illustrates that
rechromatography of Fraction D-I under similar conditions of
gradient elution resulted in a single peak of activity, identical
in its elution characteristics to the lower salt peak obtained
with the whole extract. The lower panel demonstrates that
rechromatography of Fraction D-II gave rise to a single peak
corresponding to the 2nd peak obtained by chromatographing
the tumor extract.
To establish that these 4 enzymatic activities were indeed
distinct, we subjected aliquots of D-I and D-II to sedimenta-
on "activated"
and
single-stranded calf thymus DNA was linear with respect to
time for at least 60 min, and this was also true for the
incorporation of TTP-3 H directed by the synthetic duplex
template poly rA-oligoidT)! 2-is- Moreover, the incorporation
of TTP-3 H catalyzed by each fraction using "activated" DNA
or poly rA-oligo(dT) as templates was linearly dependent on
the amount of protein added to the reaction mixture up to at
least 20£ig/assay.
Chart 2 demonstrates that Fractions D-I and D-II rechroma1212
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DNA Polymerases of Murine Myeloma
Table 3
The activities of Fractions D-I and D-II on
different DNA templates
Assays were performed on various DNA templates as described in
"Materials and Methods." The specific activity of the TTP-3H was 390
cpm/pmole. Fractions D-I and D-II were supplied at levels of 31 and 96
Mgtotal protein, respectively.
(cpm)Fractionincorporated
DNA
Native DNA
"Activated" DNATTP-3H
D-II1,044
D-I4,944
Mg)Denatured
(25
572
4,306
1,410
12,400Fraction
579
13 15 17
FRACTION
579
NUMBER
II
15 17
tion on glycerol gradients (Chart 3). When the gradient
containing Fraction D-I was assayed with "activated" DNA and
poly rA'oligo(dT)12—ig as templates, 2 principal activities
utilizing each of these templates were found at 7.5 to 8.5 S
corresponding to molecular weights of 165,000 to 185, 000.
In fraction D-II, 2 other activities at 6.5 to 7.0 S and 4 to 5 S,
with corresponding molecular weights of 125,000 to 140,000
and 55,000 to 75,000, were found. The molecular weights
were estimated by the method of Martin and Ames (8), with
bovine serum albumin as a standard.
The activities in Fractions D-I and D-II capable of using
poly rA'oligo(dT)12_1g
as template could also be dis
tinguished by their respective requirements for divalent cation.
20
FRACTION
40
60
NUMBER
Chart 2. A, rechromatography (stepwise elution). In each case, an aliquot of Fraction D-I or Fraction D-II was dialyzed for 2 hr versus 0.05 M
Tris-HCl, pH 7.8; 0.002 M MnCl2 ; 0.015 M 2-mercaptoethanol; and 30% glycerol (hereafter called TMMG buffer). The dialyzed fractions were
diluted with TMMG buffer, if necessary, such that the conductivities of the diluted fractions approximated 0.02 M KC1in this buffer. The fractions
were then applied to DEAE-cellulose (Whatman Microgranular DE 52) columns (1.2 x 2.5 cm for the experiment in the upper 2 panels, and 1.2 x
12 cm for the experiments in the lower 4 panels) previously equilibrated with TMMG buffer and washed in with several column volumes of this
buffer. The column was successively eluted with 0.1 M KO and 0.3 M KCl in TMMG buffer, and 0.05-ml aliquots were assayed as described in
"Materials and Methods" with "activated" DNA and/or poly rA-oligo(dT)i2_18 as template. Upper 2 panels, rechromatography of Fraction D-I
with enzyme activity determined by incorporation of TTP-3H (specific activity, 307 cpm/pmole) in conjunction with "activated" DNA as
template; center panels, a separate experiment in which Fraction D-I was rechromatographed and assayed with poly rA-oligo(dT)12 ig as
template (TTP-3H specific activity, 2736 cpm/pmole). Bottom 2 panels, result of rechromatographing Fraction D-II with enzyme activity detected
with both "activated" DNA (specific activity of TTP-3H, 330 cpm/pmole) and poly rA-oligo(dT)12_18 (specific activity of TTP-3H, 1871
cpm/pmole).
