AU600653B2 - Improvements in or relating to interferons - Google Patents

Improvements in or relating to interferons Download PDF

Info

Publication number
AU600653B2
AU600653B2 AU45549/85A AU4554985A AU600653B2 AU 600653 B2 AU600653 B2 AU 600653B2 AU 45549/85 A AU45549/85 A AU 45549/85A AU 4554985 A AU4554985 A AU 4554985A AU 600653 B2 AU600653 B2 AU 600653B2
Authority
AU
Australia
Prior art keywords
leu
ser
arg
ctg
gag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
AU45549/85A
Other versions
AU4554985A (en
Inventor
Gunther Adolf
Norbert Hauel
Rudolf Hauptmann
Peter Meindl
Christian Pieler
Eva Rastl-Dworkin
Peter Swetly
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE3428370A external-priority patent/DE3428370A1/en
Priority claimed from DE19853505060 external-priority patent/DE3505060A1/en
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of AU4554985A publication Critical patent/AU4554985A/en
Application granted granted Critical
Publication of AU600653B2 publication Critical patent/AU600653B2/en
Anticipated expiration legal-status Critical
Expired legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/555Interferons [IFN]
    • C07K14/56IFN-alpha
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/555Interferons [IFN]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Electrotherapy Devices (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The type I interferon peptides encoded by the genetic DNA sequences are called omega-interferons and are produced using appropriate expression vehicles and organisms.

Description

C O MMO NW E AL T H OF AUSTRALIA PATENTS ACT 1952 g COMPLETE SPECIIICATION 0 (Oziy' il a FOR OFFICE USE Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: es. e es. S
S
Priority: Related Art: This document contains the amendmtiis made undler Section 49 and is corrcct fori printing.
4 Name of Applicant: @*Address of Applicant: oeo BOEHRINGER INGELHEIM INTERNATIONAL GmbH D-6507, Ingelheim am Rhein, Federal Republic of Germany
S..
*5SS 0 SO S *5 S
S
S.
Actual Inventor(s): RUDOLF HAUPTMANN, PETER MEINDL, EVA RASTL-DWORKIN, GUNTHER ADOLF, PETER SWETLY, CHRISTIAN PIELER, NORBERT HAUEL.
DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.
Address for Service: jAi t Complete specification for the invention entitled: "IMPROVEMENTS IN OR RELATING TO INTERFERONS" The following statement is a full description of this invention, including the best method of performing it known to -1rr 14H 148-693 Improvements in or relating to interferons The present invention relates to new Type I interferons as well as to recombinant DNA methods of producing these polypeptides and products required in these methods, for example, genetic sequences, recombinant DNA molecules, expression vehicles and organisms.
Interferon is a word coired to describe a variety of proteins endogenous to human cells character- 10 ised by partly overlapping and partly diverging biological activities. These proteins modify the body's immune response and are believed to contribute substantial protection against viruses. For example, t interferons have been classified into three broad few 15 species, B, and i -Interferon. Furthermore, interferons are subd4 Lded into two types: Type I and Type II interferons. Type I interferons are I further divided into o- and 8- interferons. They S 'seem to have evolved from a common ancestor protein.
I 20 The Type II interferon is named -interferon and is not related to Type I interferons.
Only one subspecies of 8- and b-interferon is known in humans (see, for example, S. Ohno et al., Proc. Natl. Acad. Sci. 78, 5305-5309 (1981); S 25 Gray et al., Nature 295, 503-508 (1982); Taya, et al., EMBO Journal 1/8, 953-958 (1982)). On the other hand, several subtypes of o-interferon have been described (See, for example, Phil. Trans.
R. Soc. Lond. 299, 7-28 (1982)). The mature ointerferons reveal a maximum divergence of 23% among each other and are 166 or 165 amino acids long. Of note is also a report of an o-interferon having an unusually high molecular weight (26,000 by SDS polyacrylamide gel electrophoresis, described I 2 by Goren, P. et al., Virology 130, 273-280 (1983) This interferon is called IFN-o 26K. It has been found to have the highest known specific anti-viral and anti-cellular activities.
The interferons known to date appear to be effective against various diseases, but demonstrate little or no efficacy in many others (see, for example, Powledge, Bio/Technology, March 1984, 215-228, "Interferon On Trial"). Interferons have also been plagued by side effects. For example, in trials of the anti-cancer properties of recombinant a-interferon, doses of around 50 million units, which had been believed to be safe on the basis of Phase I trials have been associated with acute 15 confusional states, disabling arthragia, profound fatigue and anorexia, disorientation, seizures I, and hepatic toxicity. In 1982, the French government stopped trials with o-interferon after cancer patients receiving it suffered fatal heart attacks. At least two cardiac deaths have also been reported in recent American trials. It has become increasingly clear that at least some of the side effects, like *fever and malaise, appear to be inherent in o-interferon itself and are not due to impurities.
a 25 Due to the great hopes elicited by the interferons, and spurred by the wish to discover yet new interferonlike molecules with decreased side effects, the present inventors set out to search for and produce such new substances.
The present invention therefore relates to new interferons, and active derivatives thereof, e.g. glycosylated derivatives; to genetic sequences coding therefor, as well as to recombinant DNA molecules containing such sequences. Included within the scope of the present invention are expression vectors containing a coding sequence for a novel interferon according to the invention at an appropriate 3site for expression and microorganisms and tissue culture hosts transformed by such vectors which are capable of producing the encoded interferon.
According to one aspect, the present invention S o\ dcc= provides 4e ancmega interferon polypeptides of Type 1 coded for by the cDNA inserts of plasmids P9A2 and E76E9 (deposited at the DSM within E. coli HB 101 under the numbers DSM 3004 and 3003 respectively, the polypeptides coded for by pseudo nmega interferon genes 2, 3 or 4, further interferon polypeptides having omega interferon properties comprising 168 to 174 amino acid residues which have an amino acid divergence of at least 30 to 50% compared to alpha-interferons and a divergence of at least 70% compared to beta-interferon, and coded for by sequences Lh will hybridise with the sequence corresponding to the anega-interferon-coding sequences of plasmids P9A2 and E76E9 or S. 1' degenerate variations thereof under stringent hybridisation conditions as hereinbefore defined for detecting about 85% or higher homology, eo optionally further comprising a leader peptide, and N-glycosylated derivatives thereof with omega interferon activity.
Such interferons are hereinafter referred to as omega-interferon or IFN-omega.
According to \a frther aspect, the present invention providespolydeoxyribonucleotides comprising V .a coding sequence for an omega-interferon or comprising a pseudo gene sequence capable of hybridizing with 25 a sequence corresponding to an omega-interferoncoding sequence under stringent hybridization conditions, suitable for detecting about 85% or higher homology with the chosen omega-interferon coding sequence.
To carry out such stringent hybridization 30 tests, appropriate single-stranded polydeoxyribonucleo- .tides are hybridized in the presence of 6 x SSC (1 x SSC corresponds to 0.15 M NaCI, 0.015 M trisodium citrate, pH 5 x Denhardt's solution (1 x Denhardt's solution corresponds to 0.02% polyvinylpyrrolidone, 0.02% ficoll 40,000), 0.02% Sbovine serum albumin) and 0.1% sodium dodecylsulphate 3q at 65°C. The degree of stringency is determined in the washing step. Thus, for selection to DNA sequences with about 85% homology or more, the conditions 0.2 x SSC/0.01% 0 C are suitable and for selection to DNA sequences with about 90% homology or more, the conditions 0.1 x SSC/0.01% SDS/65 0 C are suitable.
It has been found that natural interferon omega consists of a mixture and that the size range of sequences in the mixture lie within the range 168 to 174 amino acid t residues.
ee 0 e •0 0 oo o g o S I *I f. I: t t' n r- p I_ 3 site for expression and microorganisms and tissue culture hosts transformed by such vectors which are capable of producing the encoded interferon.
According to one aspect, the present invention provides new Type 1 interferon polypeptides of 168 to 174, preferably 172, amino acids, which have a divergence of 30 to 50%, preferably 40-48%, compared to o-interferons, and a divergence of at least 70% compared to B-interferon, optionally further comprising a leader peptide, and N-glycosylated or other derivatives thereof with interferon activity.
Such interferons are hereinafter referred to as omega-interferon or IFN-omega.
According to a further aspect, the present invention provides polydeoxyribonucleotides comprising S• a coding sequence for an omega-interferon or comprising a pseudo gene sequence capable of hybridizing with a sequence corresponding to an omega-interferoncoding sequence under stringent hybridization conditions, suitable for detecting about 85% or higher homology S. with the chosen omega-interferon coding sequence.
To carry out such stringent hybridization tests, appropriate single-stranded polydeoxyribonucleotides are hybridized in the presence of 6 x SSC 25 (1 x SSC corresponds to 0.15 M NaC1, 0.015 M trisodium citrate, pH 5 x Denhardt's solution (1 x Denhardt's solution corresponds to 0.02% polyvinylpyrrolidone, 0.02% ficoll 40,000), 0.02% bovine serum albumin) and 0.1% sodium dodecylsulphate fl 30 at 651C. The degree of stringency is determined in the washing step. Thus, for selection to DNA Ssequences with about 85% homology or more, the conditions 0.2 x SSC/0.01% SDS/65 0 C are suitable Sand for selection to DNA sequences with about homology or more, the conditions 0.1 x SSC/0.01% 0 C are suitable.
-T.
4 As preferred embodiments, the present invention provides an omega-interferon (hereinafter referred to as omega (Gly)-interferon) having the following amino acid sequence: Cys
TGT
Val
GTG
Lys
AAG
Asp
GAT
Leu
CTT
Asp
GAC
Leu
CTG
Leu
CTG
Pro
CCT
His
CAC
Gin
CAG
Gin
CAA
Asn
AAC
Met
ATG
His Gly CAT GGC Leu
CTA
Ile
ATC
10 Leu Ser Arg CTT AGC AGG Asn Thr AAC ACC Leu Cys TTG TGT Arg
AGG
*a 15 Ser Gin AGC C(G Arg Arg Asp AGA AGA GAC 50 Leu Gin Lys TTG CAG AAG Gin Ile Phe CAG ATC TTC Asn Met Thr AAC ATG ACC Phe Arg TTC AGG Ala His GCC CAT Ser Leu AGC CTC Leu Leu CTC CTA Arg
AGA
Phe
TTC
Val
GTC
25 Ser
TCC
40 Pro Gin CCC CAG 55 Met Ser ATG TCT Glu
GAG
Pro Phe CCT TTC Met Val Lys ATG GTA AAA a.
a a *a a Leu
CTG
Ala
GCC
Gin
CAG
Glu
GAA
Gin
CAG
Trp
TGG
Arg
AGG
Tyr
TAC
Gin Leu CAA CTG Gly Glu GGA GAA Arg Tyr AGG TAC Ser Asp AGC GAC Leu Phe TTG TTC 95 Gln His CAA CAC 110 Ser Ala TCT GCT 125 Phe Gin TTC CAG 140 Cys Ala TGT GCC 155 Leu Ser TTA TC A 170 Leu Gly Le u
CTG
Gly
GGG
Gly
GGA
Trp
TGG
Thr
ACA
Glu
GAG
Ala
GCA
Ile
ATC
Glu
GAA
Asn
AAC
Phe His TTC CAC Asp Gln GAC CAA Thr Cys ACC TGC Ile Ser ATT AGC Arg Val CGT GTC Val Val GTT GTC Met Gin ATG CAA 70 Th AC A 85 Leu
CTC
100 Leu
TTG
115 Ser
AGC
130 Tyr
TAC
145 Arg
AGA
160 Glu
GAA
Val Leu GTC CTC Glu Arg GAG CGC His Thr CAC ACT Leu Gin CTG CAG Pro Ala CCT GCA Leu Lys CTG AAA Met Glu ATG GAA Arg Leu AGA CTG His Glu CAT GAG Ser Ser TCC TCT Gly Leu GGA CTT Val Val GTA GTG Leu Thr CTG ACC Glu Lys GAG AAG Ile Met ATC ATG Arg Ser AGA AGT Leu
TTG
Leu
CTC
Gly
GGG
Met
ATG
Ala
GCT
His
CAT
105 Gly
GGA
120 Leu
TTG
135 Lys
AAA
150 Lys
AAA
165 Lys
AAA
Ser
TCC
Asp Arg Asp Ser Ser GAT AGA GAC CTG GGC TCA TCT
I
I a second omega-interferon (hereinafter referred to as omega (Glu)-interferon), which has the same amino acid sequence as omega (Gly)-interferon except that amino acid residue 111 is glutamic acid rather than glycine, and derivatives thereof which are N-glycosylated at amino acid position 78.
