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Mouse interferon messenger RNA: Characterization of the Xenopus laevis oocyte translation product
ARCHIVES
OF BIOCHEMISTRY
Vol. 217, No. 1, August,
AND
BIOPHYSICS
pp. 273-281, 1982
Mouse Interferon
Messenger RNA: Characterization laevis Oocyte Translation Product
of the Xenopus
FAZLUL H. SARKAR,l PHYLLIS SUSSMAN-BERGER, LULU A. PICKERING2
AND
Interferon Laboratories, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021 Received December
9, 1981, and in revised form April
15, 1982
Mouse interferon messenger RNA was isolated from Newcastle disease virus-induced mouse Lpa cells and then translated in Xenopus laevis oocytes. The resulting oocyte homogenate containing translated interferon activity was unstable to treatment with 5 M urea and to repeated freeze/thaw cycles, and it was 1% cross-reactive on human cells, as was native mouse interferon. Both native mouse interferon and the mouse interferon produced by the translation of mouse interferon mRNA behaved almost similarly on CPG, poly(U)-Sepharose, and anti-mouse interferon antibody columns. When the oocyte-translated product was partially purified and analyzed on sodium dodecyl sulfate-polyacrylamide gels, it migrated as a major single band of activity at 21-22,000 daltons with a trailing edge at 22-30,000 daltons. Only minor activity was detected in the region of 35-40,000 daltons where the vast majority of the native mouse interferon migrated. Thus, the oocyte-translated mouse interferon product comigrated largely with the minor species of native mouse interferon with a little activity which corresponds with the larger molecular weight species of native mouse interferon.
It has long been recognized that mouse interferon consists of at least two molecular species with size ranges of about 35,000 to 40,000 and 22,000 to 28,000 daltons (l-4). These molecular species differ in their cross-species activity (5, 6) and their affinity for binding to lectins (7) and also appear to be antigenically distinct (8). They possess both antiviral and anticellular activities (3, 9) and their tryptic digests are similar (10). In addition, Kawakita et al. (11) have described three species of mouse interferon produced from NDV3-induced mouse Ehrlich ascites tu’ To whom correspondence should be addressed. * Present address: Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139. 3 Abbreviations used: NDV, Newcastle disease virus; SDS, sodium dodecyl sulfate; CPG, controlled pore glass; NP-40, Nonidet P-40; PBS, phosphate-
mor cells that have similar amino acid compositions (12). Although two of these species, A and B, have similar tryptic digests (12) and amino-terminal amino acid sequences (13), the third species, C, differs in both these respects. In addition, with respect to the first 20 amino acid residues in the NH,-terminal region, it appears as though mouse interferon species A and B are homologous to human fibroblast-derived p-interferon whereas mouse interferon species C is homologous to human lymphoblastoid-derived a-interferon (13). It is of interest that a low-molecularweight subspecies of mouse interferon is cross-reactive on human cells (5). There buffered saline; FCS, fetal calf serum; EDTA, ethylenediaminetetraacetate; MEM, minimum essential medium; IFN, interferon; Temed, N,N,N,N’,-tetramethylethylenediamine.
-.-
27.1
0003-9861/82/090273-09$02.00/O Copyright All rights
Q 1982 by Academic Press. Inc. of repmduction in any form reserved.
274
SARKAR,
SUSSMAN-BERGER,
also appears to be a species of human lymphoblastoid-derived a-interferon (spontaneous interferon) that is antigenically similar to mouse interferon (14). Cavalieri et al. (15, 16) demonstrated that human leukocyte-derived cY-interferons and fibroblast-derived @-interferons are products of distinct cellular genes. The translation products of human lymphoblastoid-derived a-interferon mRNA (15, 16) and of human fibroblast-derived B-interferon mRNA translated in Xenopue Laevis oocytes (15-18) have been analyzed by SDSpolyacrylamide gel electrophoresis, but the Xenopus oocyte-translated mouse interferon mRNA(s) product(s) has not been studied by this approach. In the past, the analysis of Xenopus oocyte-translated interferon mRNAs product(s) has been hampered by the low levels of interferon produced in the translated products. In view of this, the purpose of the present study was to produce large quantities of the Xenopus-translated product of mouse interferon mRNA for characterization studies to determine whether it contained all of the molecular species of interferon characteristic of native mouse interferon. MATERIALS
AND METHODS
viruses. Newcastle disease virus (NDV) was propagated in 11-day-old chick embryos. Virus was harvested as allantoic fluid, clarified by centrifugation for 20 min at 3OOOg,and stored in 5- to lo-ml aliquots at -80°C. Virus stocks were titered by plaque assay on secondary chick embryo cell monolayers and contained about 10 plaque-forming units (pfu)/ml. CeUa L929 and Lpa cells (1) were propagated as monolayers in growth medium containing MEM, 10% FCS, 2 mM glutamine, 100 rg/ml streptomycin, and 100 units/ml penicillin. Chemicala SDS (electrophoretic purity), Temed, ammonium persulfate, and Affi-Gel 202 were from Bio-Rad; poly(U)-Sepharose and protein standards (electrophoretic calibration kit) from Pharmacia; controlled-pore glass beads (CPG 350 mesh, size 120/ 200) from Accurate Chemicals and Science Corporation; and mouse L cell interferon antiserum was obtained from the National Institutes of Allergy and Infectious Diseases (Lot G-024-501-563). All other chemicals were of highest purity grade. Production of native mawe interferon Monolayers of L929 or Lpa cells in e-liter roller bottles were
AND PICKERING
primed for 2 h with 100-290 units/ml of mouse L cell interferon and then induced with NDV at about 5 pfu/cell. Following an overnight incubation at 3’7”C, the culture medium was collected and centrifuged at 20009 for lo-15 min to remove the cellular debris. The supernatant fluid containing crude interferon in growth medium supplemented with 2% FCS was adjusted to pH 2.0 to inactivate any residual virus and then readjusted to neutral pH prior to interferon assay. mRNA preparation Lpa cells were grown almost to confluency in 2-liter roller bottles (approximately 10s cells/roller bottle) and lo-30 roller bottles were used for each RNA preparation. Cell monolayers were primed for 2 h with 100-200 units/ml of mouse L cell interferon and then induced with about 5 pfu NDV/cell. At 11-15 h postinduction, cell monolayers were washed once with PBS and then scraped off the bottles with rubber policemen. Cells were washed 3 times with cold PBS and then lysed in 10 vol of hypotonic lysis buffer (10 mM Tris-HCl, pH 7.6; 10 mM NaCl; 1.5 mM MgClz) containing 0.5% NP-40. Following gentle lysis for 5 min at 4’C, cell nuclei were removed by centrifugation at 1OOOgfor 10 min. The supernatant fluid was then extracted 3-4 times using the SDS/phenol/chloroform procedure described by Palmiter (19) and the resulting aqueous phase was precipitated twice with 2 vol of 95% ethanol at -20°C. The precipitated RNA (total cytoplasmic RNA) was adjusted to 1 mg/ml (assuming 40 pg/ml of RNA per ODzso unit) and fractionated by two cycles of oligo(dT)-cellulose (type 3, Collaborative Res., Inc.) chromatography. The poly(A)+ mRNA fraction was then precipitated l-2 times with 2 vol of 95% ethanol at -20°C and resuspended at a concentration of 0.2 to 0.5 pg/pl in oocyte injection buffer (88 mM NaCl; 1 mM KCl; 15 mM Tris-HCl, pH 7.6). RNA samples were stored at -80°C until injection into X Lewis oocytes. Translation assay. Small segments of ovary were removed from X .!een+ and individual mature oocytes were separated from ovarian tissue and immature oocytes. These oocytes were placed in modified Barth’s medium (20) and either used immediately or stored at room temperature overnight for use the following day. Poly(A)+ mRNA samples at a concentration of 0.2-0.5 pg/pl were injected into oocytes at a volume of approximately 100 nl/oocyte (20-50 ng RNA/oocyte) with no regard to the animal or vegetal pole. Following injection, oocytes were incubated overnight at room temperature at a ratio of 100 ~1 of modified Barth’s medium per 10 oocytes. Usually an additional small volume of Barth’s medium was used to rinse the incubation wells and the ooeytes were homogenized to collect all of the oocyte product. The pooled medium and homogenate was then centrifuged 5 min at 12,000g in an Eppendorf centrifuge to remove the oocyte debris. The entire supernatant
