Nature Vol. 295 11 February 1982
`
`S03
`
`Expression of human immune interferon cDNA
`in E. coli and monkey cells
`Patrick W. Gray, David W. Leung, Diane Pennica, Elizabeth Yelverton,
`Richard Najarian, Christian C. Simonsen, Rik Derynck, Pamela J. Sherwood,
`Donald M. Wallace”, Shelby L. Berger*, Arthur D. Levinson & David V. Goeddel
`Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080, USA
`* Laboratory of Pathophysiology, National Cancer Institute, Bethesda, Maryland 20205, USA
`
`A cDNA sequence coding for human immune interferon (IFN-'y) has been identified in a cDNA library prepared from
`gel-fractionated IFN-y mRNA. The DNA sequence codes for a polypeptide of 166 amino acids, 20 of which could
`constitute a signal peptide. The polypeptide produced through expression of this DNA sequence in Escherichia coli or
`cultured monkey cells had properties characteristic of authentic human IFN-y.
`
`INTERFERONS (IFNs) are secreted proteins which induce an
`antiviral
`state
`in
`their
`target
`cells,
`and which have
`immunomodulatory and antitumour properties‘. On the basis of
`antigenicity and biological and chemical properties, human
`interferons have been grouped into three major classes: IFN-oz
`(leukocyte),
`IFN-B (fibroblast)
`and
`IFN-‘y
`(immune)2.
`Considerable information has accumulated on the structures
`and properties of the virus—induced acid—stable interferons
`(IFN—a and -3). These have been purified to homogeneity and
`partial amino acid sequences determined”. Analyses of cloned
`cDNA and gene sequences for IFN—B1 (refs 8-10) and the IFN-01
`multigene family“‘15 have permitted the deduction of the
`complete amino acid sequences of many of these interferons. In
`addition, efiicient synthesis of IFN-B1 (ref. 10) and several
`IFN-as‘3’16 in Escherichia coli and IFN-a1 in yeast” have now
`made possible the purification of large quantities of these pro-
`teins in biologically active form.
`Much less data are available concerning the structure and
`properties of IFN-y. IFN-y is generally produced in cultures of
`lymphocytes exposed to various mitogenic stimuli, is acid labile
`and does not cross-react with antisera prepared against IFN-an
`or IFN-B (ref. 1). Molecular weights ranging from 35,000 to
`70,000 have been reported for IFN-y (refs 18-21). Recently, a
`quite extensive, but still partial, purification of IFN-y has been
`reported“, and IFN—~y mRNA has been isolated and translated
`in Xenopus laevis oocytes”’“. A broad range of biological
`activities have been attributed to IFN-7, including potentiation
`of the antiviral activities of IFN-or and ~13 (ref. 24), and it differs
`from these interferons in its virus and cell specificities and in the
`antiviral mechanisms it induces”. However,
`in vitro studies
`performed with crude preparations suggest that the primary
`function of IFN-y may be as an immunoregulatory agent“. The
`antiproliferative effect of IFN—7 on transformed cells has been
`reported to be 10-100-fold greater than that of IFN-Ct or -[J
`(refs 25, 27), suggesting a potential use in the treatment of
`neoplasia. Indeed, murine IFN-y preparations have been shown
`to
`have
`significant
`antitumour
`activity
`against mouse
`sarcomas". However, the poor availability of purified IFN-7
`has precluded the unambiguous determination of its physico-
`chemical and biological properties.
`We describe here the first isolation and characterization of a
`recombinant plasmid containing a cDNA sequence coding for
`human IFN-7. Expression of this sequence in E. coli and
`cultured monkey cells gives rise to a polypeptide having the
`properties of authentic human IFN-y.
`
`Construction and identification of bacterial
`
`clones containing induced cDNA sequences
`Human peripheral blood lymphocytes (PBLs) from healthy
`donors were stimulated to produce IFN-7 by treatment with
`staphylococcal enterotoxin B (SEB) and desacetylthymosin ct,
`as described by Svedersky et all”. Cell cultures from individual
`
`0028-0836/8?./060503-06 $01 .00
`
`donors were collected 48 h after induction and used to prepare
`polyadenylated RNA3°’3‘ which was fractionated by electro-
`phoresis
`through denaturing agarose gels”’”.