The omega-interferons mentioned above may be fused with a leader peptide, for example, the leader peptide having the amino acid sequence: Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly.
'These omega interferons show similar effectiveness to a-interferons without many of the known 1 therapeutic disadvantages.
pi 15 As further preferred embodiments, the present invention provides polydeoxyribonucleotides comprising the omega (Gly)-interferon coding sequence shown above, the equivalent sequence coding for omega S(Glu)-interferon which differs only in that codon 20 111 is GAG (coding for glutamic acid) rather than GGG (coding for glycine) and degenerate variations thereof.
The above-mentioned sequences may be fused with a leader peptide-coding sequence, for example, the leader peptide-coding sequence shown below: I ATG GCC CTC CTG TTC CCT CTA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC DESCRIPTION OF THE FIGURES :30 Figure 1: Restriction map for clone E76E69.
Figure 2: Restriction map for clone P9A2.
Figures 3a, 3b: DNA sequence of the cDNA insert of clone P9A2.
Figures 4a, 4b: DNA sequence of the cDNA insert of clone E76E9.
Figure 5: Genomic Southern Blot Analysis 6 using DNA derived from the cDNA insert of clone P9A2 as a probe.
Figure 6: Construction of the expression vector pRHW12.
Figure 7: Amino acid and nucleotide differences between Type I interferons.
Figure 8a: Identification of the unique nucleotide positions of the IFN-aA gene.
Figure 8b: Identification of the unique nucleotide positions of the IFN-omega 1 gene.
Figure 8c: Identification of the unique nucleotide positions of the IFN-aD gene.
o •Figure 9: Schematic representation of the synthesis of a specific hybridization probe for 15 omega-l-mRNA.
Figure 10: Detection of interferon-subtype of specific mRNAs.
Figures lla, llb: DNA sequence of the IFNomegal gene.
Figures 12a, 12b: DNA sequence of the IFNpseudo-omega2 gene.
Figure 13: DNA sequence of the IFN-pseudoomega3 gene.
Figures 14a, 14b, 14c: DNA sequence of the IFN-pseudo-omega4 gene.
Figure 15: Corrected list of the IFN-omega gene sequences.
Figure 16: Homologies of the signal sequences.
Figure 17: Homologies of the "mature" protein sequences.
S Figure 18: Homologies of the 4 DNA sequences s relative to one another.
Figure 19: Antiproliferative activity of IFNomegal on human Burkitt's lymphoma cells.
Figure 20: Antiproliferative activity of IFNomegal on human cervical carcinoma cells.
The new omega interferons omega (Gly)-interferon and omega-(Glu) interferon and polydeoxyribonucleotides 7 comprising sequences coding for them may be obtained as follows: A human B-cell lymphoid line, for example, Namalwa cells (see G. Klein et al. Int. J. Cancer 10, 44 (1972)) can be stimulated for the simultaneous production of o- and 8-interferons through treatment with a virus, for example, Sendai Virus. The mRNA formed is isolated from the stimulated Namalwa cells, and this can then be used as a template for cDNA synthesis. In order to increase the yield of interferon-specific sequences during the cloning process, the mRNA preparation is separated in a sucrose density gradient into mRNA molecules of Sdifferent lengths.
15 Preferably, mRNAs of around 12S (about 800- 1,000 bases) are collected. These will include mRNAs which are specific for o-interferons and B-interferons. The mRNA collected from the gradient is concentrated through precipitation and dissolution in water and a cDNA library is then prepared.
The preparation of a cDNA library essentially involves the use of methods known in the literature (see, for example, E. Dworkin-Rastl, M.B. Dworkin and P. Swetly., Journal of Interferon Research 2/4, 575-585 (1982)). The mRNA is primed through the addition of oligo-dT. After that, by adding the four deoxynucleosidetriphosphates (dATP, dGTP, dCTP, dTTP) and the enzyme reverse transcriptase, in an appropriately buffered solution, cDNA is •i 30 synthesized at 45 0 C for 1 hour. Through chloroform extraction and chromatography via a gel column, for example, via Sephadex G50, the cDNA/mRNA hybrid is purified. The RNA is hydrolysed by means of alkali treatment (0.3 M NaOH at 50 0 C for 1 hour), and the cDNA is precipitated with ethanol after neutralising with acid sodium acetate solution.
8 Double strand synthesis is performed after addition of the four deoxynucleosidetriphosphates and E.coli DNA-polymerase I in an appropriately buffered solution.
The cDNA is used both as a template and as a primer through hairpin structure formation at its 3' end (6 hours at 15 0 C) (see also A. Eftratiadis et al., Cell 7, 279 (1976)). Following phenol extraction, Sephadex G50 chromatography and ethanol precipitation, the DNA is subjected, in a suitable solution, to treatment with endonuclease S1, which is specific for single strands. The hairpin structure as well as any cDNA that was not converted into double stranded is degraded. After chloroform extraction and precipitation with ethanol, the double-stranded DNA (dsDNA) is separated on the basis of size on Sa sucrose density gradient. In the subsequent steps of cloning, it is preferred to use only dsDNA of at least 600 bp in length in order to increase the probability of obtaining clones which contain the complete coding sequence for omega (Gly) or omega (Glu)-interferon. dsDNA with a length of more than 600 bp is concentrated out of the gradient through precipitation with ethanol and dissolution in water.
25 In order to increase the number of dsDNA molecules that have been obtained, they are first placed in an appropriate vector, e.g. an E.coli vector, and then introduced into an appropriate Shost, e.g. the bacterium E. coli. The vector used is preferably the plasmid pBR322 Bolivar et al., Gene 2, 95 (1977)). This plasmid essentially consists of a replicon and two selectable marker genes. The selectable marker genes confer on hosts resistance against the antibiotics ampicillin and tetracycline (Ap Tc). The gene for B-lactamase, which confers resistance to ampicillin (Apr), contains the recognition sequence for the restriction endonuclease PstI. pBR322 can thus be cut with PstI. The overlapping 3'-ends are extended with terminal deoxynucleotide transferase (TdT) along with a premixed quantity of dGTP in an appropriately buffered solution.
At the same time, the selected dsDNA is likewise extended with the enzyme TdT, using dCTP at the 3'-ends. The homopolymer ends of the plasmid and dsDNA are complementary and will hybridize if plasmid DNA and dsDNA are mixed in the appropriate concentration ratio and under suitable salt, buffer, and temperature conditions Nelson et al., Methods in Enzymology 68, 41-50 (1980)).
E. coli HB101 strain [genotype (r B, m B) recAl3, ara-14, proA2, lacYl, galK2, .rpsL20 (Smr), xyl-5, mtl-l, supE44, lambda-] is prepared for transformation by the recombinant vector-dsDNA molecules by washing with a CaC1 2 solution. Competent E. coli HB101 are mixed with the previously ligated DNA and, after incubation at 0°C, transformation is achieved by heat shock at 42°C for 2 minutes Dagert et al., Gene 6, S' 23-28 (1979)). The transformed bacteria are then spread on tetracycline-containing agar plates (10 pg per ml). Only E. coli HB101 cells which have received a vector or recombinant carrier molecule are resistant to tetracycline (Tc r and can thus grow on this agar. Recombinant vector-dsDNA molecules give s r a host the genotype Ap Tc because the introduction of the dsDNA into the a-lactamase gene destroys i the information for B-lactamase. Clones are then f transferred to agar plates, containing 50 ug/ml ampicillin. On-y about 3% grow, meaning that 97% of the clones contain the insertion of a dsDNA molecule. By the above process and starting with pg dsDNA, we obtained more than 30,000 clones; 28,600 clones thereof were individually transferred into the cups of microtiter plates which contained r U 10 nutrient medium, 10 pg/ml tetracycline and glycerine.
After the clones had grown, the plates were kept at -70°C for storage (cDNA library).
In order to search the cDNA library for the new interferon gene-containing clones, the clones are transferred after thawing to nitrocellulose filters. These filters rest on tetracycline containing nutrient agar. The bacterial colonies are allowed to grow and then the DNA of the bacteria is fixed on the filter.
As a probe, one can advantageously use the insert of the clone pER33 Rastl et al., Gene 21, 237-248 (1983) see also European Patent application No. 0.115.613) which contains the gene for 15 IFN-o2-Arg. By means of nick translation using DNA-polymerase I, dATP, dGTP, dTTP and a- P-dCTP, this portion of DNA is radioactively labelled.
The nitrocellulose filters are first pretreated under relaxed hybridisation conditions, without the addition of the radioactive probe, and subsequently they are hybridised for about 16 hours with the addition of the radioactive probe. The filters S**are then washed under relaxed conditions. Due to the low stringency of hybridisation and washing, 25 not only are clones obtained that contain the interferon o2-Arg gene, but also other clones containing interferon coding sequences which may differ considerably from that of the interferon o2-Arg gene. After drying, the filters are exposed on an x-ray film.
A blackening effect, which is substantially above the level of background, shows the presence of 2 clones with interferon-specific sequences.
Because the radioactivity signals are of differing quality, the positive clones or the clones that react in a manner leading to a suspicion of positive results are then cultured on a small scale.
The plasmid DNA molecules are isolated, digested 11 with the restriction endonuclease PstI, and separatea electrophoretically on an agarose gel according to size (Birnboim et al., Nucl. Acid. Res. 7, 1513 (1979)). The DNA in the agarose gel is transferred to a nitrocellulose filter according to the method of Southern M. Southern, J. Mol. Biol. 98, 503-517 (1975)). The DNA in this filter is hybridised with the radioactive, IFN o2-Arg gene-containing, denatured probe. As a positive control, the plasmid 1F7 (deposited at the DSM under the DSM no. 2362) may be used, which also contains the gene for interferon o2- Arg.
By the above procedure, we obtained an autoradio- S gram which clearly showed that two clones, E76E9 :0 15 and P9A2, contain a sequence that hybridises with S. the interferon o2-Arg gene under nonstringent, eae relaxed conditions. In order to be able to characterise 0090 more fully the dsDNA inserts of the clones E76E9 and P9A2, the plasmids of these clones were prepared on a larger scale. The DNA was digested with various restriction endonucleases, for example, with Alul, Sau3A, BglII, Hinfl, PstI, and HaeIII and the resulting fragments were separated on an agarose gel. Through comparison with size markers, for example the fragments 25 which result from digestion of pBR322 with the restriction endonuclease Hinfl or HaeIII, the sizes of the restriction fragments derived from the plasmids •E76E9 and P9A2 were determined. By means of mapping according to Smith and Birnsteil O. Smith et al., Nucl. Acid. Res. 3, 2387-2398 (1967)), the R arrangement of these fragments within E76E9 and .P9A2 was determined. From the restriction enzyme maps thus obtained (see Figures 1 and the surprising finding was made that the inserts of the clones E76E9 and P9A2 involve a hitherto unknown interferon gene, that is, the omega-interferon gene.
This information was used in order to digest the cDNA inserts of E76E9 and P9A2 with suitable 12 restriction endonucleases. The fragments were ligated into the dsDNA form (replicative form) of the bacteriophage M13 mp9 Messing et al., Gene 19, 269-276 (1982)) and were sequenced with the help of Sanger's dideoxy method Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)). This sequencing method, which is well known to those skilled in the art of recombinant DNA technology, may be summarized as follow: The single-strand DNA of recombinant M13 phages is isolated. After the binding of a synthetic oligomer, second-strand syntheses are performed in four separate preparations, using the large fragment of E. coli DNA-polymerase I (Klenow fragment). For each of 15 the four partial reactions, one of the four didexoynucleosidetriphosphates (ddATP, ddGTP, ddCTP, ddTTP) are added. This leads to statistically distributed chain breaks at those places where a base that is complementary to the particular dideoxynucleosidetriphosphate in the reaction mixture happens to be in the template-DNA. Radioactively labelled dATP is also used. After termination of the synthesis reactions, the products are denatured and the single- *strand DNA fragments are separated according to 25 size in a denaturing polyacrylamide gel Sanger et al., FEBS Letters 87, 107-111 (1978)). The ,o gel is then exposed to x-ray film. From the autoradiogram one can read off the DNA sequence of the recombinant M13 phage. The sequences of the inserts of the various recombinant phages are processed by Smeans of suitable computer programs Staden, Nucl. Acid. Res. 10, 4731-4751 (1982)).