MOUSE
INTERFERON
mRNA TRANSLATION
PRODUCT
275
Production of large quantities of oocytetranslated product. A total of 11 different mouse interferon mRNA preparations were used to produce the three pools (A, B, and C) of oocyte-translated products. Sets of oocytes including from 40 to 1106 oocytes per set were injected (it was usually possible to inject 500 oocytes within 6 h) and processed to yield different lots of oocyte homogenate. These homogenates were stored at -80°C and were then pooled RESULTS prior to its analysis. Three separate pools Stability of the oocyte-translated product of oocyte translated material were produced that had a total volume of about 240 of interferon mRNA. Prior to producing large quantities of interferon by the trans- ml. This material ranged in titer from 400 lation of interferon mRNA in oocytes, the to 3000 reference units/ml. Partial pur@kation and characterization stability of this oocyte-translated product was determined. Although the crude of oocyte-translated product. The oocytetranslated oocyte product was stable in- translated product was analyzed on several column matrices to characterize its definitely at -80°C repeated freeze/thaw chromatographic behavior in relation to cycles gradually decreased its activity. Samples were also stable for several weeks crude native mouse interferon, as well as when stored at 4°C and in addition main- to partially purify it prior to analysis by tained their activity for at least 24 h at SDS-gel electrophoresis. Chromatograroom temperature. Small volumes of the phy on either anti-mouse interferon anoocyte-translated product were also ad- tibody columns, poly(U)-Sepharose, or justed to final concentrations of 5 X 10e5 Affi-Gel 202 columns did not distinguish M P-mercaptoethanol, 5 M urea, 0.1% SDS, the oocyte-translated product from native or 1% SDS in addition to boiling for 2 min mouse interferon. In each case, the oocytetranslated product resulted in a profile to determine its stability to the conditions quite similar to the profile of native mouse for gel electrophoresis. Treatment with ,&mercaptoethanol had no effect on the interferon. Thus, as can be seen in Fig. 1, interferon activity, but treatment with 5 greater than 99% of both native mouse M urea always drastically decreased the interferon (A) and the oocyte-translation activity of the oocyte-translated product. product of mouse interferon mRNA (B) The crude oocyte-translated material was bound to anti-mouse interferon antibody as stable to boiling in the presence of 0.1% columns. The purification of both of these mouse interferons by anti-mouse interSDS as to treatment with 1% SDS without boiling. However, this was not true for feron antibody columns was substantial, partially purified oocyte translated prod- and there was a recovery of 100% of the ucts; it was quite unstable to boiling for applied activities. However, these columns 1 min even in the presence of 0.1% SDS. were not routinely used, for they were difHowever, the native mouse IFN was stable ficult to regenerate. Even after elution with 4 M urea containing 1% ethanolin all the above-mentioned conditions. The crude oocyte-translated product was amine, there was still residual interferon stable to prolonged dialysis in the pres- activity retained by the columns that ence of the high salt concentrations and could only be removed by repeated pH low pH values used in the buffers for the shocks of pH 8 to pH 2. Even then, it was various columns and could also be dia- possible that enough interferon was relyzed extensively against 0.03 M NHIHCOS. tained by the columns to contaminate However, the activity of the partially pu- samples that were subsequently chrorified material was lost upon dialysis matographed. As a result, the CPG col(especially against 0.03 M NH4HC03). umns were chosen instead to partially pufluid including the floating lipid layer was removed and stored at -80°C. Interferon assay. Samples were diluted in serial threefold dilutions and assayed by a semimicrotitration method on L929 cells. Interferon titers were determined by the ability to inhibit the cytopathic effect of vesicular stomatitis virus. Each assay also included an interferon control standardized against NIH mouse interferon reference reagent (G002-902-026).
276
SARKAR, SUSSMAN-BERGER, AND PICKERING
Fraction
number
FIG. 1. Chromatography of native and oocyte-translated mouse interferon on anti-mouse interferon antibody affinity columns. Antibody columns were prepared as described earlier (21). Mouse interferon was induced in Lpa cells by NDV, the culture medium was harvested after 24 h and applied to an anti-mouse interferon antibody column (A). Over 99% of the applied interferon activity was retained on the column. The column was washed with 0.2 M sodium acetate buffer, pH 8, containing 0.5 M NaCl (E,), and bound material was eluted with 200 mM glycine-HCl, pH 2.2 (E,), followed by further elution with 4 M urea, pH 7.2, containing 1% ethanolamine (Ea). Crude oocyte homogenates containing translated mouse interferon activity were also fractionated in the same manner (B). Void volume material containing most of the proteins in the oocyte homogenates was eluted with EO,and bound material was eluted with El. In all cases, it was possible to recover all of the loaded oocyte-translated activity in a substantially purified form in the eluted fractions that had been bound to the column.
rify oocyte-translated material prior to SDS-gel electrophoresis. These beads were easily and completely regenerated by treatment with 15 M nitric acid. As can be seen in Fig. 2, CPG columns retained all of the interferon activity from both oocyte-translated samples (B) and native mouse interferon samples (A). These columns also yielded a 100% recovery of the applied activity. However, a slight difference in the chromatographic patterns of the native mouse interferon and the oocyte translated interferon was observed. Figure 3 shows the chromatographic behavior of the oocyte-translated product (A) and native mouse interferon (B) on poly(U)-Sepharose columns. In each case, there was only partial binding of either of these mouse interferons to the poly(U)Sepharose beads. The oocyte-translated product was analyzed several times on this column matrix, and in all cases, the maximum amount of activity bound to the col-
umn never exceeded 50-60% ; there was always a substantial recovery of activity in the void volume fractions. Originally, it was believed that Affi-Gel 202 column chromatography (Fig. 4) could distinguish between the oocyte-translated product of mouse interferon mRNA (A) and native mouse interferon (B). Thus, it appeared as though none of the oocytetranslated material was bound to this column matrix, whereas all of the native mouse interferon activity was retained. However, when uninjected oocytes were homogenized in crude native mouse interferon at the same ratio of oocytes to volume of medium as that used for preparing the oocyte-translated product, then the interferon in this preparation was not able to bind to Affi-Gel202 beads (C). It is unlikely that this inhibition of binding was due to proteolysis, since homogenized oocyte samples are stable for extended periods of time at 4”C, as well as being stable
MOUSE
INTERFERON
mRNA TRANSLATION
277
PRODUCT
Analysis of the oocyte-translated product of mOuSe interferon mRNA by SDS-gel electrophoresis (22). Preliminary attempts to characterize the oocyte-translated product resulted in the complete loss of activity of the samples. It was found sub-
64 c J I 2 32 4”
-1
1.04
35
Froct~on number
FIG. 2. Chromatography of native and oocytetranslated mouse interferon on controlled pore glass bead (CPG) columns. Samples were generally dialyzed against PBS prior to being applied to the CPG columns. The majority of the loaded protein was eluted with PBS. For both native and oocyte-translated interferon samples, all of the loaded activity was bound to the CPG beads and was eluted with 400 mM glycine-HCl, pH 2.0 (El). A represents the elution profile obtained for native mouse interferon (2 ml/ fraction) and B, for oocyte-translated mouse interferon (4 ml/fraction).
