Dec 19, 1986 - Raymond Kaempfer. Department of Molecular Virology, ... in the antiviral mechanisms it induces. (Rubin and Gupta, 1980; Weil et al., 1983).
The EMBO Journal vol.6 no.3 pp.585-589, 1987
Superinduction of the human
gene
encoding immune interferon
Mario A.Lebendiker, Chloe Tal, Dror Sayar, Shlomo Pilo, Amos Eilon', Yona Banai and Raymond Kaempfer Department of Molecular Virology, The Hebrew University-Hadassah Medical School, and 'Department of Otorhinolaryngology, Bikur Cholim Hospital, Jerusalem, Israel Communicated by R.Kaempfer
Mitogen-induced interferon-ey (IFN--y) gene expression was analyzed in human tonsil cells by titration of IFN-'y activity and by quantitation of 1FN-y mRNA. Expression of the IFN-'y gene can be superinduced extensively by two distinct methods: exposure to various inhibitors of translation, or to low doses of 'y-irradiation. -y-Irradiated cells produce, after exposure to cycloheximide, up to 12-fold greater amounts of IFN--y activity. Within as little as 4 h after the addition of translation inhibitors, IFN-'y mRNA levels rise 3- to 5-fold. Superinduction acts to increase the size of the wave of IFN--y mRNA. Primary transcription of the IFN--y gene does not increase in cells superinduced by cycloheximide, nor can superinduction be explained by stabilization of IFN--y mRNA sequences. These findings show that, during normal induction, a labile protein acts post-transcriptionally to repress the accumulation of mature IFN--y mRNA sequences. The superinductive effects of cycloheximide and -y-irradiation on levels of IFN-'y are additive, suggesting that they affect different aspects of IFN--y gene expression. Superinduction by -y-irradiation also has a post-transcriptional basis and is consistent with the possibility that expression of the IFN-'y gene is normally controlled by the action of suppressor T cells. Even though the genes for human IFN--y and for interleukin-2 are both superinducible, a striking difference in the regulation of expression of these lymphokine genes is observed. Superinduction of IFN-y mRNA is not due to superinduction of interleukin-2. Key words: immunoregulation/post-transcriptional control/labile regulatory protein/-y-irradiation/interleukin-2 gene Introduction Immune interferon, or interferon--y (IFN--y), is an inducible lymphokine produced by lymphocytes in response to mitogens (Wheelock, 1965) or antigens (Perussia et al., 1980; Green et al., 1969). IFN-'y possesses antiviral activity but differs from virus-induced interferons (IFN-ax and IFN-,B) in terms of its virus and cell specificities and in the antiviral mechanisms it induces (Rubin and Gupta, 1980; Weil et al., 1983). In addition, IFN--y possesses important immunoregulatory properties. Its antiproliferative effect on transformed cells is up to two orders of magnitude greater than that of IFN-ct or (Rubin and Gupta, 1980; Blalock et al., 1980), and IFN--y is more effective than the latter species in activating natural killer cells (Claeys et al., 1982). The antiviral (Fleischmann et al., 1979) and anti-tumor effects (Fleischmann et al., 1980) of IFN-oa and -(3 can be poten-3
IRL Press Limited, Oxford, England
tiated by IFN--y. Significant anti-tumor activity has been demonstrated for murine IFN-'y (Crane et al., 1978). IFN--y possesses strong anti-tumor cell activity in synergism with lymphotoxin (Lee et al., 1984). In spite of its biological importance, little is known about the regulation of expression of the single gene (Gray and Goeddel, 1982; Taya et al., 1982) that encodes human IFN--y. Here, we have examined the regulation of IFN--y gene expression in human lymphoid cells derived from tonsils. Our finding is that expression of the IFN-'y gene can be superinduced by two distinct methods: exposure to inhibitors of translation, or to low doses of 'y-irradiation, a treatment known to prevent activation of suppressor T cells. Our results demonstrate that expression of the IFN-y gene is normally regulated by a labile protein that acts post-transcriptionally to prevent accumulation of mature IFN--y mRNA sequences. Superinduction of IFN-'y by -y-irradiation also appears to have a post-transcriptional basis and is consistent with the possibility that expression of the IFN-'y gene is under control of suppressor T cells. Although the genes for IFN-'y and for another immunoregulator, T-cell growth factor or interleukin-2 (IL-2) (Efrat and Kaempfer, 1984; Efrat et al., 1984) are both superinducible, they