Figures 1 and 2 reveal the strategy of sequencing. Figure 3 shows the DNA sequence of the insert of the clone P9A2; Figure 4 shows that of the clone E76E9. The noncoding DNA strand is shown in the direction, together with the amino acid sequence derived therefrom.
V 13 A comparison of the cDNA inserts of the clones E76E9 and P9A2 shows one important difference.
The triplet in clone E76E9 which codes for amino acid 111 is GAG and codes for glutamic acid. This triplet in clone P9A2 is GGG and codes for glycine.
DNA sequences that code for mature omega (Glu)-interferon and mature omega (Gly)-interferon are completely contained in the clones E7-E9 and P9A2 respectively. Both mature omega (Glu) interferon and mature omega (Gly) interferon start at the N-terminal end with the amino acids cysteine-aspartic acid-leucine. Quite surprisingly, these two mature omega-interferons are 172 amino acids long; this clearly deviates from the hitherto known length of other known interferons, that is, 166 (or 165) amino acids for o-interferons. Also somewhat surprisingly, the two omega-interferons have a potential N-glycosylation site at amino acid position 78, an asparagine residue.
20 The isolated cDNA of the clone E76E9 which codes for omega(Glu)-interferon is 858 base pairs long and has a 3' nontranslated region. The region which codes for mature omega(Glu)-interferon extends from nucleotide 9 to nucleotide 524. The isolated cDNA of the clone P9A2 is 877 base pairs long, the sequence which codes for mature omega (Gly) interferon extending from nucleotide 8 to nucleotide 523. The 3' nontranslated region in the case of P9A2 extends to the poly-A segment.
A comparison of the two specific omega-interferons mentioned above with the hitherto known human o- S. interferon subtypes gives the following picture: 14 omega alpha Length of protein in amino acids 172 166* Potential N-glycosylation site at position 78 0* a s* -7o 0@ 0 66 0 .0S0 S S@ 0
L
SO S *5 Interferon alpha A has only 165 amino acids.
Interferon alpha H has a potential N-glycosylation site at position 75 Goeddel et al., Nature 290, 20-26 (1981)).
E. coli HB 101 with the plosmid E76E9 and E. coli 101 with the plasmid P9A2 were deposited at the German Collection for Microorganisms (DSM G8ttigen) under the numbers DSM 3003 and 3004, respectively on 3 July 1984.
To prove that the newly discovered clones produce an activity resembling interferon, a 100 ml culture of clone E76E9 was further cultured in 20 L-broth at 370C up to an optical density of A 60 0 0.8, the bacteria were lysed and the resulting supernatant was then tested in a plaque reduction test. As expected, the supernatant tested was found to have interferon-like activity (see Example 3).
To prove that omega (Gly)- and omega (Glu)interferon are members of a new interferon family, all the DNA was isolated from Namalwa cells and digested with various restriction endonucleases.
In this way, it was possible to assess the number of genes which correspond to the cDNAs of the clones P9A2 and E76E9. For this purpose, the DNA fragments obtained were separated on agarose gel using the method of Southern Southern et al., J. Mol. Biol. 98, 503-517 (1975)), placed on nitrocellulose filters and hybridised under 15 relatively stringent conditions with radioactively labelled specific DNA of the clone P9A2.
Figure 5b illustrates the cDNA of the clone P9A2 and the fragment used for hybridisation.
The points of recognition sites of some restriction enzymes are shown (P=PstI, S=Sau3A, A=AluI). The probe employed included only two of the three possible PstI fragments (see Example In addition to the omega interferon gene probe, an interferon o2-Arg gene probe was used derived from the plasmid PER33. The results which were obtained are shown in Figure The individual lanes are marked with letters to indicate the various restriction enzymes used to digest the Namalwa cell DNA samples (E=EcoRI, H=HindIII, B=BamHI, S=SphI, P=PstI, C=ClaI). The left-hand half of the filter as hybridised with the o-interferon gene probe and the righthand half was hybridised with the omega interferon 20 gene probe derived from the clone P9A2 S. The DNA bands which hybridised with the o-interferon gene probe were different than those which hybridized with the new interferon gene probe. No crosshybridisation could be detected with the two different probes by assessing the corresponding lanes.
The hybridisation pattern obtained with the probe derived from the cDNA insert of clone P9A2 shows only one hybridising fragment with approximately 1300 base pairs which belongs to the homologous g 30 gene. The shorter fragment, 120 bp long, had run out of the gel. The band belonging to the 5' part of the gene cannot be observed since the probe does not contain this region. At least 6 different S" bands can be seen in the PstI lane. This indicates that scme other genes which are related to the sequences for omega (Gly)-interferon and omega (Glu)-interferon must be present in the human genome.
-16- From these results, one can deduce that if one or more PstI recognition sites are present in these genes, one can expect to be able to isolate at least three more additional genes. These genes may preferably be isolated by hybridisation from a human gene library contained in a plasmid vector, phage vector or cosmid vector (see Example 4e).
Thus, it will be understood in view of the foregoing that the present invention provides the omega-interferon polypeptides of Type 1 coded for by the cDNA inserts of plasmids P9A2 and E76E9, further interferon polypeptides coded for by sequences which will hybridise with the sequences corresponding to the interferon-coding sequences of plasmids P9A2 and E76E9 or degenerate variations thereof under stringent hybridisation conditions suitable for detecting about 85% or higher homology, and *N-glycosylated or other derivatives thereof with interferon activity.
20 The omega-interferons according to the invention do not only encompass the mature interferons which are specifically described but also any modifications of these polypeptides which leave IFN-omega activity.
These modifications comprise shortening of the molecules at the N- or C-termini thereof, exchanging amino acid residues for other residues without substantially affecting activity, or chemically or biochemically attaching the molecules to other molecules, which may be inert or otherwise. Among 30 the latter can be mentioned hybrid molecules made SI from one or more omega interferons and/or known S o- or B-interferons.
g In order to be able to compare the differences between the amino acid and nucleotide sequences of the new interferons, particularly of the omega(Gly) and omega(Glu)-interferon, with the amino acid and nucleotide sequences which have already been
I
17 S C S. C C. CCC S S C CCC S
CSSS
C
0@ C C CC. 5* CO C
*C
S.
050
CC..
S S CS C S. S 05 S0 published for o-interferons and B-interferon (C.
Weissmann et al., Phil. Trans. R. Soc. London 299, 7-28 (1982); A. Ullrich et al., J. Molec. Biol.
156, 467-486 (1982);, T.Taniguchi et al., Proc.
Nat. Acad. Sci. 77, 4003-4006 (1980); K. Tokodoro et al., EMBO J. 3, 669-670 (1984)), the corresponding sequences are arranged in pairs and the differences at individual positions are counted.
The results shown in Figure 7 demonstrate that the interferon coding nucleotide sequences of the clones P9A2 and E76E9 are related to the sequences of the Type I interferons (o and -interferons).
It is also shown that the differences in the amino acid sequences between the individual o-interferons and the new sequences are between 41.6% and 47.0%.
The differences between both the new sequences and those of the individual o-interferons to the one of s-interferon are about 70%. Taking into account the results of Example 4 in which the existence 20 of a whole set of related genes is demonstrated, and also taking into account the proposed nomenclature for interferons Vilceck et al., J. Gen. Virol.
669-670 (1984)), it is assumed that the cDNA inserts of the clones P9A2 and E76E9 code for a new class of Type I interferon, interferon-omega.
It has also been shown that omega-interferon gene expression occurs analogously to that of a Type I interferon gene. Transcription of the individual members of the multi gene families coding for the o- and omega-interferons may be investigated using the Sl mapping method Berk et al., Cell 12, 721 (1977)). By means of this technique, it has been demonstrated that the expression of mRNA corresponding to the omega (Glu)- and omega (Gly)interferon genes (hereinafter referred to as omega- 1-mRNA) is virus-inducible. Since the transcripts of a gene family of this kind differ by only a 18 few bases out of approximately 1000, hybridisation alone is not a sufficiently sensitive criterion to distinguish between the various IFN mRNAs.
To overcome this problem, the mRNA sequences of 9 o-interferons, omega (Gly)-interferon/omega (Glu)-interferon (hereinafter referred to as interferonomega 1) and B-interferon were aligned and capital letters were used to designate those bases which are specific to the top sequence (see Figures 8(a), and Such specific sites can easily be found using a simple computer programme. A hybridisation probe complementary to the top sequence which starts from such a specific site can only hybridise perfectly with the mRNA of the selected subtype. All other mRNAs are unable to hybridise at the specific site of the subtype. If the hybridisation probe is radioactively labelled at its specific 5'-end, only those radioactive labels which are protected from digestion with a single strand-specific 20 nuclease (preferably Sl nuciease) are those which I have hybridised with the interferon subtype mRNA for which the probe was designed.
This principle is not restricted to interferoncoding mRNAs but may be applied to any group of known sequences which have the specific sites described in Figure 8.
The above-mentioned specific sites of the subtypes are not restriction sites, in most cases, which means that the cutting of the corresponding cDNAs with restriction endonucleases is not capable of producing subtype-specific hybridisation probes.
A specific probe for omega-l-mRNA was therefore produced by extending an oligonucleotide radioactively labelled at the 5' end which is complementary to the mRNA of interferon-omegal above its specific site (see Example 7(a) and Figure 9).
Figure 10 shows that, as expected, omega- 1-mRNA can be induced in Namalwa and NC37 cells
Y
19 (see Example It should be understood that the present invention encompasses not only the genetic sequences specifically coding for the omega interferons mentioned, but also modifications obtained readily and routinely by mutation, deletion, transposition or addition.
Any sequence which codes for an omega-interferon a polypeptide having a spectrum of biological activities as indicated herein) and which is degenerate to those actually shown is included. Means for preparing such a sequence are well known to those skilled in the art of recombinant DNA technology.
Also, any sequence coding for a polypeptide having the spectrum of activities shown herein for IFNomega, and which hybridises with the sequences (or portions thereof) shown herein under stringent hybridisation conditions selecting for better than about 85%, preferably better than about homology) is also covered.
0 20 By screening a cosmid human DNA library using I an IFN omegal gene probe and stringent hybridization fe. conditions suitable for detecting about 85% or higher homology, one will find a number of cosmids which hybridise. Sequence analysis of restriction enzyme fragments isolated therefrom will give the authentic IFN-omegal gene (see Figure 11) and three other related genes which have been designated the IFN-pseudo-omega2 gene, the IFN-pseudo-omega3 I *,gene and the IFN-pseudo-omega4 gene (see Figures 12-14). The invention also relates to these and to the encoded peptides.
DNA comparisons give an approximately homology of the pseudo genes with the IFN-omegal Sgene.
Moreover, the IFN-omegal gene shows that upon transcription the mRNA contains the information for a functional interferon protein. A signal I yl~(u( LBOiil* 20 peptide 23 amino acids long, of the formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly is coded, fused to Lhe mature IFN-omegal which is 172 amino acids long.
Interferon-omega genes may be introduced into any organism under conditions which result in high yields. Suitable hosts and vectors are well known to anyone skilled in the art; by way of example, reference is made to EP-A-0.093.619.
In particular, prokaryotes are preferred for exprest on. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E.
I 15 coli X1776 (ATCC No. 31.537). The aforementioned strains, as well as E. coli W3110 lambda-, prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcesens, 20 and various pseudomonad species may be used.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a& 25 a replication site, as well as marking sequences go which are capable of providing phenotypic selection in transformed cells. For example, E. coli is C.J typically transformed u-ing pBR322, a plasmid derived from an E. coli strain (Bolivar, et al., Gene 2, S 30 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
The pBR322 plasmid or other plasmids must also contain, or be modified to contain, promoters 21 which can be used by the microbial organism for expression. Those promoters most commonly used in recombinant DNA construction include the betalactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); Itakura et al., Science 198. 1056 (1977); Goeddel et al., Nature 281, 544 (1979)) and tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980); EP-A-0.036.776). While these are the most commonly used, other microbial promoters have been discovered and jtilized. For example, the genetic sequence for IFN-omega can be placed under the control of the leftward promoter of bacteriophage Lambda This promoter is one of the strongest known promoters which can be controlled. Control is exerted by the lambda repressor, and adjacent restriction sites are known.