for at least 24 h at room temperature. Since the protein concentration of the oocyte homogenate is quite high compared to that of the native mouse interferon samples (prepared in medium containing only 2% FCS), it is quite possible that the above results could be due to the protein content of the different samples. Thus, in samples containing homogenized oocyte material, the high concentration of extraneous protein could shield the reactive side chains of the At&Gel 202 beads and thus prevent the interferon molecules from binding.
T .L % 0 I.02 $
) 01
Froctlon number
FIG. 3. Poly(U)-Sepharose column chromatography of native and oocyte-translated mouse interferon. Interferon samples were dialyzed against 10 mM Tris-HCl, pH 7.6, and then applied to the column. The column was washed with 10 mM Tris-HCl, pH 7.6, and the bound material was eluted with the same buffer containing 0.5 M NaCl (E,). For both the oocyte-translated product (A) and native mouse interferon (B), activity was recovered in the void volume fractions as well as the fractions eluted with 0.5 M NaCl. For the oocyte-translated product, 25% of the activity had bound to the column, whereas 91% of the activity from the native mouse interferon sample had bound. In all cases, only partial binding of either sample was observed, with at most 50% binding of the oocyte-translated activity.
SARKAR. SUSSMAN-BERGER, AND PICKERING
Froctlon number
FIG. 4. A&Gel 202 column chromatography of native and oocyte-translated mouse interferon and native mouse interferon mixed with oocyte homogenate. Samples were applied to the columns, and washed with 50 mM sodium acetate, pH 5.0. Bound material was then eluted with 20 mM sodium phosphate, pH 7.4 (E,), followed by further elution in 20 mM sodium phosphate, pH 7.4, buffer containing 0.5 M NaCl (Ea) and 1.0 M NaCl (E,). The elution profile for oocytetranslated mouse interferon is shown in A and for native mouse interferon, in B. C shows the elution profile of native mouse interferon that had been mixed with uninjected oocyte homogenates. Thus, oocytes were homogenized in medium containing crude mouse interferon at a ratio of 30 oocytes/400 pl medium. The homogenate was centrifuged at 12,000~for 10 min, incubated overnight at room temperature, and then applied to an Affi-Gel202 column. All fractions were 2 ml each.
sequently that the CPG-purified oocytetranslated products were usually sensitive to dialysis against 0.03 M NHIHCOB and were also unstable to boiling prior to elec-
trophoresis. To circumvent these difficulties, oocyte-translated products were dialyzed against PBS, lyophilized, and resuspended in water. They were then adjusted to contain 1% SDS as well as about 10% of a bromophenol blue-sucrose solution (60% sucrose; 0.2 M sodium phosphate, pH 7.4; 1% SDS; and 0.1% bromophenol blue). No urea was added either to the samples or to the electrophoresis buffers, and the samples were not boiled prior to electrophoresis. To ensure that these treatments were not affecting the migration behavior of the samples upon electrophoresis, native mouse interferon (Fig. 5) was either treated with 1% SDS without boiling (A) or with 1% SDS with boiling at 100°C for 1 min (B), or with 1% SDS and 5 M urea with boiling for 1 min at 100°C (C). As can be seen in this figure, there were no significant differences in the resulting electrophoretic profiles; each sample produced a heterogenous profile with bands of activity at about 38,000 (major) and 22,000 daltons (minor). Parallel gels were also run with human leukocyte samples that had been adjusted to contain 1% SDS with and without 5 M urea and with and without boiling. All of these samples also produced identical profiles upon electrophoresis (data not shown). Thus, samples treated only with 1% SDS migrated similarly upon electrophoresis as samples treated with 5 M urea and with boiling for 1 min at 100°C in addition to 1% SDS. Figure 6 shows the SDS-gel electrophoresis profile of the CPG-purified mouse interferon sample obtained by the translation in X laevis oocytes of mouse interferon mRNA. This sample migrated as a single band of activity with about 90% of the recovered activity present in a sharp band at 21-22,000 daltons. In addition, some activity was recovered in the range of 22-30,000 daltons which could represent a small degree of trailing in the gel or a heterogeneity due to unknown reason. It is possible that a small amount of activity was present in the 38,000-dalton range, but this activity was just barely at the limits of detection of the assay. Some of the oocyte-translated product was also boiled for
MOUSE
INTERFERON
mRNA TRANSLATION
A
.O-
.O-
.O-
..o -
I.0 z8.0
..O-
279
PRODUCT
the translated interferon activity characterized by chromatography on various column matrices. The oocyte-translated product of mouse interferon mRNA was retained completely on anti-mouse interferon antibody columns and on CPG columns and on poly(U)-Sepharose. Crude native mouse interferon behaved in a similar fashion when chromatographed on these column matrices. The only noticeable difference was observed in the chromatographic profile on CPG columns where the native mouse interferon bound to the columns was eluted as a double peak of activity, whereas the oocyte-translated interferon activity eluted as a single peak. It has been reported earlier that interferon activity in the homogenates of oocytes that had been injected with mouse interferon mRNA was retained on CPG columns (23). Thus, it was not possible to
t.0 04
05
06
07
0%
09
FIG. 5. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis of native mouse interferon. CPG-purified mouse interferon was made to contain 1% SDS and portions of this material were either left untreated (A), boiled 1 min at 100°C (B), or adjusted to contain 5 M urea in addition to the SDS and then boiled 1 min at 100°C (C). Following electrophoresis for 17-18 h at 7 mA/gel, gels were scanned at 280 nm and then sliced into 2.1-mm slices. Each slice was eluted overnight into 1 ml of growth medium containing 10% FCS and the eluted material was then assayed. Protein standards were run in a parallel gel and were: OA, ovalbumin (43,000 daltons); CA, carbonic anhydrase (30,000 daltons); SBTI, soybean trypsin inhibitor (20,100 daltons); and LA, /3-lactalbumin (14,400 daltons).