`Interferon
`mRNA activity was determined by injecting aliquots of each
`fraction into X. laevis oocytes“‘37 and after 24 h assaying the
`incubation medium for antiviral activity36‘37. One peak of
`activity was consistently observed which co-migrated with 18S
`RNA (Fig. 1). Using different procedures for sucrose density
`gradient centrifugation, Wallace et al.” and Taniguchi et al.”
`have reported single peaks of IFN -1 mRNA activity sediment-
`ing at 18S and 15S respectively. RNA from our most active 18S
`gel fraction (600 units ml” when 0.5 pg was injected into 15
`oocytes at a concentration of 0.5 ug pl“) was used to prepare
`double—stranded cDNA by standard procedures”’”. The cDNA
`was fractionated according to size and material longer than 800
`base pairs (bp) was extended with deoxy(C) residues, annealed
`to deoxy(G)-tailed PstI-cleaved pBR322 and used to transform
`E. coli K-12 strain 294 (ref. 40) as described previously”'”.
`283
`I83
`4S
`l
`l
`l
`
`600
`
`400
`
`Interferon
`units
`
`200 2
`
`4
`
`3
`6
`Fraction no.
`
`IO
`
`I2
`
`14
`
`I6
`
`Fig. 1 Gel electrophoresis of induced lymphocyte polyadenylated RNA.
`Peripheral blood lymphocytes (PBLs) derived from individual human
`donors by lymphophoresis were purified by centrifugation on a Ficoll—
`I-Iypaque gradient and induced for IFN-y production with staphlococcal
`enterotoxin B (1 ug ml") and desacetylthymosin-a, (0.1 ug ml“)2". Total
`RNA was extracted” from PBL cultures and poly(A)-containing RNA was
`purified by oligo(dT) cellulose chromatography“. 200 Mg of mRNA was
`fractionated by electrophoresis through a denaturing agarose gel (1.75°/o
`agarose, 0.025 M sodium citrate (pH 3.8) and 6 M urea) for 7 h at 25 mA and
`4 °C (refs 32, 33). The slab gel was sectioned with a razor blade and
`individual slices were melted at 70 “C and extracted twice with phenol and
`once with chloroform. Fractions were precipitated with ethanol and
`subsequently assayed for
`IFN-y mRNA by injection into X.
`laevis
`oocytes“‘37 at a concentration of 0.5 [Lg ul“. After 24 h incubation at room
`temperature the antiviral activity of the translation products in the incuba-
`tion medium was determined by the cytopathic efiect (CPE) inhibition assay
`using vesicular stomatitis virus or encephalomyocarditis virus on WISH
`(human amnion) cells‘. All IFN-y units are expressed with reference to the
`NIH IFN~cr standard (G—023-901-527), because no internationally accepted
`IFN-y standard was available. In this assay system 0.2 units of the standard
`results in a 50% reduction in cytopathogenic effect. A single peak of activity
`was observed which co-migrated with 18S ribosomal RNA. The positions of
`ribosomal RNA markers which were electrophoresed in an adjacent lane
`and visualized by ethidium bromide staining are labelled above the activity
`profile. The activity seems to be due to IFN--y, as no activity was observed
`when the same fractions were assayed on a bovine cell line (MDBK) which is
`not protected by IFN-y (ref. 29). Both IFN-a activity and IFN)-L3 activity
`would have been easily detectable with the MDBK assay”.
