APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1978, p. 397-402
0099-2240/78/0036-0397$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 3 Printed in U.S.A.
Chicken Egg Yolk Stabilizes the Reverse Transcriptase Activity in Type C Particles Produced by Cultured MOPC-315 Murine Myeloma Cells A. GAZIT, A. YANIV,* AND E. EYLAN
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Received for publication 16 March 1978
Type C particles released from cultured murine myeloma MOPC-315 cells were significantly protected when the purification steps were all conducted in the presence of 10% chicken egg yolk fluid. The yolk fluid also slowed down the inactivation of viral particles during incubation at 37°C and enabled full recovery of viral particles through several cycles of freezing and thawing. The purification of viral particles in the presence of yolk fluid did not affect the capability of the viral DNA polymerase to reverse-transcribe the virion RNA in vitro, nor that of the viral RNA to act as a functional template. per ml in Eagle modified medium supplemented with 10% calf serum. Culture fluids were clarified from cell debris by centrifugation at 2,000 x g for 10 min and then at 10,000 x g for an additional 10 min. Purification steps of viral particles. (i) Sedimentation through a 20% glycerol column. Clarified culture fluids were placed on a 13-ml column of 20% (vol/vol) glycerol in TNE [0.1 M tns(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4), 0.1 M NaCl, 0.001 M ethylenediaminetetraacetate] and sedimented in a Beckman SW27 rotor at 95,000 x g for 1 h at 4°C. The resulting pellet was resuspended in TNE, and samples were assayed for DNA polymerase activity. (ii) Equilibrium gradient centrifugation. The resuspended pellet, obtained after sedimentation through 20% glycerol, was layered over a preformed gradient of 20 to 55% (wt/wt) sucrose in TNE containing 3 mM dithiothreitol and centrifuged at 95,000 x g for 16 h at 4°C. The gradient was fractionated, and samples were tested for DNA polymerase activity. Densities of the fractions were calculated from refractive index measurements. (iii) Sedimentation of banded virus. Gradient fractions corresponding to the viral peak according to particles. buoyant density and DNA polymerase activity were diluted with TNE, and sedimented at 95,000 pooled, MATERIALS AND METHODS x g for 1 h. The sedimented particles were resusYolk fluid. Yolk fluid was harvested from embry- pended in 0.01 M Tris-hydrochloride (pH 8.2) and onated SPF chicken eggs on day 6 of incubation. At assayed for DNA polymerase activity. DNA polymerase assay. DNA polymerase activthis stage, the yolk fluid is not viscous and hence is amenable to convenient handling. After centrifugation ity was assayed as previously described (15), using the at 1,000 x g for 10 min to remove heavy debris, fluids synthetic template oligo(dT)1,2,8 poly(rA). [;'H]TTP were spun at 95,000 x g for 1 h. The interphase formed was added to yield 1,000 cpm/pmol. Endogenous assay of high-molecular-weight between the floating lipids and the pelleted debris was collected with a syringe and then centrifuged at RNA and RNA-instructed DNA polymerase. The 142,000 x g for 2 h to obviate sedimentation of lighter simultaneous assay for the detection of high-molecular-weight RNA and RNA-instructed DNA polymerdebris during the steps of virus purification. Source of virus. The myeloma viral particles were ase was according to the method of Schlom and Spieobtained from culture fluids harvested 48 h after seed- gelman (11), as previously described (15). Hybridization assays. Viral RNA with a sedimening MOPC-315 cells at a concentration of 2 x 105 cells 397
Recently, we have succeeded in establishing a tissue-culture-adapted cell line from murine myeloma MOPC-315 cells. The cells of this line preserved in vitro the capacity to secrete type C viral particles, which were identical in their nucleic acids and other biochemical features to the particles released by MOPC-315 tumor cells into the peritoneal fluids of tumor-bearing mice (15). The viral particles released in vitro, however, were significantly unstable, and there was substantial loss of intact viral particles in the course of the various purification steps. Stability of oncornaviruses has been reported to increase in the presence of protein-rich fluids (3-5, 12, 13). However, such fluids as calf serum, fetal calf serum, or bovine plasma albumin demonstrated only a slight protective effect on the viral particles produced by cultured murine myeloma MOPC-315 cells. The present study describes an additional protein-rich chicken egg yolk fluid which more effectively stabilizes the murine myeloma viral
398
APPL. ENVIRON. MICROBIOL.