1 min at 100°C in the presence of 1% SDS and run on a parallel gel. As expected from the stability studies, no interferon activity was recovered from this gel, probably due to the boiling (not shown). DISCUSSION
Mouse interferon mRNA preparations were translated in X luevis oocytes, and
O-
&1.01
L 0.4
0.5
0.6 0.7 Rf ValUCs
, 0.6
0.9
FIG. 6. SDS-polyacrylamide gel electrophoresis of the oocyte-translated product of mouse interferon mRNA. Crude oocyte homogenates were partially purified by CPG-column chromatography. All of the loaded activity was recovered in the bound fractions, which were then pooled and dialyzed against PBS (10,000 total units). This material was then transferred to vials, shell-frozen with liquid nitrogen, and lyophilized. The dry powder was next resuspended in water and adjusted to contain 1% SDS (recovery of 5000 units). No urea was added and the sample was not boiled. Following electrophoresis, the gels were processed as described in Fig. 5. The hatched area in the figure represents the presence of minor levels of activity just at the limits of detection of the interferon assay. Protein standards were run in a parallel gel and are listed in Fig. 5.
280
SARKAR, SUSSMAN-BERGER, AND PICKERING
distinguish between native mouse interferon and the interferon produced in oocytes by the translation of mouse interferon mRNA on the basis of the antigenie and chromatographic properties. The chromatographic behavior of the oocytetranslated product on Affi-Gel 202 gave preliminary evidence of a distinction from native mouse interferon. However, mixing of native mouse interferon with uninjetted oocyte homogenates showed that this distinction was an artifact, which could be attributed to protein concentration effects and was not further investigated. Crude oocyte homogenates containing interferon activity were partially purified by CPG column chromatography and then analyzed by SDS-gel electrophoresis. The partially purified oocyte-translated product migrated as a single major band of activity. The majority of the activity was recovered in a sharp peak at 21-22,000 daltons, but there was additional minor activity in a rather broad range from 23 to 30,000 daltons. This additional activity could represent the trailing of activity along the gel or could arise from heterogeneity. It is interesting that the majority of the oocyte-translated product comigrated with the minor species of native mouse interferon, whereas there was little or no activity comigrating with the major 35- to 40,000-dalton species. It is possible that the larger molecular weight species was synthesized in the oocyte, but was selectively inactivated, leaving only the lower molecular weight species, although it is less likely since it is the lower molecular weight species of native mouse interferon that is more sensitive to electrophoresis or to SDS than the larger species (13, 24). Alternatively, it is also possible that the native A, B, and C species of mouse IFN described earlier (12,13) are similar in size and the differences in their electrophoretic mobility are largely due to post-translational modification (e.g., glycosylation). It is conceivable that in oocytes, the IFN mRNA translation product does not undergo such post-translational modifications giving rise to interferon species which comigrate with the low-molecular-
weight species of native mouse IFN. If so, the translation of mRNA in oocytes can be used to evaluate the possible significance of post-translational modification in the biological activity and stability of interferons. Recent reports suggest that there may be four different mRNA species for mouse interferons (25) and a total of five molecular species of NDV-induced L cell interferon (26). However, it is difficult to conclude such possibilities. The molecular cloning of the cDNAs corresponding to these interferon mRNA species should ultimately resolve these issues. ACKNOWLEDGMENTS We appreciate the excellent technical assistance of Julie Rosebaum, whose skill, patience, and expertise in injecting oocytes was greatly helpful in making this study possible. We are thankful to William E. Stewart, II, for his comments and suggestions during this study. We are also thankful to Dr. M. Krim for her constant support and encouragements. Phyllis Sussman-Berger was supported by USPHS PostDoctoral Fellowship 5F32-CA-06147 from the National Cancer Institute. REFERENCES 1. STEWART, W. E., II (1974) Viro& 2. KAWADE, Y. (1973) Japan
61,80-86. J. MicrobioL 17, 129-
140. 3. DEMAEYER-GUIGNARD,
J., TOVEY, M. G., GRESSER, I., AND DEMAEYER, E. (1978) Nature (Lendon) 271,622-625. IWAKURA, Y., YONEHARA, S., AND KAWADE, Y. (1978) J. Bid Ch.em. 253,5074-5079. STEWART, W. E., II, AND HAVELL, E. A. (1980) ViroZogg 101, 315-318. STEWART, W. E., II, LEGOFF, S., AND WIRANOWSKA-STEWART, M. (1977) J Gm Vim! 37, 277-284.
7. YAMAMOTO, Y. (1979) J. Gm
Viral
42,533-539.
8. YAMAMOTO, Y., AND KAWADE, Y. (1980) Virdogy 103,80-88.
9. STEWART, W. E., II, GRESSER, I., TOVEY, M. G., BANDU, M. T., AND LEGOFF. S. (1977) Nature (Londm)262,300-302.
10. IWAKURA, Y., YONEHARA, S., AND KAWADE, Y. (1978) Bidwm.
Biophys.
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11. KAWAKITA, M., CABRER, B., TAIRA, H., REBELLO, M., SLA’ITERY, E., WEIDELI, H., AND LENGYEL, P. (1978) J. Bid Chem 253,598-602. 12. CABRER, B., TAIRA, H., BROEZE, R. J., KEMPE,
MOUSE
13.
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16. 17.
18.
INTERFERON
mRNA TRANSLATION
T. D., WILLIAMS, K., SLATTERY, E., KONIGSBERG, W. H., AND LENGYEL, P. (1979) J. BioL Ch. 254, 3681-3684. TAIRA, H., BROEZE, R. J., JAYARAM, B. M., LENGYEL, P., HUNKAPILLAR, M. W., AND HOOD, L. E. (1980) Science 207, 528-530. PICKERING, L. A., KRONENBERG, L. H., AND STEWART, W. E., II (1980) Proc. Nat. Acad. Sci. USA 77,5938-5942. CAVALIERI, R. L., HAVELL, E. A., VILCEK, J., AND PESTKA, S. (1977) Proc. Nat. Acad. Sk USA 74, 3287-3291. CAVALIERI, R. L., AND PESTKA, S. (1977) Texa Rep. BioL Med. 35, 117-125. REYNOLDS, F. H., JR., PREMKUMAR, E., AND PITHA, P. M. (1975) Proc. Nat. Acd Sci. USA 72, 4881-4885. WEISSENBACH, J., ZEEVI, M., LANDAU, T., AND REVEL, M. (1979) Eur. J. B&hem. 98, l-8.
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19. PALMITER, R. D. (1974) Biochemistrg 13, 36063615. 20. GURDON, J. B. (1968) J. EmbryoL Exp. MorphoL 20, 401-414. 21. STEWART, W. E., II, SARKAR, F. H., TAIRA, H., HALL, A., NAGATA, S., AND WEISSMANN, C. (1980) Gene 11,181-186. 22. STEWART, W. E., II, AND DESMYTER, J. (1975) Virology 67, 68-73. 23. LEBLEU, B., HUBERT, E., CONTENT, J., DE WIT, L., BRAUDE, I. A., AND DE CLERCQ, E. (1978) Bio&em. Biophys. Res. Commun 82, 665-673. 24. YONEHARA, S., IWAKURA, Y., AND KAWADE, Y. (1980) virology 100.125-129. 25. SAGAR, A. D., PICKERING, L. A., SUSSMAN-BERGER, P., STEWART, W. E., II, AND SEHGAL, P. B. (1981) NucL Acids Res. 9,149-160. 26. ERICKSON, J. S., AND PAUCKER, K. (1979) J. Gen ViroL 43, 521-529.