appear to be regulated by distinct mechanisms. Results Superinduction of IFN-'y mRNA Figure 1 depicts the kinetics of accumulation of IFN-7y mRNA in human tonsil cells stimulated with phytohemagglutinin (PHA) (Efrat et al., 1982; Efrat and Kaempfer, 1984). Hybridization with a cloned IFN--y cDNA probe detects an RNA species migrating at 1250 nucleotides, the expected size of mature IFN'y mRNA (Devos et al., 1982; Gray et al., 1982). The amount of this RNA rises during induction, reaching a maximum by 12 h, and then declines (top lanes). This finding agrees with those reported earlier (Efrat et al., 1982; Vaquero et al., 1984). A wave of IFN--y mRNA is consistently observed in individual lymphocyte cultures, but the actual time at which maximal levels of mRNA are reached is somewhat variable (cf. Figures 3 and 4a). When cycloheximide (CHX), an effective inhibitor of translation (Efrt and Kaempfer, 1984), is added 3 h before RNA is extracted, significantly greater levels of IFN-'y mRNA are observed (bottom lanes). The increase in IFN--y mRNA averages 2.5-fold. Considering that CHX was present only during the last 3 h of culture, it is clear that this inhibitor leads to a rapid and extensive superinduction of IFN-'y mRNA sequences. Superinduction ofIFN--y mRNA involves neutralization of a labile protein The assumption that CHX causes superinduction by inhibiting protein synthesis was tested by comparing its effect with that of three other specific inhibitors of translation, each acting via a different mechanism (Efrat et al., 1984): T-2 toxin, pactamycin and sparsomycin. In conditions where these agents inhibited translation to an equivalent extent (Efrat et al., 1984), each yield-
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Fig. 1. Superinduction of IFN--y mRNA sequences by CHX. Human tonsil cells were induced with PHA and at the indicated times thereafter (in h), total RNA was extracted, subjected to formaldehyde/agarose gel electrophoresis, and blot-hybridized with 32P-labeled IFN--y cDNA (top). Alternatively, CHX was added to cell cultures 3 h before RNA was extracted at the times indicated (bottom). Size markers denote nucleotide length.
Fig. 2. Superinduction of IFN--y mRNA by four translation inhibitors. In (a), Northern blots of total RNA extracted at 14 h are shown as for Figure 1, except th4t inhibitors were present from 10 h as follows (Chirgwin et al., 1979): none (lane 1), CHX (lane 2), 20 ng/ml T-2 toxin (lane 3), 10 0.1 g/ml pactamycin (lane 4), l,g/ml sparsomycin (lane 5). The extent of superinduction in lanes 2-5, determined by microdensitometry, is 5-, 3-, 4.4- and 3.2-fold, respectively. In (b), Northern blots of total RNA extracted at 20 h are shown; hybridization was to IFN--y (top) or IL-2 cDNA (bottom). Inhibitors were present from 4 h as follows: none (lane 1), T-2 toxin (lane 2), pactamycin (lane 3), sparsomycin (lane 4); concentrations were as in (a).
ed a comparable superinduction of IFN-,y mRNA of 3- to 5-fold within 4 h (Figure 2a). Thus, during normal induction, the accumulation of IFN--y mRNA is apparently inhibited by a labile protein. Differential superinduction of human IFN-y and IL-2 genes We showed previously that the addition of inhibitors of translation at 4 h leads to extensive superinduction of mRNA for another lymphokine, IL-2 (Efrat and Kaempfer, 1984; Efrat et al., 1984). Figure 2b shows that IFN-'y and IL-2 gene expression are affected in a strikingly different manner by the addition of translation inhibitors at 4 h instead of at 10 h as in Figure 2a. IL-2 mRNA, barely detectable in the control culture (lane 1), is superinduced extensively (lanes 2-4). By contrast, the accumulation of IFN--y mRNA, quantitated separately in the same gel, is inhibited relative to the control. Thus, although IFN-'y and IL-2 genes can both be superinduced by inhibitors of translation, they 586
Fig. 3. Differential effect of CHX on levels of IFN--y and IL-2 mRNA. In (a), Northern blots of total RNA extracted at the times indicated (in h) are shown as for Figure 1, except that the 32P-labeled probe was a mixture of IFN--y and IL-2 cDNAs with the same specific radioactivity. Positions of IFN--y and IL-2 mRNAs are indicated; marker lane on left displays predominantly IL-2 mRNA. CHX was absent (top) or present from 4 h onwards (bottom). In (b), CHX was present from the times indicated (in h after PHA addition); the gel was hybridized separately to IFN--y and IL-2 cDNA probes.