A temperature sensitive allele of this repressor gene can be placed on the vector that contains 20 the complete IFN-omega sequence. When the temperature is raised to 42 0 C, the repressor is inactivated, oo and the promoter will be expressed at its maximum level. The amount of mRNA produced under these conditions should be sufficient to result in a cell which contains about 10% newly synthesised RNA originating from the PL promoter. In this Sway, it is possible to establish a bank of clones S in which a functional IFN-omega sequence is placed *adjacent to a ribosome binding sequence, and at varying distances from the lambda PL promoter.
These clones can then be screened and the one giving the highest yield selected.
The expression and translation of an IFNomega sequence can also be placed under control of other regulons which may be "homologous" to the organism in its untransformed state. For example, lactose dependent E. coli chromosomal DNA comprises 22 a lactose or lac operon which permits lactose digestion by expressing the enzyme beta-galactosidase.
The lac control elements may be obtained from bacteriophage lambda plac5, which is infective for E. coli. The phage's lac operon can be derived by transduction from the same bacterial species.
Regulons suitable for use in the process of the invention can be derived from plasmid DNA native to the organism. The lac promoter-operator system can be induced by isopropyl-8-D-thiogalacto-pyranoside
(IPTG).
Other promoter-operator systems or portions thereof can be employed as well: for example, the arabinose-operator, Colicine E 1 -operator, galactose- .operator, alkaline phosphatase-operator, trp-operator, xylose A-operator and tac-promoter/operator.
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiae is the most commonly used among eukaryotic 20 microorganisms, although a number of other species are commonly available. For expression in Saccharomyces, plasmid YRp7 (Stinchcomb, et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper, et al., Gene 10, 157 (1980)) and plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)) are, for o example, commonly used. The plasmid YRp7 contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076.
The presence of the TRP1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, the plasmid YEpl3 contains the yeast LEU2 gene which can be used to complement a LEU2 minus mutant strain.
'I 23 Suitable promoting sequences for yeast vectors include the 5'-flanking regions of the genes for ADH I (Ammerer, Methods of Enzymology 101, 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman, et al., J.
Biol. Chem. 255, 2073 (1980)) and other glycolytic enzymes (Kawasaki and Fraenkel, BBRC 108, 1107- 1112 (1982)), such as enolase, glyceraldehyde-3phosphate dehydrogenase, hexokinase, pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase, and glucokinase.
In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector at the 3' end of the sequences to be expressed, to .provide polyadenylation of the mRNA and termination.
Other suitable promoters, which like the aforementioned promoter region of the glyceraldehyde- S' 3-phosphate gene have the additional advantage of enabling transcription control by growth conditions, 20 are the promoter regions of the genes for alcohol i'r dehydrogenase-2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism and enzymes responsible for maltose and galactose utilisation. Promoters which are regulated by the yeast mating type locus, such as the promoters of the genes BARI, MFol, STE2, STE3, STE5 can be used for temperature regulated systems by using temperature dependent siv mutations (Rhine, Ph.D.
Thesis, University of Oregon, Eugene, Oregon (1979), So• 30 Herskowitz and Oshima, The Molecular Biology of S the Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutations directly influence the expressions of the silent mating type cassettes of yeast, and therefore indirectly the mating type dependent promoters. Generally, however, any plasmid vector containing a yeastcompatible promoter, originating replication and termination sequences is suitable.
24 In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture may be employed, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, Editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of -replication, a promoter located in front of the gene to be expressed, along with any necessary else ribosome binding sites, RNA splice sites, polyadeny- A lation site, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters So.. *are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late end promoters of SV40 are particularly useful because both are obtained easily from the virus S0". as a fragment which also contains the SV40 viral *origin of replication (Fiers et al., Nature 273, S" 1123 (1978)). Smaller or larger SV40 fragments 1 ;may also be used, provided there is included the approximately 250 bp sequence extending from the °4 Hind III site toward the Bgl I site location in so-* the viral origin of replication. Further, it is also possible, and often desirable, to utilise promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
I: i -4 V I 25 An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from or another viral source Polyoma, Adeno, VSV, BPV, etc.) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
The genes may, however, preferably be expressed in the expression plasmid pER103 Rastl-Dworkin et al., Gene 21, 237-248 (1983) and EP-A-0.115- 613 deposited at the DSM under the number DSM 2773 on 20th December 1983), since this vector contains all the regulons necessary for a high expression rate of the cloned genes in E. coli.
According to a preferred embodiment of the present invention, therefore, we provide an expression o vector derived from plasmid PBR322 wherein the shorter EcoRI/BamHI fragment belonging to plasmid 20 pBR322 is replaced by a polydeoxyribonucleotide comprising the sequence: EcoRI Sau3A gaattcacgctGATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTCGCGA 59 mRNA-Start Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RBS HindIII Cys Asp TGT GAT C IFN-omega-coding sequence--- SSau3A In order to construct an expression vector of this type, the following procedure may, for example, be used, which is illustrated in Figure 6.
26 Preparation of the individual DNA fragments required: Fragment (a) In order to produce fragment a plasmid which contains an IFN-omega coding sequence, e.g.
the plasmid P9A2, is digested with the restriction endonuclease AvaII. After chromatography and purification of the resulting cDNA insert, the latter is twice redigested with the restriction endonucleases NcoI and Alul and then isolated by chromatography and electroelution. This fragment contains the majority of the corresponding omega-interferon gene. Thus, for example, the omega(Gly) interferon gene fragment derived from the clone P9A2 has the following sequence: 10 His Gly Leu Leu Ser Arg Asn Thr Leu c CAT GGC CTA CTT AGC AGG AAC ACC TTG 28
NI
Ncol 20 25 Val Leu Leu His Gin Met Arg Arg Ile Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 '0 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly 25 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 55 Ser Gin Leu Gin Lys Ala His Val Met Ser Val Leu His Glu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 65 70 3 0 Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 80 85 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 3 100 105 Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Val Val Gly CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 27 110 115 120 Glu Gly Glu Set Ala Gly Ala Ile Set Ser Pro Ala Leu Thr Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 125 130 135 Arg Arg Tyr Phe Gin Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys AGO AGG TAC TTC CAG GGA AIC COT GTC TAC CTG AAA GAG AAG AAA 388 140 145 150 Tyr Ser Asp Cys Ala Trp Glu Val Val Arg Met Giu Ile Met Lys 1 0 TAC AGC GAC TGT 0CC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Arg Ser Lys WCC TTG T'rC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 170 ,~Asp Arg Asp Leu Giy Ser Ser GAT AGA GAC CTG GOC TC A TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 **g 1AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707 s*0 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAAC AAATGTTTGCC 766 TTTACATTGTATTAAGATAACAAAACATGTTCAG~t 802 S AluI 0 0 00 Fragment (b) In order to isolate fragment from the 6 01130plasmid P9A2 or plasmid E76E9, the chosen piasmid is dgesed iththerestriction endonuclease AvaIl.
After chromaLography and purification of thereutn fragment of 189bp is isolated by chromatography and electroelution. it has the following sequence: mm
ENEWL-
28 10 Asp Leu Pro Gin Asn His Giy Leu Leu Sec Arg Asn Thr Lou GAT CTG CCT GAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 42 Su3A NcoI 25 Val Leu Leu His Gin Met Arg Arg Ile Ser Pro Phe Lou Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATG TCC CCT' TTG TTG TGT CTC 87 40 Lys Asp Arg Arg Asp Phe Ary Phe Pro Gin Giu Met Val Lys Gly AAG GAG AGA AGA GAG TTC AGG TTC CCtCGAG GAG ATG GTA AAA GGG 132 .50 55 Ser Gin Leu Gin Lys Ala His Val Met Ser Val Leu His Glu Met ACG GAG TTG GAG AAG GGG CAT GTC ATO TCT GTC GTC CAT GAG ATG .177 Leu Gin Gin Ile GAG C batc18 S.7 Su3A Fragment (c) order to prepare fragment the plasmid
S*
pER33 (see E. Rastl-Dworkin et al., Gene 21, 237-248 (1983) and EP-A-0.115.613) is digested twice with the restriction enzymes EcoRI and PvuII. The 389bp fragment which is obtained after agarose gel fractionation and purification and which contains the Trp promotor, the ribosomal binding site and the starting codon, is. subsequently digested with Sau3A. The desired fragment of lO8bp is obtained by agarose gel electrophoresis, electroelution and elutip column purification (carried out with an Elutip column available from
I
-29 Messrs. Schleicher and Schuell). it has the following sequence: EcoRI ISau3A g aa ttca cg c tIATC GCTAAAACATTGTGCAA'AAAGAGGGTTGACTTTGCCTTrCGCGA 59 [mA-Sta rtMe ACCAGTTAACTAGTACACAAGTTCACGGCA\CGGTAAGGAGGTT"ITAAGCTTAAAG ATG 116 P135 HindIII Cys Asp TGT Igat c 123 Sau3A Ligation of fragments and The fragments and Cc) are liqated with T4 ligase and, after destruction of the enzyme, j cut with HindIII. This ligated fragment has the :::following structure: [idI :a3
NO
AGCTTAAAG ATGTGTGATC TGCC'ICAGAA CCATGGCCTA CTTAGCAGGA ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAA CITCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTICCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAG;GCCCATG TCATGTCTGT CCTCCATGAG ATGCTIGCAGC 200 AGATCACACA TCTTT Ct qau3A IlidII I Alternatively, this DNA fragment necessary :*:for the production of the plasmid pRI11lO may also be produced by using two synthetically produced oligonucleotides: The oligonucleotide of formula -ACTAAAGATGTGr-31 remains dephosphorylated at its 5' end.
The oligonucleotide of formula 5'-GATCACACATCTTTA-3' is phosphorylated at the 5' end by using T 4 polynucleotide kinase and ATP.
When the two oligonucleotides are hybridised, the following short DNA fragment is obtained: 3' ATTTCTACACACTAGp This produces at one end the 5' overlap typical of HindIII and at the other end the 5' overlap typical of Sau3A.
I Fragment is dephosphorylated using calves' 20 intestine phosphatase. Fragment and the fragment described above are combined and joined together by means of T 4 ligase.
Since the ligase requires at least one end containing 5'-phosphate, only the synthetic piece of DNA can be joined to fragment or 2 synthetic fragments may be connected at their Sau3A ends.
Since the two resulting fragments are of different lengths, they may be separated by selective isopropanol precipitation. The desired fragment thus purified 30 is phosphorylated using T 4 polynucleotide kinase S: and ATP.
Preparation of the expression plasmids a) Preparation of plasmid The expression plasmid.pER103 (E Rastl-Dworkin et al., Gene 21, 237-284 (1983) and EP-A-0.115.613, filed at the DSM under DSM Number 2773) is linearised with HindIII and then treated with calves' intestine 31 phosphatase. After isolation and purification of the DNA thus obtained, it is dephosphorylated and then ligated with the fragment obtainable by liating fragments and followed by digestion with HindIII as described above. Then, E.coli HB 101 is transformed with the resulting ligation mixture and cultivated on LB agar plus 50 pg/ml of ampicillin. The resulting plasmid designated pRHW 10, (see Figure after replication is used as an intermediate for preparing the desired expression plasmids.
b) Preparation of the expression plasmid pRHW12: The Klenow fragment of DNA polymerase I and *the 4 deoxynucleoside triphosphates are added to the plasmid pRHW10 cut with BamHI. The linearised blunt-ended plasmid obtained after incubation is purified and then cut with NcoI. The larger fragment, which is obtained using agarose gel electrophoresis, 20 electroelution and elutip purification (carried 009' out with an Elutip column available from Messrs.
Schleicher and Schuell), is ligated with fragment E. coli HB 101 is then transformed with the ligation mixture and cultivated on LB agar plus 25 50 pg/ml of ampicillin. The resulting plasmid which expresses omega(Gly)-interferon has been designated pRHW12 (see Figure 6).