OF BIOCHEMISTRY
Vol. 217, No. 1, August,
AND
BIOPHYSICS
pp. 273-281, 1982
Mouse Interferon
Messenger RNA: Characterization laevis Oocyte Translation Product
of the Xenopus
FAZLUL H. SARKAR,l PHYLLIS SUSSMAN-BERGER, LULU A. PICKERING2
AND
Interferon Laboratories, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021 Received December
9, 1981, and in revised form April
15, 1982
Mouse interferon messenger RNA was isolated from Newcastle disease virus-induced mouse Lpa cells and then translated in Xenopus laevis oocytes. The resulting oocyte homogenate containing translated interferon activity was unstable to treatment with 5 M urea and to repeated freeze/thaw cycles, and it was 1% cross-reactive on human cells, as was native mouse interferon. Both native mouse interferon and the mouse interferon produced by the translation of mouse interferon mRNA behaved almost similarly on CPG, poly(U)-Sepharose, and anti-mouse interferon antibody columns. When the oocyte-translated product was partially purified and analyzed on sodium dodecyl sulfate-polyacrylamide gels, it migrated as a major single band of activity at 21-22,000 daltons with a trailing edge at 22-30,000 daltons. Only minor activity was detected in the region of 35-40,000 daltons where the vast majority of the native mouse interferon migrated. Thus, the oocyte-translated mouse interferon product comigrated largely with the minor species of native mouse interferon with a little activity which corresponds with the larger molecular weight species of native mouse interferon.
It has long been recognized that mouse interferon consists of at least two molecular species with size ranges of about 35,000 to 40,000 and 22,000 to 28,000 daltons (l-4). These molecular species differ in their cross-species activity (5, 6) and their affinity for binding to lectins (7) and also appear to be antigenically distinct (8). They possess both antiviral and anticellular activities (3, 9) and their tryptic digests are similar (10). In addition, Kawakita et al. (11) have described three species of mouse interferon produced from NDV3-induced mouse Ehrlich ascites tu’ To whom correspondence should be addressed. * Present address: Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Mass. 02139. 3 Abbreviations used: NDV, Newcastle disease virus; SDS, sodium dodecyl sulfate; CPG, controlled pore glass; NP-40, Nonidet P-40; PBS, phosphate-
mor cells that have similar amino acid compositions (12). Although two of these species, A and B, have similar tryptic digests (12) and amino-terminal amino acid sequences (13), the third species, C, differs in both these respects. In addition, with respect to the first 20 amino acid residues in the NH,-terminal region, it appears as though mouse interferon species A and B are homologous to human fibroblast-derived p-interferon whereas mouse interferon species C is homologous to human lymphoblastoid-derived a-interferon (13). It is of interest that a low-molecularweight subspecies of mouse interferon is cross-reactive on human cells (5). There buffered saline; FCS, fetal calf serum; EDTA, ethylenediaminetetraacetate; MEM, minimum essential medium; IFN, interferon; Temed, N,N,N,N’,-tetramethylethylenediamine.
-.-
27.1
0003-9861/82/090273-09$02.00/O Copyright All rights
Q 1982 by Academic Press. Inc. of repmduction in any form reserved.
274
SARKAR,
SUSSMAN-BERGER,
also appears to be a species of human lymphoblastoid-derived a-interferon (spontaneous interferon) that is antigenically similar to mouse interferon (14). Cavalieri et al. (15, 16) demonstrated that human leukocyte-derived cY-interferons and fibroblast-derived @-interferons are products of distinct cellular genes. The translation products of human lymphoblastoid-derived a-interferon mRNA (15, 16) and of human fibroblast-derived B-interferon mRNA translated in Xenopue Laevis oocytes (15-18) have been analyzed by SDSpolyacrylamide gel electrophoresis, but the Xenopus oocyte-translated mouse interferon mRNA(s) product(s) has not been studied by this approach. In the past, the analysis of Xenopus oocyte-translated interferon mRNAs product(s) has been hampered by the low levels of interferon produced in the translated products. In view of this, the purpose of the present study was to produce large quantities of the Xenopus-translated product of mouse interferon mRNA for characterization studies to determine whether it contained all of the molecular species of interferon characteristic of native mouse interferon. MATERIALS
AND METHODS
viruses. Newcastle disease virus (NDV) was propagated in 11-day-old chick embryos. Virus was harvested as allantoic fluid, clarified by centrifugation for 20 min at 3OOOg,and stored in 5- to lo-ml aliquots at -80°C. Virus stocks were titered by plaque assay on secondary chick embryo cell monolayers and contained about 10 plaque-forming units (pfu)/ml. CeUa L929 and Lpa cells (1) were propagated as monolayers in growth medium containing MEM, 10% FCS, 2 mM glutamine, 100 rg/ml streptomycin, and 100 units/ml penicillin. Chemicala SDS (electrophoretic purity), Temed, ammonium persulfate, and Affi-Gel 202 were from Bio-Rad; poly(U)-Sepharose and protein standards (electrophoretic calibration kit) from Pharmacia; controlled-pore glass beads (CPG 350 mesh, size 120/ 200) from Accurate Chemicals and Science Corporation; and mouse L cell interferon antiserum was obtained from the National Institutes of Allergy and Infectious Diseases (Lot G-024-501-563). All other chemicals were of highest purity grade. Production of native mawe interferon Monolayers of L929 or Lpa cells in e-liter roller bottles were
AND PICKERING
primed for 2 h with 100-290 units/ml of mouse L cell interferon and then induced with NDV at about 5 pfu/cell. Following an overnight incubation at 3’7”C, the culture medium was collected and centrifuged at 20009 for lo-15 min to remove the cellular debris. The supernatant fluid containing crude interferon in growth medium supplemented with 2% FCS was adjusted to pH 2.0 to inactivate any residual virus and then readjusted to neutral pH prior to interferon assay. mRNA preparation Lpa cells were grown almost to confluency in 2-liter roller bottles (approximately 10s cells/roller bottle) and lo-30 roller bottles were used for each RNA preparation. Cell monolayers were primed for 2 h with 100-200 units/ml of mouse L cell interferon and then induced with about 5 pfu NDV/cell. At 11-15 h postinduction, cell monolayers were washed once with PBS and then scraped off the bottles with rubber policemen. Cells were washed 3 times with cold PBS and then lysed in 10 vol of hypotonic lysis buffer (10 mM Tris-HCl, pH 7.6; 10 mM NaCl; 1.5 mM MgClz) containing 0.5% NP-40. Following gentle lysis for 5 min at 4’C, cell nuclei were removed by centrifugation at 1OOOgfor 10 min. The supernatant fluid was then extracted 3-4 times using the SDS/phenol/chloroform procedure described by Palmiter (19) and the resulting aqueous phase was precipitated twice with 2 vol of 95% ethanol at -20°C. The precipitated RNA (total cytoplasmic RNA) was adjusted to 1 mg/ml (assuming 40 pg/ml of RNA per ODzso unit) and fractionated by two cycles of oligo(dT)-cellulose (type 3, Collaborative Res., Inc.) chromatography. The poly(A)+ mRNA fraction was then precipitated l-2 times with 2 vol of 95% ethanol at -20°C and resuspended at a concentration of 0.2 to 0.5 pg/pl in oocyte injection buffer (88 mM NaCl; 1 mM KCl; 15 mM Tris-HCl, pH 7.6). RNA samples were stored at -80°C until injection into X Lewis oocytes. Translation assay. Small segments of ovary were removed from X .!een+ and individual mature oocytes were separated from ovarian tissue and immature oocytes. These oocytes were placed in modified Barth’s medium (20) and either used immediately or stored at room temperature overnight for use the following day. Poly(A)+ mRNA samples at a concentration of 0.2-0.5 pg/pl were injected into oocytes at a volume of approximately 100 nl/oocyte (20-50 ng RNA/oocyte) with no regard to the animal or vegetal pole. Following injection, oocytes were incubated overnight at room temperature at a ratio of 100 ~1 of modified Barth’s medium per 10 oocytes. Usually an additional small volume of Barth’s medium was used to rinse the incubation wells and the ooeytes were homogenized to collect all of the oocyte product. The pooled medium and homogenate was then centrifuged 5 min at 12,000g in an Eppendorf centrifuge to remove the oocyte debris. The entire supernatant