respond distinctly, indicating differences in their regulation. In the experiment of Figure 3a, RNA was isolated from induced lymphocytes at time intervals and analyzed by hybridization analysis with a mixture of labeled IFN--y and IL-2 cDNA probes. IFN--y mRNA is resolved from the more rapidly migrating IL-2 mRNA, allowing direct comparison of their levels. Both mRNA species increase with similar kinetics, reaching a maximum around 16 h in this experiment (top lanes), but again, their amounts are affected in an opposite manner by the addition of CHX at 4 h (bottom lanes). IL-2 mRNA levels increase strongly relative to the controls, while, in contrast, IFN--y mRNA levels decrease significantly. While in the CHX-free control, IFN--y mRNA is the dominant species seen, IL-2 mRNA predominates in RNA from the culture that received CHX at 4 h. In the experiment of Figure 3b, CHX was added at 4 or 10 h and RNA was analyzed at 14 h after induction. Significant superinduction of IFN--y mRNA is observed if CHX is added at 10 h, while in contrast, superinduction of IL-2 mRNA is less pronounced than when CHX is added at 4 h. Superinduction of IFN--y mRNA by -y-irradiation Figure 4a depicts IFN-'y mRNA levels in normally induced human lymphocytes (bottom lanes) to those in cells of the same population that had been exposed to a low dose of 'y-irradiation before the addition of PHA (top lanes). -y-Irradiation causes a significant increase in IFN-'y mRNA. Figure 4b shows that superinduction of IFN--y mRNA by -y-irradiation (lanes 1 and 2) is prevented if CHX is introduced at 4 h after induction (lane 3). Hence, the effect of -y-irradiation on IFN--y mRNA levels is not based on a bypass of the requirement for protein synthesis early in induction. Superinduction of human IFN--y IFN--y activity in the medium of induced cultures of normal or 'y-irradiated cells is compared in Figure 5. Levels of IFN-7y produced by -y-irradiated cells exceeded those produced by normal cells throughout the induction period, up to 2.5-fold. To demonstrate superinduction by CHX of IFN-'y activity in cell culture medium, we measured IFN-'y levels in cultures that had been incubated for 22 h with PHA and then transferred to fresh medium lacking PHA. As in the case for IL-2 (Efrat and
Superinduction of the human y-interferon gene A
4
A-
_Q
A i-7!!-4
-0
C0
~~x
x
I-RAY
-
C- 2 z
Fig. 4. Superinduction of IFN--y mRNA by -y-irradiation. Northern blots of total RNA extracted at the times indicated (in h) and hybridized to IFN--y cDNA are shown in (a), for human tonsil cells induced with PHA directly (control) or after exposure to 1500 rads of -y-irradiation (-y-ray). The extent of superinduction is 3-fold or greater, as judged by densitometry. In (b), tonsil cell cultures were -y-irradiated as in (a), or received CHX at 4 h, as indicated; total RNA was extracted at 24 h.
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,
,
Y-IRRADIATED
0
CHX
Y-RAY
10-10 ++-
4410-10 -
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Fig. 6. Superinduction of IFN--y by CHX, -y-irradiation, or both. Human tonsil cells were induced with PHA directly or, where indicated, after exposure to 1500 rads of -y-irradiation. CHX was added at the indicated times (in h) after PHA addition. All cultures were transferred to PHA-free medium at 22 h. Medium was assayed for IFN--y activity by radioimmunoassay (a) or antiviral activity (b) and up to 120 h; average titers between 70 and 90 h are depicted. (a) and (b) represent different experiments.
cmJ O0 15-J
z z 5 Li-
0
100 50 TIME (H)
Fig. 5. Superinduction of IFN-'y by 'y-irradiation. Medium from cultures used to prepare RNA for Figure 4 as collected at the times indicated after addition of PHA, and IFN--y titers were determined by radioimmunoassay.