`
`© 1982 Macmillan Journals Ltd
`
`Merck Ex. 1062, pg 1466
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`Merck Ex. 1062, pg 1466
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`© Nature Publishing Group1982
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`Merck Ex. 1062, pg 1467
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`© Nature Publishing Group1982
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`Merck Ex. 1062, pg 1468
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`© Nature Publishing Group1982
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`Merck Ex. 1062, pg 1469
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`© Nature Publishing Group1982
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`Merck Ex. 1062, pg 1470
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`508
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`Nature Vol. 295 11 February 1982
`
`amino acids constitute the signal peptide, the molecule expres-
`no additional genomic homologues. It remains to be deter-
`sed in E. coli may not exhibit the same specific activity as natural
`mined, however, whether additional nonhomologous genes
`IFN-7. If one assumes that authentic mature IFN-y is a 146-
`exist which code for proteins meeting the criteria required for
`classification as IFN-y (refs 1, 2).
`amino acid polypeptide, its molecular weight, minus carbo-
`We thank Dr Lloyd Svedersky and Anna Hui for processing
`hydrate, would be 17,110. The difference between this value
`and the published molecular weights of 35,000—70,000 for
`PBLs, Eileen Cunningham for performing Xenopus oocyte
`IFN--y probably cannot be accounted for by glycosylation alone.
`injections, Laurie May for interferon assays, Dr Nowell Steb-
`It is conceivable that authentic IFN-y is associated with carrier
`bing for his support of those aspects of the work which were
`proteins in serum or exists as a multimer of identical subunits.
`performed in the Biology Department at Genentech, Mark
`The purification and sizing of IFN-y synthesized by E. coli and
`Vasser for deoxyoligonucleotide synthesis, Shirley Norton for
`plasmid DNA preparations, Dr Peter Seeburg for help with the
`monkey cells should help resolve this discrepancy in molecular
`DNA sequencing, Dr Ernst Rinderknecht and Vince Anicetti
`weight.
`The human IFN-y gene corresponding to the cDNA sequence
`for preparing IFN—y antiserum and Dr Yakov Gluzman for
`described here seems to contain at least one intron and to have
`providing the COS-7 cell line.
`Received 16 November; accepted 7 December 1981.
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`
`
`
`LETTERS TO N TURE
`
`Is the remnant of
`SN1006 composed of iron?
`
`A. C. Fabian & G. C. Stewart
`
`Institute of Astronomy, Madingley Road, Cambridge CB3 OHA, UK
`
`W. Brinkmann
`
`MPI fiir Extraterrestrische Physik, D-8046 Garching bei Miinchen,
`FRG
`
`The remnant of the supernova explosion of AD 1006 (the
`brightest in recorded history) has a shell-like appearance at both
`radio and X-ray wavelengths. Einstein X-ray observations by
`Pye et al.‘ show a soft (photon energy 5 s % keV) filled interior
`surrounded by a harder (it 2 1 keV) unresolved ring of emission
`of radius ~15 arc min. Superficially it appears to be similar to
`most supernova remnants for which many studies have shown
`that the X-ray emission is thermal radiation from gas shock-
`heated by high-velocity ejecta. The spectrum2 of parts of the
`
`0028-0836/82/060508—02$()l .00
`
`edge of the remnant in the range 0.75$e S 10 keV shows,
`however, no evidence for line emission. The spectral shape is
`consistent with a power-law perhaps due to a non-thermal
`mechanism. Moreover, the total mass of gas‘ required exceeds
`~10 Me if it is of normal cosmic abundance. This is much
`greater than that expected for a remnant several hundred
`parsecs above the galactic plane. As no pulsar (or Crab-like
`morphology) is evident in the remnant of SN1006, Reynolds
`and Chevalier’ have proposed that relativistic particles are being
`efficiently accelerated in shocked gas, and provide the major
`source of X rays through synchrotron emission. SN1006 is then
`an important region for the study of such acceleration processes.
`Here we reinvestigate a thermal interpretation and show that
`the element abundances associated with a Type I supernova“‘7
`may provide an alternative explanation.
`First we note that the observed lack of line emission from the
`edge of the remnant requires that sulphur, silicon and iron be at
`less than half their cosmic value relative to hydrogenz. We
`suggest that SN 1006 was a Type I supernova of a few solar
`masses exploding into its own stellar wind (outer density p 0C
`
`© 1982 Macmillan Journals Ltd
`Merck Ex. 1062, pg 1471
`
`Merck Ex. 1062, pg 1471