MOUSE
INTERFERON
mRNA TRANSLATION
PRODUCT
275
Production of large quantities of oocytetranslated product. A total of 11 different mouse interferon mRNA preparations were used to produce the three pools (A, B, and C) of oocyte-translated products. Sets of oocytes including from 40 to 1106 oocytes per set were injected (it was usually possible to inject 500 oocytes within 6 h) and processed to yield different lots of oocyte homogenate. These homogenates were stored at -80°C and were then pooled RESULTS prior to its analysis. Three separate pools Stability of the oocyte-translated product of oocyte translated material were produced that had a total volume of about 240 of interferon mRNA. Prior to producing large quantities of interferon by the trans- ml. This material ranged in titer from 400 lation of interferon mRNA in oocytes, the to 3000 reference units/ml. Partial pur@kation and characterization stability of this oocyte-translated product was determined. Although the crude of oocyte-translated product. The oocytetranslated oocyte product was stable in- translated product was analyzed on several column matrices to characterize its definitely at -80°C repeated freeze/thaw chromatographic behavior in relation to cycles gradually decreased its activity. Samples were also stable for several weeks crude native mouse interferon, as well as when stored at 4°C and in addition main- to partially purify it prior to analysis by tained their activity for at least 24 h at SDS-gel electrophoresis. Chromatograroom temperature. Small volumes of the phy on either anti-mouse interferon anoocyte-translated product were also ad- tibody columns, poly(U)-Sepharose, or justed to final concentrations of 5 X 10e5 Affi-Gel 202 columns did not distinguish M P-mercaptoethanol, 5 M urea, 0.1% SDS, the oocyte-translated product from native or 1% SDS in addition to boiling for 2 min mouse interferon. In each case, the oocytetranslated product resulted in a profile to determine its stability to the conditions quite similar to the profile of native mouse for gel electrophoresis. Treatment with ,&mercaptoethanol had no effect on the interferon. Thus, as can be seen in Fig. 1, interferon activity, but treatment with 5 greater than 99% of both native mouse M urea always drastically decreased the interferon (A) and the oocyte-translation activity of the oocyte-translated product. product of mouse interferon mRNA (B) The crude oocyte-translated material was bound to anti-mouse interferon antibody as stable to boiling in the presence of 0.1% columns. The purification of both of these mouse interferons by anti-mouse interSDS as to treatment with 1% SDS without boiling. However, this was not true for feron antibody columns was substantial, partially purified oocyte translated prod- and there was a recovery of 100% of the ucts; it was quite unstable to boiling for applied activities. However, these columns 1 min even in the presence of 0.1% SDS. were not routinely used, for they were difHowever, the native mouse IFN was stable ficult to regenerate. Even after elution with 4 M urea containing 1% ethanolin all the above-mentioned conditions. The crude oocyte-translated product was amine, there was still residual interferon stable to prolonged dialysis in the pres- activity retained by the columns that ence of the high salt concentrations and could only be removed by repeated pH low pH values used in the buffers for the shocks of pH 8 to pH 2. Even then, it was various columns and could also be dia- possible that enough interferon was relyzed extensively against 0.03 M NHIHCOS. tained by the columns to contaminate However, the activity of the partially pu- samples that were subsequently chrorified material was lost upon dialysis matographed. As a result, the CPG col(especially against 0.03 M NH4HC03). umns were chosen instead to partially pufluid including the floating lipid layer was removed and stored at -80°C. Interferon assay. Samples were diluted in serial threefold dilutions and assayed by a semimicrotitration method on L929 cells. Interferon titers were determined by the ability to inhibit the cytopathic effect of vesicular stomatitis virus. Each assay also included an interferon control standardized against NIH mouse interferon reference reagent (G002-902-026).
276
SARKAR, SUSSMAN-BERGER, AND PICKERING
Fraction
number
FIG. 1. Chromatography of native and oocyte-translated mouse interferon on anti-mouse interferon antibody affinity columns. Antibody columns were prepared as described earlier (21). Mouse interferon was induced in Lpa cells by NDV, the culture medium was harvested after 24 h and applied to an anti-mouse interferon antibody column (A). Over 99% of the applied interferon activity was retained on the column. The column was washed with 0.2 M sodium acetate buffer, pH 8, containing 0.5 M NaCl (E,), and bound material was eluted with 200 mM glycine-HCl, pH 2.2 (E,), followed by further elution with 4 M urea, pH 7.2, containing 1% ethanolamine (Ea). Crude oocyte homogenates containing translated mouse interferon activity were also fractionated in the same manner (B). Void volume material containing most of the proteins in the oocyte homogenates was eluted with EO,and bound material was eluted with El. In all cases, it was possible to recover all of the loaded oocyte-translated activity in a substantially purified form in the eluted fractions that had been bound to the column.
rify oocyte-translated material prior to SDS-gel electrophoresis. These beads were easily and completely regenerated by treatment with 15 M nitric acid. As can be seen in Fig. 2, CPG columns retained all of the interferon activity from both oocyte-translated samples (B) and native mouse interferon samples (A). These columns also yielded a 100% recovery of the applied activity. However, a slight difference in the chromatographic patterns of the native mouse interferon and the oocyte translated interferon was observed. Figure 3 shows the chromatographic behavior of the oocyte-translated product (A) and native mouse interferon (B) on poly(U)-Sepharose columns. In each case, there was only partial binding of either of these mouse interferons to the poly(U)Sepharose beads. The oocyte-translated product was analyzed several times on this column matrix, and in all cases, the maximum amount of activity bound to the col-
umn never exceeded 50-60% ; there was always a substantial recovery of activity in the void volume fractions. Originally, it was believed that Affi-Gel 202 column chromatography (Fig. 4) could distinguish between the oocyte-translated product of mouse interferon mRNA (A) and native mouse interferon (B). Thus, it appeared as though none of the oocytetranslated material was bound to this column matrix, whereas all of the native mouse interferon activity was retained. However, when uninjected oocytes were homogenized in crude native mouse interferon at the same ratio of oocytes to volume of medium as that used for preparing the oocyte-translated product, then the interferon in this preparation was not able to bind to Affi-Gel202 beads (C). It is unlikely that this inhibition of binding was due to proteolysis, since homogenized oocyte samples are stable for extended periods of time at 4”C, as well as being stable
MOUSE
INTERFERON
mRNA TRANSLATION
277
PRODUCT
Analysis of the oocyte-translated product of mOuSe interferon mRNA by SDS-gel electrophoresis (22). Preliminary attempts to characterize the oocyte-translated product resulted in the complete loss of activity of the samples. It was found sub-
64 c J I 2 32 4”
-1
1.04
35
Froct~on number
FIG. 2. Chromatography of native and oocytetranslated mouse interferon on controlled pore glass bead (CPG) columns. Samples were generally dialyzed against PBS prior to being applied to the CPG columns. The majority of the loaded protein was eluted with PBS. For both native and oocyte-translated interferon samples, all of the loaded activity was bound to the CPG beads and was eluted with 400 mM glycine-HCl, pH 2.0 (El). A represents the elution profile obtained for native mouse interferon (2 ml/ fraction) and B, for oocyte-translated mouse interferon (4 ml/fraction).