Kaempfer, 1984), IFN--y levels were not affected by the removal of PHA (control not shown). In Figure 6a, it is seen that the addition of CHX at 10 h and its removal, together with PHA, at 22 h, led to a doubling of IFN--y activity when assayed at -80 h; a similar difference was observed at other times. In another experiment (Figure 6b), a > 5-fold superinduction was evident when CHX was added at 10 h. By contrast, no superinduction of IFN--y occurred if CHX was present from 4 h onwards. IFN-'y levels in the medium rose commensurately in cultures that were 'y-irradiated and in ones that received CHX between
Fig. 7. Decay of IFN--y mRNA sequences in control and superinduced lymphocyte cultures. Aliquots of a human tonsil cell population were induced with PHA. Northern blots of total RNA extracted at the times indicated (in h) and hybridized to IFN--y cDNA are shown for cells cultured in the absence (control) or presence of CHX from 11 h (+CHX). Culture was in the absence (top lanes) or presence (bottom lanes) of DRB from 14.5 h onwards (+DRB). Lanes 1 and 2 present blots as above, but for cultures in a different experiment that received PHA alone or together with DRB, respectively; total RNA was extracted at 20 h.
10 and 22 h (Figure 6). However, the combination of both treatments increased the extent of superinduction of IFN-'y significantly, up to 5-fold (a) or 12-fold (b) over normal values. Apparently, the superinductive effects of CHX and -y-irradiation are additive, suggesting that they affect different aspects of regulation of IFN-y gene expression. IFN--y mRNA stability To study the possibilty that CHX might act to stabilize IFN-'y mRNA and thus cause superinduction, we examined IFN--y
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Primary transcription of the IFN--y gene is not altered perceptibly when IFN--y gene expression is superinduced by CHX or by Py-irradiation. Superinduction of the human IFN--y gene thus
Fig. 8. Quantitation of nascent IFN-,y transcripts during normal induction and superinduction. RNA was labeled with [a-32P]UTP in nuclei prepared from cells induced for 13 h, with CHX present where indicated, from 10 h (left) and from control or, where indicated, -y-irradiated cells (see Figure 4) induced for 24 h (right). The indicated amounts of labeled RNA (in c.p.m. x 106) were dot-hybridized with excess IFN-y or chicken ,B-tubulin (,3-tub) cDNA, as indicated. Film densitometry showed that no spot was overexposed. Control hybridizations to pBR322 plasmid DNA gave no detectable film blackening (not shown).
mRNA sequences in lymphocyte cultures in which synthesis of RNA was inhibited by 5,6-dichloro-1-,B-D-ribofuranoysl benzimidazole (DRB) (Efrat and Kaempfer, 1984). When DRB was added at the onset of induction, formation of IFN-y mRNA was not detected (Figure 7, lanes 1 and 2). The kinetic data of Figure 7 show IFN-7y mRNA levels in a normally induced culture and in one that received CHX at 11 h. Upon addition of DRB to both cultures at 14.5 h, IFN-'y mRNA sequences declined immediately and at approximately the same rate, whether or not CHX was present.