For example, 1 litre of the bacterial culture thus obtained (optical density: 0.6 at 600 nm) 30 contains 1 x 10 International Units of interferon.
c) Preparation of the expression plasmid pRHW11: The Klenow fragment of DNA polymerase I and the 4 deoxynucleoside triphosphates are added to the plasmid pRHW10 cut with BamHI. The linearised blunt-ended plasmid obtained after incubation is purified and then cut with NcoI. The larger fragment, which is obtained using agarose gel electrophoresis, 32 electroelution and elutip purification, is ligated with fragment obtained analogously as in (I) above from the plasmid E76E9, the IFN-coding sequence of which differs from that of plasmid P9A2 only in that the GGG codon coding for the amino acid Gly is replaced by the GAG codon coding for the amino acid Glu at codon position 111. Subsequently the resulting ligation mixture is used to transform E. coli HB101 which is then cultivated on LB agar plus 50 pg/ml of ampicillin. The resulting plasmid which expresses omega(Glu)-interferon has been designated pRHW11.
Transformation of cells with vectors can be effected by a number of procedures. For example, the transformation procedure may comprise either washing cells in magnesium and adding DNA to the cells suspended in calcium or exposing the cells to a coprecipitate of DNA and calcium phosphate.
Following gene expression, the cells are plated 20 on media which select for transformants.
After appropriate transformation of the host, expression of the gene therein and fermentation or cell culture under conditions where IFN-omega is expressed, the product can normally be extracted 25 by means of well known chromatographic separation procedures to yield a material comprising IFN-omega with or without leading and trailing sequences.
The IFN-omega may be expressed with a leading sequence at the N-terminus thereof (to yield pre-IFN-omega), S 30 which may be removed in some of the host cells.
S. If not removed, it may be necessary to cleave the leading polypeptide (if any is present) to yield the mature IFN-omega. Alternatively, the IFN-omega clone can be modified in such a way that the mature protein will be directly produced in the microorganism instead of pre-IFN-omega. In this respect, the precursor sequence of the yeast mating pheromone MF-alpha-1 can be used for precise maturation of 33 the fused protein, and for secretion of the products into the growth medium or periplasmic space. The DNA sequence corresponding to functional or mature IFN-omega can be connected to a portion of the leader sequence of the MF-alpha-1 gene at the sequence coding for the cathepsin-like cleavage site (after lys-arg) at position 256 from the initiation codon ATG (Kurjan, Herskowitz, Cell 30, 933-943 (1982)).
On the basis of their biological actions, the new interferons according to the invention are suitable for the treatment of any condition for which the known interferons have been used.
These include conditions such as herpes, rhinovirus, AIDS infections and certain cancers. The new interferons of the present invention can be used by themselves or in S* combination with other known interferons or other biologically active products, such as IFN-alpha, IFN-gamma (see Example 12D), IL-2 and other immune modulators.
According to yet another aspect of the present 20 invention, we therefore provide a pharmaceutical composition comprising at least one interferon polypeptide according to the present invention and/or at least one derivative of such an interferon S* polypeptide with interferon activity in association S. 25 with a pharmaceutically acceptable carrier or excipient.
Suitable carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin, to which reference is expressly made. The chosen active ingredient comprising 30 at least one IFN-omega and/or at least one IFNomega derivative is mixed together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for effective administration to the patient. The preferred mode of administration is parenteral.
IFN-omega may be parenterally administered to subjects requiring antitumour or antiviral treatment, and to those exhibiting immunosuppressive 34 conditions. Dosage and dose rate may parallel those currently in use in clinical investigations of o-IFN materials, e.g. about (1-10) x 106 units daily, and in the case of materials of purity greater than up to e.g. 5 x 107 units daily.
As one example of an appropriate dosage form for essentially homogenous bacterial IFN-omega in parenteral form, 3 mg IFN-omega may be dissolved in 25 ml of 5 N human serum albumin, the solution is passed through a bacteriological filter and the filtered solution aseptically subdivided into 100 vials, each containing 6 x 106 units pure IFNomega suitable for parenteral administration.
The vials are preferably stored in the cold (-200C) prior to use.
The following Examples, which are not exhaustive, illustrate the present invention in greater detail.
fa *o0 o* 2- 35 Example 1 Finding IFN-sequence-specific clones a) Preparation of a cDNA Library mRNA from Sendai-virus-stimulated cells was used as starting material for the establishment of a cDNA library according to methods known in the literature Dworkin-Rastl et al., Journal of Interferon Research Vol. 2/4, 575-585 (1982)).
The 30,000 clones obtained were individually transferred into the wells of microtiter plates. The following medium was used for growing and freezing the colonies: g trypton g Yeast Extract 5 g NaC1 0.51 g Na-Citrate x 2 H20 2 7.5 g K 2
HPO
4 x 2 1.8 g KH 2
PO
4 0.09 g MgSO 4 x 7 20 0.9 g (NH 4 2 S0 4 44 g glycerine 0.01 g tetracycline x HC1 ad 1 1 25 The microtiter plates with the individual clones were incubated overnight at 37 0 C and were then stored at -70 0
C.
b) Hybridisation test As the starting material for the hybridisation test was used the recombinant plasmid pER33 (E.
.Dworkin-Rastl et. al., Gene 21, 237-248 (1983)).
This plasmid contains the coding region for the mature interferon IFN-o2 arg plus 190 bases of the 3' nontranslated region. 20 ug pER33 were incubated with 30 units of Hind III restriction endonuclease in 200 ul reaction solution (10 mM Tris-HCl, pH-7.5, 10 mM MgCl 2 1 mM Dithiothreitol 36 (DTT), 50 mM NaCl) for 1 hour at 37°C. The reaction was terminated by the addition of 1/25 vol M ethylenedinitrilotetraacetic acid (EDTA) and heating to a temperature of 70 0 C for 10 minutes.
After the addition of 1/4 vol 5 x buffer (80% glycerine, mM Tris acetate, pH 7.8, 50 mM EDTA, 0.05% Sodium dodecylsulphate (SDS), 0.1% bromophenol blue), the resulting fragments were separated electrophoretically according to size in a 1% agarose gel. [Gel and electrophoresis buffer (TBE): 10.8 g/l trishydroxymethylaminomethane (Tris-Base), 5.5 g/l boric acid, 0.93 g/l EDTA]. After the incubation of the gel in a 0.5 pg/ml ethidium bromide solution, the DNA strips were made visible in UV-light and the gel .area, which contained the IFN-gene-containing DNA piece (about 800 bp long), was cut. The DNA was electroeluted into 1/10 x TBE buffer. The DNA solution was extracted once with phenol and four times with ether, and the DNA was precipitated 1 20 by adding 1/10 vol 3 M sodium acetate (NaAc) pH 5.8 and 2.5 vol EtOH from the aqueous solution at -20°C. After centrifuging, the DNA was washed with 70% ethanol and was dried in a vacuum for minutes. The DNA was dissolved in 50 ul of water 25 (about 50 pg/ul). The DNA was marked radioactively by means of nick translation (modified according to T. Maniatis et al., Molecular Cloning, Ed. CSH).
0* Furthermore, 50 pl incubation solution contained the following: 50 mM Tris pH 7.8, 5 mM MgCl 2 10 mM mercaptoethanol, S 100 ng DNA insert from pER33, 16 pg DNaseI, 25 pM dATP, 25 pM dGTP, 25 pM dTTP, 20 pCi o 3 2 P-dCTP (73,000 Ci/mMol), as well as 3 units of DNA polymerase I (E.coli). Incubation was performed at 14 0 C for minutes. The reaction was terminated through the addition of 1 vol 50 mM EDTA, 2% SDS, 10 mM Tris pH=7.6 solution and heating to 70 0 C for -37minutes. The DNA was separated by means of chromatography using Sephadex G-100 into TE buffer (10 mM Tris pH=8.0, 1 mM EDTA) from non-incorporated radioactivity. The radioactively labelled sample had a specific radioactivity of about 4 x 107 cpm/pg.
c) Screening the clones for IFN gene-containing inserts The bacterial cultures, which were kept frozen in the wells of the microtiter plates, were thawed A piece of nitrocellulose filter of corresponding size (Schleicher and Schull, BA 85, 0.45 pm pore size) was placed on LB-agar (LB-agar: 10 g/l Trypton, g/l yeast extract, 5 g/l NaCl, 15 g/l Bacto Agar, mg/l tetracycline-HCl). By means of a plunger adapted to the microtiter plates, the individual clones were transferred to the nitrocellulose filter.
The bacteria grew overnight at 37 0 C to form colonies with a diameter of about 5 mm. To destroy the S. bacteria and to denature the DNA, the nitrocellulose 20 filters were, one after the other, placed on a of.: stack of Whatman 3MM Filter which had been soaked with the following solutions: 8 minutes at M NaOH, 2 minutes at 1 M Tris pH=7.4, (3) 2 minutes at 1 M Tris pH-7.4 and 4 minutes 25 at 1.5 M NaC1, 0.5 M Tris pH=7.4. The filters were dried in air and were then kept at 80 0 C for 2 hours. The filters were pretreated for 4 hours at 65°C in the hybridisation solution, consisting of 6 x SSC (1 x SSC corresponds to 0.15 M NaCl; 0.015 M trisodium citrate; pH 5 x Denhardt's solution (1 x Denhardt's solution corresponds to S 0.02% PVP (polyvinylpyrrolidone); 0.02% ficoll (MW: 40,000D); 0.02% BSM (bovine serum albumin)) and 0.1% SDS (sodium dodecylsulphate). About 1 x 106 cpm per filter of the sample made in were denatured by boiling and added to the hybridisation solution.
Hybridisation was performed at 65 0 C for a period of 16 hours. The filters were washed four times I I 38 1 hour at 65 0 C with 3 x SSC/0.1% SDS. The filters were dried in air, were covered with Saran Wrap-, and exposed on Kodak X-OmatS" film.
Example 2 Southern Transfer to confirm IFN gene-containing recombinant plasmids ml cultures of the positively reacting colonies or those colonies that were suspected of reacting positively, were grown in L-broth (10 g/l trypton, 5 g/l yeast extract, 5 g/l NaCl, 20 mg/1 tetracycline x HC1) at 37 0 C overnight. The plasmid- DNA was isolated using a modified protocol according to Birnboim and Doly (Nucl. Acid. Res. 7, 1513 (1979)). The cells in 1.5 ml suspension were centrifuged (Eppendorf Centrifuge) and resuspended at 0 0 C in 100 pl lysozyme solution consisting of 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH=8.0 and 4 mg/ml of lysozyme. After 5 minutes of incubation 20 at room temperature, 2 vol of ice-cold 0.2 M NaOH, 1% SDS solution were added, and incubation continued for another 5 minutes. Then 150 p1 of ice-cold sodium acetate solution pH=4 8 was added and incubated for 5 minutes. The precipitated cell components 25 were centrifuged. The DNA solution was extracted with 1 vol phenol/CHC13 and the DNA was precipitated by the addition of 2 vol ethanol.
After centrifuging, the pellet was washed once with 70% ethanol, and dried in a vacuum for 5 minutes.
The DNA was dissolved in 50 ul (TE)-buffer. Of that amount, 10 pl were digested in 50 ul reaction solution (10 wM Tris-HCl pH=7.5, 10 mM MgCl 2 50 mM NaC1, 1 mM DTT) with 10 units PstI-restriction endonuclease for 1 hour at 37 0 C. After the addition of 1/25 vol 0.5 M EDTA as well as 1/4 vol 5 x buffer (see Example it was heated for 10 minutes and the DNA was then separated electrophoretically in a 1% agarose gel (TBE-buffer). The DNA in the 39 agarose gel was transferred to a nitrocellulose filter according to the method of Southern (E.
M. Southern, J. Mol. Biol. 98, 503-517 (1975)).
TLe DNA in the gel was denatured for 1 hour by incubating the gel in a 1.5 M NaCl/0.5 M NaOH solution.
This was followed by neutralisation for 1 hour with a 1 M Tris x HC1 pH=8/1.5 M NaCI solution.
The DNA was transferred to the nitrocellulose filter with 10 x SSC (1.5 M NaC1, 0.15 M sodium citrate, pH=7.0). After completion of transfer (about 16 hours), the filter was briefly rinsed in 6 x SSC buffer and then dried in air; it was finally baked at 80 0 C for 2 hours. The filter was pretreated for 4 hours with a 6 x SSC/5 x Denhardt's solution.