GAZIT, YANIV, AND EYLAN 4
tation coefficient of 60-70S and 'H-labeled complementary DNA (cDNA) product were prepared from purified virus as previously described (15). Annealing reaction between 'H-labeled cDNA (1,000 cpm) and viral 70S RNA (0.2 ,tg) was assayed by resistance to Si nuclease (8).
RESULTS Optimal concentration of yolk fluid for maximal protection of viral particles. An experiment was performed to determine the concentration of yolk fluid that would provide maximal protection of viral particles while sedimenting through 20% glycerol in TNE columns. To this end, MOPC-315 viral suspensions were sedimented through 20% glycerol columns containing various concentrations of yolk fluid, ranging from 0 to 20%. The data presented in Fig. 1 demonstrate that the recovery of viral particles possessing DNA polymerase activity is directly proportional to the concentration of the yolk fluid, reaching maximal level with 10% yolk fluid. The yolk fluid alone did not reveal any DNA polymerase activity, nor did it increase the rate of DNA polymerase activity when added to the reaction mixture. Protective activity of clarified yolk fluid. We suspected that the heterogeneous composition (1) and heavy opacity of the crude yolk fluid might interfere with and decrease its protective capacity. To test this possibility, we assayed the stabilizing activity of the yolk fluid after various methods of clarification, including ether extraction, autoclaving, and filtration through a variety of Diaflo membranes (XM 300, XM 100, and XM 50). The clarified yolk fluids were included at a 10% final concentration in the 20% glycerol columns through which the viral particles were sedimented. The results (Fig. 2) show that clarification of the crude yolk fluid did not render it more protective. On the contrary, the recovery of viral particles decreased to some extent, particularly after the Diaflo membrane ultrafiltrations. Protective effect of yolk fluid on MOPC315 viral particles during equilibrium gradient centrifugation and sedimentation of banded virus. To determine whether the protective capacity of the yolk fluid is expressed also during the further steps of virus purification, we observed the effect of the yolk fluid on the recovery of intact particles throughout isopycnic gradient centrifugation. Viral suspension (with a total of 460,000 cpm of virion-associated polymerase activity) obtained after centrifugation through 20% glycerol columns supplemented with 10% yolk fluid was divided into two portions
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2.5 5 10 15 20 CONCENTRATION OF YOLK FLUID (%) FIG. 1. Optimal concentration of yolk fluid required for maximal recovery of viral particles. Viral suspension containing 50,000 cpm of DNA polymerase activity was sedimented at 95,000 x g for I h through 20% glycerol columns containing the indicated concentrations ofyolk fluid. Pellets were resuspended in 0.01 M Tris-hydrochloride (pH 8.2) and assayed for templated DNA polymerase activity for 30 min at 37°C as described in the text. Data presented were corrected for nonspecific background radioactivity.
and layered onto isopycnic sucrose gradients with or without 10% yolk fluid. After centrifugation as described in Materials and Methods, fractions were tested for DNA polymerase activity. Figure 3 demonstrates graphically the significant protection provided by the yolk fluid during the sucrose equilibrium centrifugation. To quantitate the extent of protection, the fractions corresponding to the peak of viral DNA polymerase activity (1.15 to 1.19 g/ml) were pooled and assayed for DNA polymerase activity. From the results summarized in Table 1, it can be seen that after isopycnic centrifugation of viral particles in sucrose gradients lacking yolk fluid, only 14.7% of the viral input is recovered, whereas in the presence of 10% yolk fluid the viral recovery was 57.3%. To assay the stabilizing effect of the yolk fluid during the sedimentation of banded viral particles, the pooled gradient fractions were centrifuged in the presence or absence of 10% yolk fluid, and the obtained viral pellet was tested for DNA polymerase activity. The data (Table 1)
VOL. 36, 1978
399
EGG YOLK STABILIZES MYELOMA TYPE C VIRUSES
4
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118 ,
0099-2240/78/0036-0397$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 3 Printed in U.S.A.