Superinduction is not accompanied by increased primary transcription In Figure 8, nuclear run-on transcription was quantitated in nuclei isolated from cells that were induced in the absence or presence of CHX, or after 7y-irradiation. The extent of hybridization of IFN-,y RNA molecules, labeled in nuclei from normally induced cells, increases linearly with input radioactivity. IFN--y RNA sequences labeled in nuclei from cells that were superinduced wtih CHX hybridze within this linear range. Their amount is not increased perceptibly over the corresponding control (0.2); an increase of 2-fold would have been detected easily (cf. control 0.4). This agrees with a recent report that the time course of transcription is unaffected by CHX (Kronke et al., 1985). Likewise, levels of IFN-'y RNA labeled in nuclei from 'yirradiated cells did not increase over the control (Figure 8). Quantitation of ,B-tubulin RNA sequences served as internal control and revealed no effect of either CHX treatment or -y-irradiation. Apparently, superinduction of the human IFN--y gene involves escape from a post-transcriptional control mechanism. Discussion The powerful immunoregulatory and antiviral properties of IFN-,y and its potential anti-tumor activity emphasize the importance of understanding how IFN--y production is controlled in man. In this study, we have examined the regulation of IFN--y gene expression in human lymphoid cells derived from tonsils. We show that expression of the IFN--y gene can be superinduced extensively by two distinct methods: exposure to inhibitors of translation, or to low doses of -y-irradiation. Four independently acting inhibitors of translation all yield a comparable superinduction of IFN-'y mRNA, supporting the concept that accumulation of IFN--y mnRNA is normally repressed by a labile protein. 588
appears to have a post-transcriptional basis. IFN-'y mRNA levels increase 3- to 5-fold within only 4 h after the addition of a translation inhibitor to induced lymphocytes (Figures 1, 2a and 7), attesting to a rapid and vigorous superinduction of the IFN--y gene. Yet, if protein synthesis is blocked early within the wave of IFN-'y mRNA, from 4 h onwards, accumulation of IFN--y mRNA is actually inhibited to well below normal values (Figures 2 and 3). This requirement for translation in the earlier phases of induction is probably not due to a lack of interleukin-1, for, in the case of IL-2 production, levels of this activating factor reach saturation by 3-4 h in induced tonsil cell cultures (Efrat and Kaempfer, 1984). Although IL-2 drives the proliferation of cells that produce IFN--y (Efrat and Kaempfer, 1984), our results clearly rule out the interpretation that superinduction of the IFN--y gene is due to superinduction of IL-2 protein, since superinduction of IFN-'y mRNA is observed in conditions (Efrat and Kaempfer, 1984) where IL-2 synthesis is inhibited. A wave of IFN--y mRNA is generated upon stimulation of lymphocytes (Figures 1, 3, 4 and 7). The shape of this wave is preserved when IFN-'y gene expression is superinduced by CHX (Figure 1) or by 'y-irradiation (Figure 4a). Hence, the regulatory mechanisms that determine the shape of the IFN--y mRNA wave are resistant to these treatments, while those that determine the amplitude of this wave are sensitive. Expression of the human IL-2 gene can also be superinduced extensively in the presence of translation inhibitors (Efrat and Kaempfer, 1984; Efrat et al., 1984). However, the results of Figures 2 and 3 reveal a striking difference in the regulation of expression of the human IFN-'y and IL-2 genes. The IL-2 gene can be strongly superinduced by CHX in conditions where IFN-y gene expression is actually inhibited and conversely, superinduction of IFN--y mRNA is maximal in conditions where superinduction of IL-2 mRNA is not. An effect of CHX on RNA pool size can be rejected as an explanation for the observed superinduction, because the decrease in RNA pool size is insignificant within 4 h (Efrat and Kaempfer, 1984), because levels of active IFN-'y in the culture medium rise considerably (Figure 6), and because of the distinct behavior of the IFN--y and IL-2 genes. To explain the observed superinduction of 3- to 5-fold within 3 or 4 h on the basis of IFN--y mRNA stabilization would demand a half-life of IFN--y mRNA of the order of 2.5 h, and an increase of at least 3- to 4-fold, to 2 9 h, in the presence of CHX. The analysis of Figure 7 shows an apparent half-life of -9 h in the presence of