B, rechromatography (gradient elution). Bottom 2 panels, we prepared 2.0 ml of Fraction D-II and 0.5 ml of Fraction D-I for rechromatography
as described in A. The dialyzed fractions were applied to the DEAE columns (1.2 x 12 cm) previously equilibrated with TMMG buffer and washed
in with TMMG adjusted to 0.02 M KCl. The columns were eluted with linear KCl gradients, 0.02 M to 0.35 M KCl in TMMG buffer, and 0.05-ml
aliquots were removed for assay. Upper panel, DEAE-cellulose chromatography, essentially carried out in this manner, of an extract of 8 g of
tumor tissue processed as described in "Materials and Methods."
The fraction sizes for all experiments were approximately 1.4 to 1.6 ml. Assays were performed as described in "Materials and Methods" with
"activated" DNA as template. Specific activity of the TTP-3H ranged from 284 to 311 cpm/pmole.
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1213
F. J. Persico, D. E. Nicholson, and A. A. Gottlieb
D-I
26
24
b 22
- 20
52
48
44
40
36
32
28
24
20
16
12
8
4
poly rA oligo dT
§18
16
14
12
10
activated
DNA
8
6
4
2
20
10
30
40
D-n
26
24
b22
T 20
activated
DNA
I 18
" 16
14
12
poly rA-oligo
dT
IO
8
6
4
2
10
20
30
40
FRACTION NUMBER
Chart 3. Glycerol gradient centrifugation. A 0.5-ml aliquot of the D-I or D-II fraction was
layered on a 10 to 30% (v/v) glycerol gradient prepared in 0.05 M Tris-HCl (pH 7.8),
containing 0.15 M potassium chloride and 0.001 M 2-mercaptoethanol. The gradient was
centrifuged for 32 hr at 4°at 36,000 rpm in an SW 40 rotor. Fractions of approximately 0.3
ml were collected dropwise from the bottom of the tube. From every other fraction, a
0.05-ml sample was assayed for polymerase activity with "activated" DNA or poly
rA-oligo(dT)i2—is as template. A, profile given by Fraction D-I; B, profile given by
Fraction D-II. Left and right ordinales refer to activity on "activated" DNA and poly
rA-oIigo(dT), respectively.
Both of these enzymes preferred Mn** to Mg4* ion. The
optimal Mn* ion concentration for this activity in Fraction
D-I was 0.3 mM while that of the analogous activity in
Fraction D-II was 1.0 mM. For Mg4* ion, the respective optima
were 1.0 mM for Fraction D-I and 4.0 mM for Fraction D-II.
The levels of TTP-3H incorporation
directed by poly
rA-oligo(dT)12-i8 w'*h Fractions D-I and D-II in conjunction
with these optimal concentrations of Mg4* ion were 20.4 and
15.3%, respectively, of that observed under optimal concen
trations of Mn4* ion.
DISCUSSION
Our method of isolation of these myeloma DNA polymerases achieves a separation of these enzymes into 2 fractions.
1214
Each of these fractions contains an activity that is active on
"nicked" DNA and another activity that is capable of
transcribing
the
ribopolymer
strand
of poly
rAoligo(dT)12.i8The activities within Fraction D-I can be
separated from each other by chromatography on phosphocellulose (11). The same type of separation can be achieved with
Fraction D-II (our unpublished results). Our method does not
achieve recovery of the small-molecular-weight DNA polym
erase present in the nucleus which has been described by
Chang and Bollum (2). An activity analogous to this
"minipolymerase"
exists in this myeloma line, but that
enzyme does not absorb to DEAE-cellulose under the
conditions used in our isolation procedure (unpublished
results).