SDS (see Example Ic) at 65 0 C. About 2 x 106 cpm of the hybridisation probe (see Example lb) were denatured by means of heating to a temperature of 100 0 C and were then added to the hybridisation solution. The duration of hybridisation was 16 20 hours at 65 0 C. Then the filter was washed 4 x 1 hours at 65 0 C with a 3 x SSC/0.1% SDS solution.
After air-drying, the filter was covered with Saran SWrap and was exposed on Kodak X-OmatS film.
25 Example 3 Detection of interferon activity in the clone E76E9 1 A 100-ml culture of clone E76E9 was cultured in L-broth (10 g/l trypton, 5 g/l yeast extract, g/l NaC1, 5 g/l glucose, 20 mg tetracycline x _1 30 HC1 per 1) at 37 0 C up to an optical density of o 'A 6 0 0 The bacteria were centrifuged for minutes at 7,000 rpm, they were washed once with a 50 mM Tris x HC1 pH=8.0, 30 mM NaCl solution and then were resuspended in 1.5 ml washing solution.
After incubation with 1 mg/ml of lysozyme at 0°C for half an hour, the bacterial suspension was frozen and thawed five times. The cells were pelleted by means of centrifugation at 40,000 rpm for 1 40 hour. The supernatant was sterile filtered and was tested for interferon activity. The test used was the Plaque Reduction Test with V3 cells and Vesicular Stomatitis Virus R. Adolf et al., Arch. Virol. 72, 169-178 (1982)). Surprisingly, the clone produced up to 9,000 IU of interferon per litre of initial culture.
Example 4 Genomic Southern Blot for determining the number of genes associated with the new sequences a) Isolating the DNA from Namalwa cells 400 ml of a Namalwa cell culture are centrifuged at 1000 rpm in a JA21 centrifuge in order to pellet .the cells. The resulting pellets are carefully washed by resuspending them in NP40 buffer buffer: 140 mM NaCl, 1.5 mM MgCl 2 10 mM Tris/Cl pH=7.4) and pelleting them again at 1000 rpm.
The pellets obtained are again suspended in 20 ml 20 of NP40 buffer and mixed with 1 ml of a 10% solution in order to destroy the cell walls. After standing for 5 minutes in an ice bath the intact cell nuclei are pelleted by centrifuging at 1000 rpm and the supernatant is discarded. The cell nuclei 25 are resuspended in a 10 ml of a solution consisting of 50 mM Tris/Cl pH=8.0, 10 mM EDTA and 200 mM NaC1, and then 1 ml of 20% SDS are added to eliminate the proteins. The resulting viscous solution is extracted twice with the same quantity of phenol 30 (saturated with 10 mM Tris/Cl pH=8.0) and twice S **"with chloroform. The DNA is precipitated by the addition of ethanol and by centrifuging. Then the resulting DNA pellet is washed once with ethanol, dried for 5 minutes in vacuo and dissolved in 6 ml of TE buffer (TE buffer: 10 mM Tris/Cl 1 mM EDTA). The concentration of the DNA is 0.8 mg/ml.
41 b) Restriction endonuclease digestion of the DNA from Namalwa cells The restriction endonuclease digestion was carried out in accordance with the conditions specified by the manufacturer (New England Biolabs). 1 pg of DNA is digested with 2 units of the suitable restriction endonuclease in a volume of 10 ul at 37 0 C for 2 hours or longer. The restriction endonucleases EcoRI, HindIII, BamHI, SphI, PstI and Clal were used. 20 pg of DNA are used for each digestion.
In order to monitor the completeness of the digestion, pl (aliquot parts) are taken out at the start of the reaction and mixed with 0.4 pg of lambda phage-DNA. After 2 hours incubation, these aliquot portions are monitored by agarose gel electrophoresis and the completeness of digestion is assessed with the aid of a sample of the stained lambda phage DNA fragments.
After these checks have been carried out 20 the reactions are stopped by adding EDTA to a final concentration of 20 mM and heating the solution to 70 0 C for 10 minutes. The DNA is precipitated by the addition of 0.3 M NaAc, pH=5.6 and 2.5 vols of ethanol. After 30 minutes incubation at -70 0
C
25 the DNA is pelleted in an Eppendorf centrifuge, S* washed once with 70% ethanol and dried. The resulting DNA is taken up in 30 pl of TE buffer.
S*
c) Gel electrophoresis and Southern Transfer t 30 The digested DNA samples are fractionated according to their size in a 0.8% agar gel in TBE buffer (10 2 g/l Tris base, 5.5 g/l boric acid, 0.93 9/l EDTA). For this purpose, 15 pl of the DNA sample are mixed with 4 pV of loading buffer (0.02% SDS, 5 x TBE buffer, 50 mM EDTA, 50% glycerine, 0.1% bromophenol blue), heated briefly to and loaded onto the troughs rrovided in the gel.
Lambda-DNA which has been cut with EcoRI and HindIII 42 is loaded separately and serves as a marker for the size of the DNA. Gel electrophoresis is carried cut for 24 hours at about 1 V/cm. Then the DNA is transferred to a nitrocellulose filter using the method of Southern (Schleicher and Schuell, using 10 x SSC (1 x SSC: 150 mM trisodium citrate, 15 mM NaCI, pH=7.0). After the filter has been dried at ambient temperature it is heated for 2 hours to 80 0 C in order to bind the DNA to it.
d) Hybridisation probe pg of the plasmid P9A2 are cut with AvaIl, thereby generating a fragment approximately 1100 bp -long which contains the entire cDNA insert. This DNA fragment is again cut with Sau3A and AluI and the largest DNA fragment is isolated after agarose gel electrophoresis (see Figure 5b) by electroelution and elutip column chromatography. The resulting DNA (1.5 pg) is dissolved in 15 ul of water.
i 20 pg of the plasmid pER33 are cut with HindIII, and this expression plasmid for IFN-a2-Arg (E.
Rastl.-Dworkin et al. Gene 21, 237-248 (1983)) is cut twice. The smaller DNA fragment contains 25 the gene for interferon-a2-Arg and is isolated in the same way as the desired fragment from the ScDNA insert of plasmid P9A2.
Both DNA's are nick-translated using the method proposed by P.W.J. Rigby et al. Mol.
Biol. 113, 237-251 (1977)). The nick translation S is carried out with 0.2 ug of DNA in a solution of 50 pi, consisting of 1 x nick buffer (1 x nick buffer: 50 mM Tris/Cl pH=7.2, 10 mM MgSO 4 0.1 mM DTT, 50 pg/ml BSA), 100 pmol each of dATP, dGTP and dTTP, 150 uCi a- 3 2 P-dCTP (Amersham, 3000 Ci/mMol) and 5 units of DNA polymerase I (Boehringer-Mannheim, nick translation quality). After 2 hours at 14 0
C
the reaction is stopped by adding the same amount 43 of an EDTA solution (40 mMol) and the unreacted radioactive material is separated off by means of G50 column chromatography in TE buffer. The remaining specific radioactivity is approximately 100 x 106 cpm/pg DNA.
e) Hybridisation and autoradiography The nitrocellulose filter is cut into two halves. Each half contains an identical set of lanes with Namalwa DNA which have has treated with one of the restriction enzymes mentioned in Example 4a. The filters are pre-hybridised in a solution containing 6 x SSC, 5 x Denhardt's (1 x Denhardt's: 0.02% bovine serum albumin (BSA), 0.02% polyviny]- .pyrrolidone (PVP), 0.02% Ficoll 400), 0.5% SDS, 0.1 mg/ml of denatured calf thymus DNA and 10 mM EDTA for 2 hours at 65 0 C. Hybridisation is carried out in a solution containing 6 x SSC, 5 x Denhardt's, o 6 10 mM EDTA, 0.5% SDS and approximately 10 x 10 cpm of nick translated DNA for 16 hours at 65 0 C. One I half of the filter is hybridised with interferono2-Arg-DNA and the other half with interfe.on-DNA which has been isolated from the plasnid P9A2.
I After hybridisation, both filters are wished at ambient temperature, four lires with a solution consisting of 2 x SSC and 0.J% 3DS and twice at 65 0 C for 45 minutes with a soluti',, consisting of 0.2 x SSC and 0.01% SOS. The tilters are then "dried and exposed to a Kodak X-Omat S film.
Example Preparation of the expression plasmids pRHW 12 and pRHW 11 Preliminary comment: The preparation of the expression plasmids is illustrated in Figure 6 (not to scale), and also all the restriction enzyme digestions are carried out in accordance with the instructions of the enzyie manufacturers.
44 a) Preparation of the plasmid pRHW 100 pg of the plasmid P9A2 are digested with 100 units of the restriction endonuclease AvaII (New England Biolabs). After digestion, the enzyme is deactivated by heating to 70 0 C and the fragments obtained are fractionated on a 1.4% agarose gel with TBE buffer (TBE buffer: 10.8 g/l Tris base, g/l boric acid, 0.93 g/1 EDTA) according to their size. The band which contains the entire cDNA insert is electroeluted and purified "sing an elutip column (Schleicher Schuell). Of the H 20 pg obtained, 6 pg are further digested with the restriction endonuclease Sau3a (20 units in a total of 100 pl of solution). The fragments -are separated using 2% agarose gel in TBE buffer.
After staining with ethidium bromide (EtBr) the S DNA fragment 189 bp long is electroeluted and purified as described above fragment b in Figure 6).
In order to link the interferon gene with a promoter, a ribosomal binding site and a starting codon, the corresponding DNA fragment is isolated II from the expression plasmid pER 33 Rastl-Dworkin et al., Gene 21, 237-248 (1983)). For this purpose pg of pER 33 are digested twice with the restriction 25 enzymes EcoRI and PvuII and the resulting fragments are fractionated according to their size on a 1.4% 0 e agarose gel in TBE buffer. The DNA fragment which is 389 bp long and contains the trp promotor, the ribosomal binding site and the starting codon, is electroeluted and purified using an elutip column.
The fragment thus obtained is then digested with Sau3A and the desired fragment 108 bp is obtained '.by agarose gel electrophoresis, electroelution and elutip column purification in a yield of approximately 1.00 ng fragment c in Figure 6).
ng of fragment are ligated with 20 ng of fragment c in a volume of 40 pl using 10 units of T4 ligase in a solution containing 50 mM Tris/Cl ~~y;rr 45 10 mM MgC1 2 1 mM DTT and 1 mM ATP, at 14 0 C for 18 hours. The enzyme is then destroyed by heating to 70 0 C and the resulting DNA is cut with HindIII in a total volume of 50 pi.
10 pg of the expression plasmid pER 103 (E.
Rastl-Dworkin et al., Gene 21, 237-248 (1983)) is linearised with HindIII in a total volume of 100 pl. After 2 hours at 37 0 C 1 volume of 2 x phosphatase buffer (20 mM Tris/Cl pH=9.2, 0.2 mM EDTA) together with one unit of calves intestine phosphatase (CIP) are added. After 30 minutes at 45 0 C, a further unit of CIP is added and the incubation is continued for 30 minutes. The DNA thus obtained is purified by extracting twice with phenol, once with chloroform .and precipitating by the addition of 0.3 M sodium acetate (pH=5.5) and 2.5 vol ethanol. It is then dephosphorylated in order to prevent religation of the vector during the next ligation step.
100 ng of the linearised pER 103 and the ligated fragment and (after HindIII digestion) S* are added to a solution of luO pl which contains ligase buffer and ligated using T4-DNA ligase for 18 hours at 14 0
C.
200 p1 of competent E. coli HB 101 Dworkin et al., Dev. Biol. 76, 435-448 (1980)) are mixed with 20 pl of the ligation mixture and incubated for 45 minutes on ice. Then the uptake of DNA is brought about by a heat shock for 2 minutes at 40 0 C. The cell suspension is incubated for L 30 a further 10 minutes on ice and finally applied to LB agar (10 g/1 trypton, 5 g/l yeast extract, S 5 g/l NaCl, 1.5% agar), containing 50 mg/l ampicillin.
The plasmids from the 24 resulting colonies obtained are isolated using the method of Birnboim and Doly (see Nucl. Acid. Res. 7, 1513-1523 (1979)). After digestion with various restriction enzymes one plasmid has the desired structure. This is designated pRHW 10 (see Figure 6).