for at least 24 h at room temperature. Since the protein concentration of the oocyte homogenate is quite high compared to that of the native mouse interferon samples (prepared in medium containing only 2% FCS), it is quite possible that the above results could be due to the protein content of the different samples. Thus, in samples containing homogenized oocyte material, the high concentration of extraneous protein could shield the reactive side chains of the At&Gel 202 beads and thus prevent the interferon molecules from binding.
T .L % 0 I.02 $
) 01
Froctlon number
FIG. 3. Poly(U)-Sepharose column chromatography of native and oocyte-translated mouse interferon. Interferon samples were dialyzed against 10 mM Tris-HCl, pH 7.6, and then applied to the column. The column was washed with 10 mM Tris-HCl, pH 7.6, and the bound material was eluted with the same buffer containing 0.5 M NaCl (E,). For both the oocyte-translated product (A) and native mouse interferon (B), activity was recovered in the void volume fractions as well as the fractions eluted with 0.5 M NaCl. For the oocyte-translated product, 25% of the activity had bound to the column, whereas 91% of the activity from the native mouse interferon sample had bound. In all cases, only partial binding of either sample was observed, with at most 50% binding of the oocyte-translated activity.
SARKAR. SUSSMAN-BERGER, AND PICKERING
Froctlon number
FIG. 4. A&Gel 202 column chromatography of native and oocyte-translated mouse interferon and native mouse interferon mixed with oocyte homogenate. Samples were applied to the columns, and washed with 50 mM sodium acetate, pH 5.0. Bound material was then eluted with 20 mM sodium phosphate, pH 7.4 (E,), followed by further elution in 20 mM sodium phosphate, pH 7.4, buffer containing 0.5 M NaCl (Ea) and 1.0 M NaCl (E,). The elution profile for oocytetranslated mouse interferon is shown in A and for native mouse interferon, in B. C shows the elution profile of native mouse interferon that had been mixed with uninjected oocyte homogenates. Thus, oocytes were homogenized in medium containing crude mouse interferon at a ratio of 30 oocytes/400 pl medium. The homogenate was centrifuged at 12,000~for 10 min, incubated overnight at room temperature, and then applied to an Affi-Gel202 column. All fractions were 2 ml each.
sequently that the CPG-purified oocytetranslated products were usually sensitive to dialysis against 0.03 M NHIHCOB and were also unstable to boiling prior to elec-
trophoresis. To circumvent these difficulties, oocyte-translated products were dialyzed against PBS, lyophilized, and resuspended in water. They were then adjusted to contain 1% SDS as well as about 10% of a bromophenol blue-sucrose solution (60% sucrose; 0.2 M sodium phosphate, pH 7.4; 1% SDS; and 0.1% bromophenol blue). No urea was added either to the samples or to the electrophoresis buffers, and the samples were not boiled prior to electrophoresis. To ensure that these treatments were not affecting the migration behavior of the samples upon electrophoresis, native mouse interferon (Fig. 5) was either treated with 1% SDS without boiling (A) or with 1% SDS with boiling at 100°C for 1 min (B), or with 1% SDS and 5 M urea with boiling for 1 min at 100°C (C). As can be seen in this figure, there were no significant differences in the resulting electrophoretic profiles; each sample produced a heterogenous profile with bands of activity at about 38,000 (major) and 22,000 daltons (minor). Parallel gels were also run with human leukocyte samples that had been adjusted to contain 1% SDS with and without 5 M urea and with and without boiling. All of these samples also produced identical profiles upon electrophoresis (data not shown). Thus, samples treated only with 1% SDS migrated similarly upon electrophoresis as samples treated with 5 M urea and with boiling for 1 min at 100°C in addition to 1% SDS. Figure 6 shows the SDS-gel electrophoresis profile of the CPG-purified mouse interferon sample obtained by the translation in X laevis oocytes of mouse interferon mRNA. This sample migrated as a single band of activity with about 90% of the recovered activity present in a sharp band at 21-22,000 daltons. In addition, some activity was recovered in the range of 22-30,000 daltons which could represent a small degree of trailing in the gel or a heterogeneity due to unknown reason. It is possible that a small amount of activity was present in the 38,000-dalton range, but this activity was just barely at the limits of detection of the assay. Some of the oocyte-translated product was also boiled for
MOUSE
INTERFERON
mRNA TRANSLATION
A
.O-
.O-
.O-
..o -
I.0 z8.0
..O-
279
PRODUCT
the translated interferon activity characterized by chromatography on various column matrices. The oocyte-translated product of mouse interferon mRNA was retained completely on anti-mouse interferon antibody columns and on CPG columns and on poly(U)-Sepharose. Crude native mouse interferon behaved in a similar fashion when chromatographed on these column matrices. The only noticeable difference was observed in the chromatographic profile on CPG columns where the native mouse interferon bound to the columns was eluted as a double peak of activity, whereas the oocyte-translated interferon activity eluted as a single peak. It has been reported earlier that interferon activity in the homogenates of oocytes that had been injected with mouse interferon mRNA was retained on CPG columns (23). Thus, it was not possible to
t.0 04
05
06
07
0%
09
FIG. 5. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis of native mouse interferon. CPG-purified mouse interferon was made to contain 1% SDS and portions of this material were either left untreated (A), boiled 1 min at 100°C (B), or adjusted to contain 5 M urea in addition to the SDS and then boiled 1 min at 100°C (C). Following electrophoresis for 17-18 h at 7 mA/gel, gels were scanned at 280 nm and then sliced into 2.1-mm slices. Each slice was eluted overnight into 1 ml of growth medium containing 10% FCS and the eluted material was then assayed. Protein standards were run in a parallel gel and were: OA, ovalbumin (43,000 daltons); CA, carbonic anhydrase (30,000 daltons); SBTI, soybean trypsin inhibitor (20,100 daltons); and LA, /3-lactalbumin (14,400 daltons).