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DNA Polymerases of Murine Myeloma
Thus, these activities are distinguishable by physical and
biochemical criteria. It is not likely that any of these enzymes
arise from microbial contamination of the myeloma, since
cultures of the tumor for Mycoplasma (kindly performed by
Dr. L. Hayflick) have been negative, and none of the
enzymatic activities described herein are affected by specific
antibody directed against Escherichia coli DNA polymerase I
under conditions that produce over 95% inhibition of the E.
coli enzyme (our unpublished observations).
The enzyme in Fractions D-I and that in Fraction D-II
which are active on "nicked" DNA appear to be related to the
the separation of nuclear and cytoplasmic constituents, it is
not possible to specify the site of origin of each of these
enzymes in the cell. As mentioned above, our method of
isolation, in which an extraction buffer of low ionic strength is
used, minimizes the recovery of an activity resembling the
minipolymerase of Chang and Bollum (2).
The functions of these various enzymes in situ remain to be
determined. We have presented a method by which separation
of these enzymes can be achieved. The opportunity is now
available to compare these DNA polymerases with similar
enzymes from other cells and hopefully to elucidate the
function of these enzymes in normal and malignant cells.
6 to 8 S polymerase described by Chang and Bollum (3), with
S values of 7.5 to 8.5 and 6.5 to 7.0, respectively. It is possible
that Bollum's 6 to 8 S polymerase is a mixture of the 2
DNA-dependent enzymes present in this fraction. Our observa
tions regarding the sedimentation
coefficients of these ACKNOWLEDGMENTS
DNA-dependent activities in Fractions D-I and D-II are
consistent with this interpretation. Functionally, the activity
in Fraction D-I appears to exhibit a greater preference for
We are grateful to Dr. P. Cassidy, Dr. F. Kahan, and Dr. L. Loeb for
helpful discussions and supplies of certain critical materials. We wish to
single-stranded DNA as compared with native DNA.
thank Seaton Bowers and E. Pankuch for very able technical assistance
The distinction between the enzyme in Fraction D-I capable
and O. M. Barr for preparation of the manuscript.
of transcribing poly rA'oligoidT^a-is
and tne analogous
activity in Fraction D-II is quite evident. Both are separable
from each other chromatographically, and the enzyme in D-I
appears to be considerably heavier than the corresponding
enzyme in D-II. We will report elsewhere that BALB/c mice
REFERENCES
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but not the enzyme in Fraction D-II. This suggests that the
Deoxyribonucleic Acid Polymerase with Rat Liver Ribosomes and
Smooth Membranes-Purification and Properties of the Enzymes.
enzyme in D-I may be related to the DNA polymerase of the
Biochemistry, 10: 1981-1992, 1971.
Rauscher murine leukemia virus and may derive from a type C
2.
Chang, L. M. S., and Bollum, F. J. Low Molecular Weight
particle in this tumor line. Indeed, such type C particles have
Deoxyribonucleic Acid Polymerase from Rabbit Bone Marrow.
been described in lines of the MOPC-21 tumor (13). The
Biochemistry, 11: 1264-1272, 1972.
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di
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Kuff (15) and Kuff et al. (5) obtained from type A particles of
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than manganese and is active in solutions of 250 mM KC1 (15),
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while neither of our enzymes are active under conditions of
7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
high ionic strength.
Protein Measurement With the Folin Phenol Reagent. J. Biol.
Until very recently, the demonstration of more than 1
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mammalian DNA polymerase has been limited to the
8. Martin, R. G., and Ames, B. A Method for Determining the
observation of a mitochondrial enzyme (4,9). However, there
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Mixtures. J. Biol. Chem., 236: 1372-1379, 1961.
are now several reports of multiple DNA polymerases in
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mammalian cells. In particular, Baril et al. (1) now report the
Mitochondria. Partial Purification of a Distinct DNA Polymerase
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CANCER
RESEARCH
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VOL.
33
Isolation and Partial Characterization of Multiple DNA
Polymerases of the Murine Myeloma, MOPC-21
Francis J. Persico, Diarmuid E. Nicholson and A. Arthur Gottlieb
Cancer Res 1973;33:1210-1216.
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