46 b) Preparation of the plasmid pRHW 12 About 10 pg of the plasmid pRHW 10 are cut with BamHI. Then the Klenow fragment of DNA polymerase I and the 4 deoxynucleoside triphosphates are added and incubated for 20 minutes at ambient temperature.
The linear blunt-ended plasmid fragment obtained is purified by phenol extraction and precipitation and then cut with the restriction endonuclease NcoI in a volume of 100 pl. The larger fragment is obtained by agarose gel electrophoresis, electroelution and elutip purification. Fragment (a) (see Figure 6) is obtained by digestion of 4 pg of AvaII fragment which contains the P9A2-cDNA insert (see above) with NcoI and AluI, thereby obtaining about 2 pg of fragment In the final ligation step, fragment (a) and the pRHW 10 added thereto, which has twice been digested with BamHI/NcoI, is ligated in a volume of 10 pI, using 10 ng of each DNA. Ligation 20 of a filled in BamHI site to a DNA cut by Alul restores a BamHI recognition site. Competent E.
coli HB 101 are transformed with the resulting ligation mixture as described above. Of the 40 colonies obtained, 25 one is selected; this is designated pRHW 12.
The plasmid is isolated and the EcoRI/BamHI insert is sequenced using the method of Sanger Sanger et al., Proc. Nat. Acad. Sci 74, 5463- 5467 (1979)). This has the expected sequence.
S c) Preparation of the plasmid pRHW 11 .This is carried out analogously to Example Sb. 1 ug of the plasmid pRHW 10 is digested with BamHI. The sticky ends of the resulting DNA are converted to blunt ends using the Klenow fragment of DNA polymerase I and the 4 deoxynucleoside triphosphates and then the linearised DNA is cut with NcoI. The larger fragment is obtained by agarose 47 gel electrophoresis, electroelution and elutip column chromatography.
The NcoI-AluI fragment is isolated from the clone E76E9 analogously to Example 5b. Then 10 ng of the vector part is ligated with 10 ng of the cDNA part in a volume of 10 pl under suitable conditions using 1 unit of T4 ligase. After transformation of the resulting DNA mixture in E. coli HB 101 and selection of the 45 resulting colonies on LB plates containing impicillin, a clone is selected, which is designated pRHW 11. After the corresponding clone has been cultivated, the plasmid DNA is isolated.
Its structure is proved by the presence of several specific restriction endonuclease cutting sites (Alul, EcoRI, HindIII, NcoI, PstI).
d) Expression of interferon activity by E. coli HB 101 containing the plasmid pRHW 12 100 ml of the bacterial culture are incubated 20 up to an optical density of 0.6 at 600 nm in M9 too minimal medium which contains all the amino acids with the exception of tryptophan (20 pg/ml per amino acid), 1 pg/ml of thiamine, 0.2% glucose and 20 pg/ml of indol-(3)-acrylic acid (IAA), the 25 inductor of the tryphtophan operon. Then the bacteria are pelleted by centrifuging (10 minutes at 7000 rpm), washed once with 50 mM Tris/Cl pH=8, 30 mM NaCl S. and finally suspended in 1.5 ml of the same buffer.
After 30 minutes incubation with 1 mg/ml of lysozyme Sp 30 on ice the bacteria are frozen and thawed five S times. The cell debris are eliminated by centrifuging for 1 hour at 40,000 rpm. The supernatant is filtered Ssterile and tested for interferon activity in a plaque reduction assay using human A549 cells and encephalo-myocarditis virus.
Result: 1 litre of the bacterial culture produced contains 1 x 10 international units of interferon Billiau, Antiviral Res. 4, 75-98 (1984)).
48 Example 6 Summary of the differences between the amino acid and nucleotide sequences of Type I interferons a) Comparison of the amino acid sequences A pairwise comparison of amino acid sequences is obtained by aligning the first cysteine residue of mature o-interferon with the first cysteine residue of the amino acid sequences which are coded for by the cDNA inserts of the plasmids P9A2 and E76E9. The two sequences are shown in Figure 7 as IFN-omega, since no differences could be detected between the specific sequences of the P9A2 or E76E9 clones in the values obtained. The only correction made was the insertion of a gap at position of the interferon-oA, which was counted as mismatch.
If the sequence of the omega-interferon is a partner, 'the comparison is carried out taking into account the usual 166 amino acids. This value is shown in Figure 7 together with the additional 6 amino acids coded for by the clones P9A2 and E76E9.
The percentage differences are obtained by dividing the differences by the number 1.66. An additional amino acid thus represents a percentage of 0.6.
This gives 3.6% for the 6 additional amino acids of IFN-omega which are already contained in the Si* percentage.
The comparisons with B-interferon are carried out by aligning the 3rd amino acid of the mature B-interferon with the first amino acid of the mature o-interferon or the first cysteine which is coded for by the plasmids P9A2 and E76E9. The longest *l comparison structure of an o-interferon with Binterferon is thus over 162 amino acids, which gives 2 additional amino acids each for the o-interferon and B-interferon. These are counted as errors and are shown separately in Figure 7 but they are included in the percentage. The listing of B-interferon with the amino acid sequences of the clones P9A2 T 49 or E76E9 is carried out in the same way. However, this gives a total of 10 additional amino acids.
b) Comparison of the nucleotide sequences The sequences which are to be compared are listed analogously to Example 6a. The first nucleotide of the DNA of the mature o-interferon is the first nucleotide of the triplet of mature o-interferon coding for cysteine. The first nucleotide from the DNA of the plasmids P9A2 or E76E9 is also the first nucleotide of the codon for cysteine-l.
The first nucleotide from the DNA of B-interferon is the first nucleotide of the third triplet.
The comparison is made over a total of 498 nucleotides if the individual DNAs of the o-interferons are o-mpared with the DNA of B-interferon, and over 516 nucleotides if the DNA sequences of the individual o-interferons or of the B-interferon are compared S. with those of the plasmids P9A2 and E76E9. The S 20 absolute number of gaps is given in the left-hand part of the Table in Figure 7 and then the corresponding percentages are given in brackets.
Example 7 Virus-inducible expression of omega-l-mRNA in Namalwa cells and NC37 cells O* a) Synthesis of a specific hybridisation probe for omega-l mRNA 10 pMol of the oligonucleotide d(TGCAGGGCTGCTAA) 3 2 are mixed with 12 pMol of gamma-3P-ATP (specific activity: 7 5000 Ci/mMol) and 10 units of polynucleotide S kinase in a total volume of 10 pl (70 mM Tris/Cl pH 7.6, 10 mM MgC1 2 50 mM DTT) and left to stand for one hour at 37 0 C. The reaction is then stopped by heating to 70 0 C for 10 minutes. The resulting radioactively labelled oligonucleotide is hybridised with 5 pMol of M13pRHW 12 ssDNA (see Figure 9) in a total volume of 35 pl (100 mM NaC1) by standing for one hour at 50 0
C.
50 After cooling to ambient temperature, nick translation buffer, the 4 deoxynucleoside triphosphates and 10 units of Klenow polymerase are added to give a total volume of 50 pl (50 mM Tris/Cl pH 7.2, 10 mM MgCl 2 50 pg/ml BSA, 1 mM per nucleotide).
Polymerisation is carried out at ambient temperature for one hour and stopped by heating to 70 0 C for minutes.
During the reaction, a partially double-stranded circular DNA is obtained. This is then cut in a total volume of 500 p1 with 25 units of AvaIl, using the buffer described by the manufacturer.
The double stranded regions are cut to uniform sizes. The reaction is then stopped by heating to 70 0 C for 5 minutes.
b) Preparation of RNA from virus-infected cells *6 6 i 100 x 10 cells (0.5 x 10 /ml) are treated with 100 uMol of dexamethasone for 48 to 72 hours 20 the control contains no dexamethasone. To induce interferon, the cells are suspended in serum-free medium in a concentration of 5 x 10 /ml and infected with 21 units/ml of Sendai virus. Aliquot parts of the cell culture supernatants are tested for IFN activity in a plaque reduction assay (Example 5d) The cells are harvested 6 hours after the virus infection by centrifuging (1000 g, 10 minutes), washed in 50 ml of NP40 buffer (Example 4a), resuspended ;in 9.5 ml of ice cold NP40 buffer and lysed by the addition of 0.5 ml of 10% NP40 for 5 minutes Son ice. After the nuclei have been removed by centrifuging (1000 x g, 10 minutes) the supernatant is adjusted to pH=8 with 50 mM Tris/Cl, 0.5% sarcosine and 5 mM EDTA and then stored at -200C. In order to isolate the total RNA from the supernatant, it is extracted once with phenol, once with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol. The aqueous phase is layered on top of 51 a 4 ml 5.7 molar CsC1 cushion and centrifuged in an SW40 rotor (35 krpm, 20 hours) in order to free the extract from DNA and remaining proteins. The resulting RNA pellet is resuspended in 2 ml of TE, pH=8.0, and precipitated with ethanol. The RNA precipitated is then dissolved in water at a concentration of 5 my/ml.
c) Detection of interferon-omega mRNA 0.2 pl of the hybridisation probe prepared in Example 7(a) are precipitated together with 20-50 pg of the RNA prepared according to Example 7(b) by the addition of ethanol. As a control, transfer RNA (tRNA) or RNA originating from E. coli transformed with the plasmid pRHW 12 (Example 5) is added instead of cellular RNA.
The resulting pellets are washed free from salt with 70% ethanol, dried and dissolved in 25 ul of formamide (100 mM PIPES pH=6.8, 400 mM NaC1, 10 mM 20 EDTA). Then the samples are heated to 100 0 C for 5 minutes in order to denature the hybridisation sample, adjusted directly to 52 0 C and incubated for 24 hours at this temperature. After hybridisation, the samples are placed on ice and 475 pl of Sl reaction mixture (4 mM Zn(Ac) 2 30 mM NaAc, 250 mM NaC1, 5% glycerine, 20 ug ss calf thymus DNA, 100 units Sl nuclease) are added. After digestion at 37°C for 1 hour the reaction is stopped by ethanol *l precipitation.
The pellets are dissolved in 6 pl formamide buffer and separated essentially like samples from DNA sequencing reactions on a 6% acrylamide gel containing 8 M urea Sanger et al., Proc. Nat.
Acad. Sci. 74, 5463-5467 (1979)).
For autoradiography, the dried gel is exposed to a DuPont Cronex X-ray film using the Kodak Lanex regular intensifying screen at -700C.
Y*U
~~IE
AZ
*4 a. @0S es 6 6S 0Or 5
S
S r S. *r
S.
S S OS S 52 Legend relating to Figure Lanes A to C represent the controls.
Lane A: 20 pg tRNA Lane B: 10 pg RNA from pER33 coli expression strain for interferon-o2-Arg) Lane C: 1 ng RNA from pRHW12 coli expression strain for interferon-omega 1) Lane D: 50 pg RNA from untreated Namalwa cells Lane E: 50 pg RNA from virus-infected Namalwa cells Lane F: 50 pg RNA from Namalwa cells pretreated with dexamethasone and infected with virus Lane G: 20 pg of RNA from untreated NC 37 cells.
.Lane H: 20 pg RNA from virus-infected NC 37 cells Lane I: 20 pg RNA from NC 37 cells pretreated with dexamethasone and infected with virus Lane M: Size marking (pBR322 cut with Hinfl).
Lanes B and C show that the expected signal can only be detected when an omegal-specific RNA is among the RNA molecules. They also show that even a large excess of the wrong RNA does not cause a background signal (see lane Furthermore, the tRNA used as a hybridisation partner does not produce a signal either (see lane A).
Lanes G to I show the induction of the omegalspecific RNA in virus-infected NC 37 cells. The 30 pretreatment with dexamethasone enhances this effect.
Lanes D to F show that fundamentally the same result is obtained with Namalwa cells as with NC37 cells. However, the induction of omegal-specific RNA is not as great as in NC 37 cells. This result is parallel to the interferon titres which were measured in the corresponding cell supernatants.
This behaviour of interferon-omegal gene expression is thus as would be expected from an interferon Type I gene.