DRB, whether or not CHX is present. This experiment is sufficiently sensitive to detect either complete, or nearly complete, stabilization of IFN--y mRNA in the presence of CHX, or a 2.5-h half-life in the absence of CHX, but in fact neither is observed. Instead, the data show similar decline rates in the absence or presence of CHX, whether or not DRB is present. Thus, if stabilization of IFN-7y mRNA does occur in the presence of CHX, the extent of stabilization needed to explain the observed superinduction is not seen in Figure 7, yet it should have been detectable. Our findings thus support the concept that a labile protein acts post-transcriptionally to repress the formation of mature IFN--y mRNA sequences. Complete lymphocyte populations contain a subset of T cells that are capable of suppressing the response of other T cells to
Superinduction of the human -y-interferon gene
mitogen or antigen. In both human and murine systems, such suppressor T cells exhibit a pronounced radiosensitivity (Schwartz et al., 1983; Gullberg and Larsson, 1982). Apparently, the activation of suppressor T cells requires DNA synthesis (Mayumi et al., 1979). The IFN-y asays of Figures 5 and 6 show that cells that produce IFN-'y are, by contrast, relatively radioresistant, for they actually produce significantly higher levels of IFN-'y upon mild irradiation. These findings are consistent with the possibility that expression of the IFN--y gene is normally controlled by the action of suppressor T cells. The effect of -y-irradiation on IFN-'y mRNA levels is not based on a bypass of the requirement for translation beyond 4 h of induction (Figure 4b). The finding that the superinductive effects of CHX and -y-irradiation are additive (Figure 6) indicates that these treatments affect different aspects of the regulation of IFN-'y gene expression. Yet, both act beyond primary transcription. A tempting speculation is that activation of suppressor T cells may be required to elicit production of the labile protein that, in turn, acts post-transcriptionally to prevent formation of mature IFN-y mRNA sequences. Materials and methods Materials pBR327-yO DNA, containing a 950-bp insert of human IFN-'y cDNA (Devos et al., 1982) was provided by Dr W.Fiers; p3-16 DNA, containing a 650-bp insert of human IL-2 cDNA (Taniguchi et al., 1983) was provided by Dr T.Taniguchi; IFN--y radioimmunoassay kits were from Centocor, Inc.; [cs-32P]UTP (3000 Ci/mmol) and ca-labeled dNTPs were from Amersham; the source of other reagents was as reported (Efrat et al., 1982; Efrat and Kampfer, 1984). Culture of lymphocytes Human tonsillar lymphocytes were cultured, induced with PHA, and transferred to PHA-free medium, as detailed (Efrat et al., 1982; Efrat and Kaempfer, 1984). When added, concentrations of CHX and DRB were 20 14g/ml and 40 mM, respectively (Efrat and Kaempfer, 1984). Assay for IFN--y Radioimmunoassay was performed as directed by the supplier. Antiviral activity was assayed by measuring the cytopathic effect of mengovirus on human amnion cells (Efrat et al., 1982). Hybridization analysis of RNA RNA was extracted by the guanidinium thiocyanate-CsCl method (Chirgwin et al., 1979). 2.2 M formaldehyde/1.5 or 1.75% agarose gels were used to separate RNA for Nothem blots (Lehrach et al., 1977). Total RNA (50 itg/1ane) was heated for 10 min at 60°C before loading. RNA was transferred from gels to nitrocellulose paper (Thomas, 1980). Papers were prehybridized overnight at 43°C with 50% formamide, 0.75 M NaCl, 0.075 M Na-citrate, 0.02% of each bovine serum albumin, polyvinylpyrrolidone and Ficoll, 0.5% SDS and 250 jg/ml of heatdenatured salmon sperm DNA. Hybridization was for 48 h at 43°C with the same solution, containing, in addition, 2 x 107 c.p.m. 32p in nick-translated, heatdenatured plasmid DNA probe (sp. act. -6 x 108 c.p.m./pg). Autoradiographs were scanned with an integrating microdensitometer. Short exposures were also scanned to ensure that the film was not overexposed. Size markers were OX174 RF DNA HaeIH fragments (New England Biolabs). Elongation of nascent RNA chains in isolated nuclei Nuclei were prepared for in vitro transcription experiments as described (Schibler et al., 1983), except that 0.05% (v/v) of Triton X-100 was included in the cell lysis buffer. Nuclei were treated wtih pancreatic RNase and used for in vitro elongation reactions at -4 x 107 nuclei/ml. RNA was labeled by the incorporation of [a-32P]UTP. Labeled RNA was extracted and hybridized to filters carrying plasmid DNA.
