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[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. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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 JUNE 1973 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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. JUNE 1973 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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). CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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 bearing myeloma tumors carry a factor in their sera that is capable of inhibiting this ribopolymer-transcribing enzyme in Fraction D-I and the Rauscher murine leukemia virus enzyme 1. Baril, E. F., Brewer, O. E., Jenkins, M. D., and Laszlo, F. 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. sedimentation behavior of the poly rA-oligo(dT)12-i8 di 3. Chang, L. M. S., and Bollum, F. J. Antigenic Relationships in rected enzyme in Fraction D-I is larger than that of the Mammalian DNA Polymerase. Science, 175: 1116-1117, 1972. 4. Kalf, G. F., and Ch'ih, F. F. Purification and Properties of Rauscher viral enzyme, indicating that this particular DNA polymerase in tais myeloma line is distinct from that of the Deoxyribonucleic Acid Polymerase from Rat Liver Mitochondria. J. Biol. Chem., 243: 4904-4916, 1968. Rauscher murine leukemia virus. Moreover, neither of the ribopolymer-transcribing polymerases, described in this report, 5. Kuff, E. L., Wivel, N. A., and Lenders, K. K. The Extraction of Intracisternal A-Particles from a Mouse Plasma-Cell Tumor. Cancer appears to be similar to the enzyme described by Wilson and Res., 28: 2137-2143,1968. Kuff (15) and Kuff et al. (5) obtained from type A particles of 6. Loeb, L. A. Purification and Properties of Deoxyribonucleic Acid another myeloma line. That enzyme prefers magnesium rather Polymerase from Nucleic of Sea Urchin Embryos. J. Biol. Chem., than manganese and is active in solutions of 250 mM KC1 (15), 244: 1672-1681,1969. 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 Chem., 193: 265-275, 1951. 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 Sedimentation Behavior of Enzymes: Application to Protein Mixtures. J. Biol. Chem., 236: 1372-1379, 1961. are now several reports of multiple DNA polymerases in 9. Meyer, R. R., and Simpson, M. V. DNA Biosynthesis in mammalian cells. In particular, Baril et al. (1) now report the Mitochondria. Partial Purification of a Distinct DNA Polymerase presence of membrane-associated, ribosome-associated, and from Isolated Rat Liver Mitochondria. Proc. Nati. Acad. Sci. U.S., nuclear DNA polymerases in rat liver and Weissbach et al. (14) 61: 130-137, 1968. have demonstrated the existence of 1 cytoplasmic and 2 10. Persico, F. J., and Gottlieb, A. A. Separation of Two DNA nuclear DNA polymerases in HeLa cells. In both of these Synthesizing Activities in a Murine Myeloma Tumor. Federation studies, a cytoplasmic enzyme closely resembled a nuclear Pioc.,30: 650, 1971. enzyme, and Chang and Bollum (2) have suggested that only 11. Persico, F. J., and Gottlieb, A. A. DNA Polymerases of Myeloma: the 3.3 S minipolymerase is associated with the nucleus. Since Template and Primer Specificities of Two Enzymes. Nature New BioL,2J9: 173-176,1972. our method involves the grinding of whole cells, rather than JUNE 1973 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 1215 F. J. Persico, D. E. Nicholson, and A. A. Gottlieb 12. Stein, H., and Hausen, P. A Factor from Calf Thymus Stimulating DNA-Dependent RNA Polymerase Isolated from This Tissue. European J. Biochem., 14: 270-277, 1970. 13. Watson, J., Ralph, P., Sarkai, S., and Conn, M. Leukemia Viruses Associated with Mouse Myeloma Cells. Proc. Nati. Acad. Sei. U. S., 66: 344-351, 1968. 1216 14. Weissbach, A., Schlabach, A., FridK ider, B., and Bolden, A. DNA Polymerases from Human Cells. Nature New Biol., 231: 167-170, 1971. 15. Wilson, S. H., and Kuff, E. L. A. Novel DNA Polymerase Activity Found in Association with Intracisternal A-Type Particles. Proc. Nati. Acad. Sei. U. S., 69: 1531-1536, 1972. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/6/1210 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1973 American Association for Cancer Research.