Chicken Egg Yolk Stabilizes the Reverse Transcriptase Activity in Type C Particles Produced by Cultured MOPC-315 Murine Myeloma Cells A. GAZIT, A. YANIV,* AND E. EYLAN
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Received for publication 16 March 1978
Type C particles released from cultured murine myeloma MOPC-315 cells were significantly protected when the purification steps were all conducted in the presence of 10% chicken egg yolk fluid. The yolk fluid also slowed down the inactivation of viral particles during incubation at 37°C and enabled full recovery of viral particles through several cycles of freezing and thawing. The purification of viral particles in the presence of yolk fluid did not affect the capability of the viral DNA polymerase to reverse-transcribe the virion RNA in vitro, nor that of the viral RNA to act as a functional template. per ml in Eagle modified medium supplemented with 10% calf serum. Culture fluids were clarified from cell debris by centrifugation at 2,000 x g for 10 min and then at 10,000 x g for an additional 10 min. Purification steps of viral particles. (i) Sedimentation through a 20% glycerol column. Clarified culture fluids were placed on a 13-ml column of 20% (vol/vol) glycerol in TNE [0.1 M tns(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4), 0.1 M NaCl, 0.001 M ethylenediaminetetraacetate] and sedimented in a Beckman SW27 rotor at 95,000 x g for 1 h at 4°C. The resulting pellet was resuspended in TNE, and samples were assayed for DNA polymerase activity. (ii) Equilibrium gradient centrifugation. The resuspended pellet, obtained after sedimentation through 20% glycerol, was layered over a preformed gradient of 20 to 55% (wt/wt) sucrose in TNE containing 3 mM dithiothreitol and centrifuged at 95,000 x g for 16 h at 4°C. The gradient was fractionated, and samples were tested for DNA polymerase activity. Densities of the fractions were calculated from refractive index measurements. (iii) Sedimentation of banded virus. Gradient fractions corresponding to the viral peak according to particles. buoyant density and DNA polymerase activity were diluted with TNE, and sedimented at 95,000 pooled, MATERIALS AND METHODS x g for 1 h. The sedimented particles were resusYolk fluid. Yolk fluid was harvested from embry- pended in 0.01 M Tris-hydrochloride (pH 8.2) and onated SPF chicken eggs on day 6 of incubation. At assayed for DNA polymerase activity. DNA polymerase assay. DNA polymerase activthis stage, the yolk fluid is not viscous and hence is amenable to convenient handling. After centrifugation ity was assayed as previously described (15), using the at 1,000 x g for 10 min to remove heavy debris, fluids synthetic template oligo(dT)1,2,8 poly(rA). [;'H]TTP were spun at 95,000 x g for 1 h. The interphase formed was added to yield 1,000 cpm/pmol. Endogenous assay of high-molecular-weight between the floating lipids and the pelleted debris was collected with a syringe and then centrifuged at RNA and RNA-instructed DNA polymerase. The 142,000 x g for 2 h to obviate sedimentation of lighter simultaneous assay for the detection of high-molecular-weight RNA and RNA-instructed DNA polymerdebris during the steps of virus purification. Source of virus. The myeloma viral particles were ase was according to the method of Schlom and Spieobtained from culture fluids harvested 48 h after seed- gelman (11), as previously described (15). Hybridization assays. Viral RNA with a sedimening MOPC-315 cells at a concentration of 2 x 105 cells 397
Recently, we have succeeded in establishing a tissue-culture-adapted cell line from murine myeloma MOPC-315 cells. The cells of this line preserved in vitro the capacity to secrete type C viral particles, which were identical in their nucleic acids and other biochemical features to the particles released by MOPC-315 tumor cells into the peritoneal fluids of tumor-bearing mice (15). The viral particles released in vitro, however, were significantly unstable, and there was substantial loss of intact viral particles in the course of the various purification steps. Stability of oncornaviruses has been reported to increase in the presence of protein-rich fluids (3-5, 12, 13). However, such fluids as calf serum, fetal calf serum, or bovine plasma albumin demonstrated only a slight protective effect on the viral particles produced by cultured murine myeloma MOPC-315 cells. The present study describes an additional protein-rich chicken egg yolk fluid which more effectively stabilizes the murine myeloma viral
398
APPL. ENVIRON. MICROBIOL.