References Blalock,J.E., Georgiades,J.A., Langford,M.P. and Johnson,H.M. (1980) Cell Immun., 49, 390-394. Chirgwin,J.M., Przybyla,A.E., MacDonald,R.J. and Rutter,W.J. (1979) Biochemistry, 18, 5294-5299. Claeys,H., Van Damme,J., De Ley,M., Vermylen,C. and Billiau,A. (1982) Br. J. Haematol., 50, 85-94. Crane,J.L.,Jr, Glasgow,L.A., Kern,E.R. and Youngner,J.S. (1978) J. Natl. Cancer Inst., 61, 871-874. Devos,R., Cheroutre,H., Taya,Y., Degrave,W., Van Heuverswyn,H. and Fiers,W. (1982) Nucleic Acids Res., 10, 2487-2501. Efrat,S. and Kaempfer,R. (1984) Proc. Natl. Acad. Sci. USA, 81, 2601-2605. Efrat,S., Pilo,S. and Kaempfer,R. (1982) Nature, 297, 236-239. Efrat,S., Zelig,S., Yagen,B. and Kaempfer,R. (1984) Biochem. Biophys. Res. Commun., 123, 842-848. Fleischmann,W.R.,Jr, Georgiades,J.A., Osbome,L.C. and Johnson,H.M. (1979) Infect. Immun., 26, 248-253. Fleischmann,W.R.,Jr., Kleyn,K.M. and Baron,S. (1980) J. Natl. Cancer Inst., 65, 963-966. Gray,P.W. and Goeddel,D.V. (1982) Nature, 298, 859-863. Gray,P.W., Leung,D.W., Pennica,D., Yelverton,E., Najarian,R., Simonsen,C.C., Derynck,R., Sherwood,P.J., Wallace,D.M., Berger,S.L., Levinson,A.D. and Goeddel,D.V. (1982) Nature, 295, 503-508. Green,J.H., Copperband,S.R. and Kibrick,S. (1969) Science, 164, 1415-1417. Gullberg,M. and Larsson,E.-L. (1982) J. Immunol., 128, 746-750. Kronke,M., Leonard,W.J., Depper,J.M. and Greene,W.C. (1985) J. Exp. Med., 161, 1593-1598. Lee,S.H,. Aggarwal,B.B., Rinderknecht,E, Assisi,F. and Chu,H. (1984) J. Immunol., 133, 1083-1086. Lehrach,H., Diamond,D., Wozney,J.M. and Boedtker,H. (1977) Biochemistry, 16, 4743-4751. Mayumi,M., Yoshida,T., Shinomiya,K., Nishikawa,S.I., Hirata,T., Lzumi,T. and Mikawa,H. (1979) J. Immunol., 123, 772-777. Perussia,B., Mangoni,L., Engers,H.D. and Trinchieri,G. (1980) J. Immunol., 125, 1589-1595. Rubin,B.Y. and Gupta,S.L. (1980) Proc. Natl. Acad. Sci. USA, 77, 5928-5932. Schibler,U., Hagenbuchle,O., Wellauer,P.K. and Pittet,A.C. (1983) Cell, 33, 501-508. Schwartz,J.L., Darr,J.C. and Gaulden,M.E. (1983) Mutat. Res., 107, 413-425. Taniguchi,T., Matsui,H., Fujita,T., Takaoka,C., Kashima,N., Yoshimoto,R. and Hamuro,J. (1983) Nature, 302, 305-310. Taya,Y., Devos,R., Tavernier,J., Cheroutre,H., Engler,G. and Fiers,W. (1982) EMBO J., 1, 953-958. Thomas,P. (1980) Proc. Natl. Acad. Sci. USA, 77, 5201-5205. Vaquero,C., Sanceau,J., Sondermeyer,P. and Falcoff,R. (1984) Nucleic Acids Res., 12, 2629-2640. Weil,J., Epstein,C.J., Epstein,L.B., Sedmak,J.J., Sabran,J.L. and Grossberg,S.E. (1983) Nature, 301, 437-439. Wheelock,E.F. (1965) Science, 149, 310-311.
Received on September 19, 1986; revised on December 19, 1986
Acknowledgements We thank Drs W.Fiers and R.Devos for pBR327-yO and Dr T.Taniguchi for p3-16 plasmid DNA, and Dr Shimon Efrat, Avivah Yeheskel, Rivkah Gonsky, Ayelet Reshef and Mali Ketzinel for their help. The research was supported by grants from the National Council for Research and Development of Israel, the Israel Academy of Sciences and the Cancer Research Institute, New York.
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