1 min at 100°C in the presence of 1% SDS and run on a parallel gel. As expected from the stability studies, no interferon activity was recovered from this gel, probably due to the boiling (not shown). DISCUSSION
Mouse interferon mRNA preparations were translated in X luevis oocytes, and
O-
&1.01
L 0.4
0.5
0.6 0.7 Rf ValUCs
, 0.6
0.9
FIG. 6. SDS-polyacrylamide gel electrophoresis of the oocyte-translated product of mouse interferon mRNA. Crude oocyte homogenates were partially purified by CPG-column chromatography. All of the loaded activity was recovered in the bound fractions, which were then pooled and dialyzed against PBS (10,000 total units). This material was then transferred to vials, shell-frozen with liquid nitrogen, and lyophilized. The dry powder was next resuspended in water and adjusted to contain 1% SDS (recovery of 5000 units). No urea was added and the sample was not boiled. Following electrophoresis, the gels were processed as described in Fig. 5. The hatched area in the figure represents the presence of minor levels of activity just at the limits of detection of the interferon assay. Protein standards were run in a parallel gel and are listed in Fig. 5.
280
SARKAR, SUSSMAN-BERGER, AND PICKERING
distinguish between native mouse interferon and the interferon produced in oocytes by the translation of mouse interferon mRNA on the basis of the antigenie and chromatographic properties. The chromatographic behavior of the oocytetranslated product on Affi-Gel 202 gave preliminary evidence of a distinction from native mouse interferon. However, mixing of native mouse interferon with uninjetted oocyte homogenates showed that this distinction was an artifact, which could be attributed to protein concentration effects and was not further investigated. Crude oocyte homogenates containing interferon activity were partially purified by CPG column chromatography and then analyzed by SDS-gel electrophoresis. The partially purified oocyte-translated product migrated as a single major band of activity. The majority of the activity was recovered in a sharp peak at 21-22,000 daltons, but there was additional minor activity in a rather broad range from 23 to 30,000 daltons. This additional activity could represent the trailing of activity along the gel or could arise from heterogeneity. It is interesting that the majority of the oocyte-translated product comigrated with the minor species of native mouse interferon, whereas there was little or no activity comigrating with the major 35- to 40,000-dalton species. It is possible that the larger molecular weight species was synthesized in the oocyte, but was selectively inactivated, leaving only the lower molecular weight species, although it is less likely since it is the lower molecular weight species of native mouse interferon that is more sensitive to electrophoresis or to SDS than the larger species (13, 24). Alternatively, it is also possible that the native A, B, and C species of mouse IFN described earlier (12,13) are similar in size and the differences in their electrophoretic mobility are largely due to post-translational modification (e.g., glycosylation). It is conceivable that in oocytes, the IFN mRNA translation product does not undergo such post-translational modifications giving rise to interferon species which comigrate with the low-molecular-
weight species of native mouse IFN. If so, the translation of mRNA in oocytes can be used to evaluate the possible significance of post-translational modification in the biological activity and stability of interferons. Recent reports suggest that there may be four different mRNA species for mouse interferons (25) and a total of five molecular species of NDV-induced L cell interferon (26). However, it is difficult to conclude such possibilities. The molecular cloning of the cDNAs corresponding to these interferon mRNA species should ultimately resolve these issues. ACKNOWLEDGMENTS We appreciate the excellent technical assistance of Julie Rosebaum, whose skill, patience, and expertise in injecting oocytes was greatly helpful in making this study possible. We are thankful to William E. Stewart, II, for his comments and suggestions during this study. We are also thankful to Dr. M. Krim for her constant support and encouragements. Phyllis Sussman-Berger was supported by USPHS PostDoctoral Fellowship 5F32-CA-06147 from the National Cancer Institute. REFERENCES 1. STEWART, W. E., II (1974) Viro& 2. KAWADE, Y. (1973) Japan
61,80-86. J. MicrobioL 17, 129-
140. 3. DEMAEYER-GUIGNARD,
J., TOVEY, M. G., GRESSER, I., AND DEMAEYER, E. (1978) Nature (Lendon) 271,622-625. IWAKURA, Y., YONEHARA, S., AND KAWADE, Y. (1978) J. Bid Ch.em. 253,5074-5079. STEWART, W. E., II, AND HAVELL, E. A. (1980) ViroZogg 101, 315-318. STEWART, W. E., II, LEGOFF, S., AND WIRANOWSKA-STEWART, M. (1977) J Gm Vim! 37, 277-284.
7. YAMAMOTO, Y. (1979) J. Gm
Viral
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8. YAMAMOTO, Y., AND KAWADE, Y. (1980) Virdogy 103,80-88.
9. STEWART, W. E., II, GRESSER, I., TOVEY, M. G., BANDU, M. T., AND LEGOFF. S. (1977) Nature (Londm)262,300-302.
10. IWAKURA, Y., YONEHARA, S., AND KAWADE, Y. (1978) Bidwm.
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11. KAWAKITA, M., CABRER, B., TAIRA, H., REBELLO, M., SLA’ITERY, E., WEIDELI, H., AND LENGYEL, P. (1978) J. Bid Chem 253,598-602. 12. CABRER, B., TAIRA, H., BROEZE, R. J., KEMPE,
MOUSE
13.
14.
15.
16. 17.
18.
INTERFERON
mRNA TRANSLATION
T. D., WILLIAMS, K., SLATTERY, E., KONIGSBERG, W. H., AND LENGYEL, P. (1979) J. BioL Ch. 254, 3681-3684. TAIRA, H., BROEZE, R. J., JAYARAM, B. M., LENGYEL, P., HUNKAPILLAR, M. W., AND HOOD, L. E. (1980) Science 207, 528-530. PICKERING, L. A., KRONENBERG, L. H., AND STEWART, W. E., II (1980) Proc. Nat. Acad. Sci. USA 77,5938-5942. CAVALIERI, R. L., HAVELL, E. A., VILCEK, J., AND PESTKA, S. (1977) Proc. Nat. Acad. Sk USA 74, 3287-3291. CAVALIERI, R. L., AND PESTKA, S. (1977) Texa Rep. BioL Med. 35, 117-125. REYNOLDS, F. H., JR., PREMKUMAR, E., AND PITHA, P. M. (1975) Proc. Nat. Acd Sci. USA 72, 4881-4885. WEISSENBACH, J., ZEEVI, M., LANDAU, T., AND REVEL, M. (1979) Eur. J. B&hem. 98, l-8.
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19. PALMITER, R. D. (1974) Biochemistrg 13, 36063615. 20. GURDON, J. B. (1968) J. EmbryoL Exp. MorphoL 20, 401-414. 21. STEWART, W. E., II, SARKAR, F. H., TAIRA, H., HALL, A., NAGATA, S., AND WEISSMANN, C. (1980) Gene 11,181-186. 22. STEWART, W. E., II, AND DESMYTER, J. (1975) Virology 67, 68-73. 23. LEBLEU, B., HUBERT, E., CONTENT, J., DE WIT, L., BRAUDE, I. A., AND DE CLERCQ, E. (1978) Bio&em. Biophys. Res. Commun 82, 665-673. 24. YONEHARA, S., IWAKURA, Y., AND KAWADE, Y. (1980) virology 100.125-129. 25. SAGAR, A. D., PICKERING, L. A., SUSSMAN-BERGER, P., STEWART, W. E., II, AND SEHGAL, P. B. (1981) NucL Acids Res. 9,149-160. 26. ERICKSON, J. S., AND PAUCKER, K. (1979) J. Gen ViroL 43, 521-529.