GAZIT, YANIV, AND EYLAN 4
tation coefficient of 60-70S and 'H-labeled complementary DNA (cDNA) product were prepared from purified virus as previously described (15). Annealing reaction between 'H-labeled cDNA (1,000 cpm) and viral 70S RNA (0.2 ,tg) was assayed by resistance to Si nuclease (8).
RESULTS Optimal concentration of yolk fluid for maximal protection of viral particles. An experiment was performed to determine the concentration of yolk fluid that would provide maximal protection of viral particles while sedimenting through 20% glycerol in TNE columns. To this end, MOPC-315 viral suspensions were sedimented through 20% glycerol columns containing various concentrations of yolk fluid, ranging from 0 to 20%. The data presented in Fig. 1 demonstrate that the recovery of viral particles possessing DNA polymerase activity is directly proportional to the concentration of the yolk fluid, reaching maximal level with 10% yolk fluid. The yolk fluid alone did not reveal any DNA polymerase activity, nor did it increase the rate of DNA polymerase activity when added to the reaction mixture. Protective activity of clarified yolk fluid. We suspected that the heterogeneous composition (1) and heavy opacity of the crude yolk fluid might interfere with and decrease its protective capacity. To test this possibility, we assayed the stabilizing activity of the yolk fluid after various methods of clarification, including ether extraction, autoclaving, and filtration through a variety of Diaflo membranes (XM 300, XM 100, and XM 50). The clarified yolk fluids were included at a 10% final concentration in the 20% glycerol columns through which the viral particles were sedimented. The results (Fig. 2) show that clarification of the crude yolk fluid did not render it more protective. On the contrary, the recovery of viral particles decreased to some extent, particularly after the Diaflo membrane ultrafiltrations. Protective effect of yolk fluid on MOPC315 viral particles during equilibrium gradient centrifugation and sedimentation of banded virus. To determine whether the protective capacity of the yolk fluid is expressed also during the further steps of virus purification, we observed the effect of the yolk fluid on the recovery of intact particles throughout isopycnic gradient centrifugation. Viral suspension (with a total of 460,000 cpm of virion-associated polymerase activity) obtained after centrifugation through 20% glycerol columns supplemented with 10% yolk fluid was divided into two portions
;: 3k D. I. ....:
i.. .......
..--.....
Ea. C)
.... .;
2
-..
-:...
.i. .-. :: ::::::
..X.. -:
i- -. iD :.
fi:
:::: .. ..
::::
:: .:: ::::: :::
*: ::
..-i:. ff
0
-
H
.:j: .., .0, -
-
U
2.5 5 10 15 20 CONCENTRATION OF YOLK FLUID (%) FIG. 1. Optimal concentration of yolk fluid required for maximal recovery of viral particles. Viral suspension containing 50,000 cpm of DNA polymerase activity was sedimented at 95,000 x g for I h through 20% glycerol columns containing the indicated concentrations ofyolk fluid. Pellets were resuspended in 0.01 M Tris-hydrochloride (pH 8.2) and assayed for templated DNA polymerase activity for 30 min at 37°C as described in the text. Data presented were corrected for nonspecific background radioactivity.
and layered onto isopycnic sucrose gradients with or without 10% yolk fluid. After centrifugation as described in Materials and Methods, fractions were tested for DNA polymerase activity. Figure 3 demonstrates graphically the significant protection provided by the yolk fluid during the sucrose equilibrium centrifugation. To quantitate the extent of protection, the fractions corresponding to the peak of viral DNA polymerase activity (1.15 to 1.19 g/ml) were pooled and assayed for DNA polymerase activity. From the results summarized in Table 1, it can be seen that after isopycnic centrifugation of viral particles in sucrose gradients lacking yolk fluid, only 14.7% of the viral input is recovered, whereas in the presence of 10% yolk fluid the viral recovery was 57.3%. To assay the stabilizing effect of the yolk fluid during the sedimentation of banded viral particles, the pooled gradient fractions were centrifuged in the presence or absence of 10% yolk fluid, and the obtained viral pellet was tested for DNA polymerase activity. The data (Table 1)
VOL. 36, 1978
399
EGG YOLK STABILIZES MYELOMA TYPE C VIRUSES
4
, 3
122
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