53 Example 8 Isolation of the gene coding for IFN-omegal or genes related thereto: a) Cosmid screening A human cosmid bank (human DNA (male) cloned in the cosmid vector pcos2 EMBL Ponstka, H.-R.
Rockwitz, Frischauf, B. Hohn, H. Lehrach Proc. Natl. Acad. Sci. 81, 4129-4133 (1984)) with a complexity of 2 x 106) was screened for the IFNomega gene or related genes. E. coli DH1 (rK-, mK+, rec.A; gyrA96, sup.E) was used as the host.
First of all, Mg cells ("plating bacteria") were prepared. E. coli DH1 grows overnight in L broth g/1 trypton, 5 g/l yeast extract, 5 g/l NaC1) .supplemented with 0.2% maltose. The bacteria are removed by centrifuging and taken up in a 10 mm MgSO 4 solution to give an optical density 6 0 0 2. 5 ml of this cell suspension are incubated 1 6 with 12.5 x 10 colony forming units of packed cosmids for 20 minutes at 37 0 C. Then 10 vol of a j LB are added and the suspension is kept at 37 0
C
for one hour for the purpose of expression of the kanamycin resistance coded for by the cosmid.
The bacteria are then removed by centrifuging, resuspended in 5 ml of LB and spread over nitrocellui* lose filter in 200 pl aliquots (BA85, Schleicher and SchUll, 132 mm diameter) placed on LB agar agar in L broth) plus 30 pg/ml kanamycin.
S About 10,000 to 20,000 colonies grow on each filter.
The colonies are replica-plated on further nitrocellulose filters which are kept at 4 0
C.
A set of the colony filters is processed as described in Example Ic), i.e. the bacteria are denatured, and the single strand DNA is fixed to the nitrocellulose. The filters are washed for 4 hours at 65 0 C in a 50 mM Tris/HCl, 1 M NaC1, 1 mM EDTA, 0.1% SDS solution. The filters are then incubated at 65 0 C for 2 hours in a 5 x Denhardt's 54 (see Example Ic), 6 x SSC, 0.1% SDS solution and hybridised with about 50 x 106 cpm of nick-translated denatured IFN-omegal DNA (HindIII-BamHI insert of the clone pRHW12, see Figure 6) for 24 hours at 65 0 C in the same solution. After hybridisation, the filters are washed first 3 x 10 minutes at ambient temperature in a 2 x SSC, 0.01% SDS solution and then 3 x 45 minutes at 65 0 C in a 0.2 x SSC, 0.01% SDS solution. The filters are dried and exposed to Kodak X-Omat S film using an intensifier film at -70 0 C Positively reacting colonies are localised on the replica filters, scratched off and resuspended in L broth kanamycin (30 pg/ml).
Of this suspension, a few pl are spread out on .LB agar 30 pg/ml kanamycin. The resulting colonies are replica-plated on nitrocellulose filters.
These filters are hybridised with 32P-labelled IFN-omegal-DNA as described above. From each hybridising colony, the cosmid is isolated using the method 20 described by Birnboim Doly (Nucl. Acids Res.
I 7, 1513 (1979)). With this cosmid DNA preparation, N E. coli DH1 was transformed and the transformants were selected on LB agar 30 pg/ml kanamycin.
32 The transformants were again tested with P-radioactively labelled IFN-omegal DNA for positively reacting clones. One clone in eac, case starting from the original material isolated is selected *ee and the cosmid thereof is produced on a larger scale (Clewell, D.B. and Helinski, Biochemistry 30 9, 4428 (1970)). Three of the isolated cosmids are designated cos9, coslO and cosB.
Sub-cloning of hybridising fragments in PUC8 1 pg of cosmids cos9, cosl0 and cosB were cut with HindII under the conditions recommended by the manufacturer (New England Biolabs). The fragments are separated on 1% agarose gels in TBE buffer by electrophoresis and transferred to nitro-
-^I
55 cellulose filters according to Southern (Example 4c). The two filters are hybridised with nicktranslated IFN-omegal DNA as described in Example 4d, and washed and exposed. About 20 pg of each cosmid are cut with HindIII and the fragments formed are separated by gel electrophoresis. The fragments hybridising with IFN-omegal-DNA in the preliminary tests are electroeluted and purified via elutip columns (Schleicher Schuell). These fragments are ligated with HindIII-linearised dephosphorylated pUC8 (Messing, Vieira, Gene 19, 269-276 (1982)) and E. coli JM101 (supE, thi, (lac-proAB), traD36, pro AB, lac q Z M15] P.L. Biochemicals) is transformed with the ligase reaction solution.
The bacteria are spread on LB agar containing 50 ug/ml of ampicillin, 250 ug/ml of 5-bromo-4-chloro-3indolyl-B-D-galactopyranoside (BCIG, Sigma) and o 250 ug/ml of isopropyl-$-D-thiogalacto-pyranoside (IPTG, Sigma). A blue colour of the resulting 20 -olonies indicates the absence of an insert in pUC8. The plasmid DNAs were isolated on a small 'scale from some white clones, then cut with HindIII and separated on 1% agarose gels. The DNA fragments were transferred to nitrocellulose filters and 32 hybridised with P-IFN-omegal-DNA as above. Starting from cos9 and cosl0, a subclone was selected in each case. These clones have been designated pRHW22 and pRH57. From cosB, two DNA fragments which hybridise well with the IFN-omegal DNA probe were e.g 30 subcloned. The resulting clones have been designated pRH51 and pRH52.
S" c) Sequence analysis The DNA inserted in pUC8 is separated from the vector part by cutting with HindIII and subsequent gel electrophoresis. This DNA, about 10 ug, is ligated in 50 pl of reaction solution using T 4 DNA ligase, the volume is adjusted to 350 pl with d 56 nick translation buffer (Example 4d) and then decomposed using ultrasound, whilst cooling with ice (MSE 100 Watt ultrasonic disintegrator, maximum output at 20 kHz, five times 30 seconds). Then the ends of the fragments are repaired by adding 1/100 vol of 0.5 mM dATP, dGTP, dCTP and dTTP and units of Klenow fragment of the DNA polymerase I for 2 hours at 14 0 C. The resulting DNA fragments having blunt ends are separated according to their size on a 1% agarose gel. Fragments of sizes between 500 and 1000 bp were isolated and subcloned in the dephosphorylated phage vector M13 mp8 cut with SmaI. The single strand DNA of recombinant phages is isolated and sequenced using the method developed by Sanger (Sanger, F. et al., Proc. Natl. Acad.
Sci 74, 5463-5467 (1976)). The individual sequences are put together to form the total sequence by means of computers (Staden, Nucl. Acids Res.
10, 4731-4751 (1982)) S* d) Sequence of the subclone pRH57 (IFN-omegal) .i The sequence is shown in Figure 11. This fragment which is 1933 bp long contains the gene for interferon-omegal. The region coding for protein comprises the nucleotides 576 to 1163. The sequence is totally identical to that of the cDNA insert Sof clone P9A2. The nucleotide portion 576 to 674 codes for a signal peptide of 23 amino acids.
The TATA box is at a distance characteristic of interferon Type I genes in front of the starting codon ATG (positions 476-482). The gene has a j number of signal sequences for polyadenylation during transcription (ATTAAA at positions 1497- 1502, or 1764-1796; AATAAA at positions 1729-1734 or 1798-1803), the first of which is present in the clone P9A2.
57 e) Sequence of the subclone pRHW22 (IFN-pseudoomega2) Figure 12 shows the sequence, 2132 bp long, of the HindIII fragment from the cosmid cos9 which hybridises with the IFN-omegal-DNA probe. An open reading frame exists from nucleotide 905 to nucleotide 1366. The amino acid sequence derived therefrom is shown. The first 23 amino acids are similar to those of the signal peptide of a typical Type I interferon. The following 131 amino acids show a similarity to interferon omegal, up to amino acid 65, whilst tyrosine is notable as the first amino acid of the mature protein.
Following amino acid position 66 is the sequence -of a potential N-glycosylation site (Asn-Phe-Ser).
From this point onwards the amino acid sequence is different from that of a Type I interferon.
S- However, it can be demonstrated that similarity to IFN-omegal can be established by suitable insertions 20 and the resulting displacement of the protein reading frame (see Example Thus, from the standpoint of Type I interferons, the isolated gene is a pseudogene: I IFN-pseudo-omega2.
f) Sequence of the subclone pRH51 (IFN-pseudo-omega3) The HindIII fragment originating from cosmid B and about 3500 bp long which hybridises with the IFN-omegal DNA probe is partially sequenced (Figure 13). An open reading frame is obtained from nucleotide position 92 to 394. The first 23 amino acids display the features of a signal i peptide. The subsequent sequence starts with tryptophan and shows similarity to IFN-omegal up to amino acid 42. Thereafter, the derived sequence is different from IFN-omegal and ends after amino acid 78.
The sequence can be altered by insertions so that it is then greatly homologous to IFN-omegal (Example The gene is designated IFN-pseudo-omega3.
-IF
II r m I 58 g) Sequence of the insert of pRH52 (IFN-pseudoomega4) The sequence of the HindIII fragment which is 3659 bp long, is isolated from cosmid B and which hybridises with IFN-omegal-DNA, is shown in Figure 14. An open reading frame, the translation product of which is partially homologous to IFNomegal, is located between nucleotide positions 2951 and 3250. After a signal peptide of 23 amino acids, the further amino acid sequence begins with phenylalanine. Homology to IFN-omegal is interrupted only after the 16th amino acid, continues at the 22nd amino acid and ends at the 41st amino acid.
Translation would be possible up to amino acid .77. Analogously to Example 8e) and 8f), good homology can be established with IFN-omegal by the introduction of insertions (Example The pseudo gene isolated here is designated IFN-pseudo-omega4.
20 Example 9 o, Evaluation of the genes for 4 members of the IFNomega family Figure 15 is a listing of the genes for IFNomegal to IFN-pseudo-omega4 together with the amino S* *25 acid translation. To establish better analogy, gaps are inserted in the individual genes which are indicated by dots. No bases are omitted.
The numbering of the bases includes the gaps.
The amino acid translation of IFN-omegal is retained at positions 352-355: "C.AC" codes for His) S In the case of the pseudo genes, translation into San amino acid is given only where this is unambigiously possible. This list immediately shows that the S4 isolated genes are related to one another. Thus, for example, the potential N-glycosylation site (nucleotide positions 301 to 309) is obtained in all 4 genes.
I
59 Similarly, apart from the case of IFN-pseudoomega4, at nucleotide positions 611 to 614, there is a triplet which represents a stop codon and which, in the case of IFN-omegal, terminates a mature protein with a length of 172 amino acids. There are premature stop codons in IFN-pseudo-omega2 (nucleotide positions 497 to 499) and in IFN-pseudo-omega4 (nucleotide positions 512 to 514) using this arrangement.
The degree of relationship between the genes or the amino acid translations can be calculated from the arrangement shown in Figure 15. Figure 16 shows the DNA homologies between the members of the IFN-omega gene family. In the comparison in pairs, those positions where one of the two -partners or both partners have a gap are not included in the counting. The comparison gives a homology S" of about 85% between IFN-omegal-DNA and the sequences of the pseudo genes. IFN-pseudo-omega2-DNA is
S*
e about 82% homologous to the DNAs of IFN-pseudo- 20 omega3 and IFN-pseudo-omega4. Figure 17 shows the results of comparisons of the signal sequences and Figure 18 shows the results of the comparisons of the "mature" proteins. The latter vary between 72 and 88%. However, this homology is substantially 25 greater than that between IFN-omegal and the o-IFNs and B-xFN (Example The fact that the individual 'members of the IFN-omega family are more distant from one another than the members of the o-IFN SOfamily can be explained by the fact that three 30 of the four isolated IFN-omega-genes are pseudo S genes and are not subject to the same selection pressure as functional genes.
-F-
11-- 11 60 Example Fermentation Strain storage: A single colony of the strain E. coli HBl01/pRHW12 on LB-agar (25 mg/l ampicillin) is inoculated into trypton-soya-broth (OXOID CM129) containing 25 mg/l ampicillin and incubated at 370C by shaking at 250 RPM until an optical density at 546 nm of about is reached (log-phase). 10% of sterile glycerol are added to the culture, which is then placed in sterile ampoules in 1.5 ml portions and frozen at -70 0
C.
Inoculum stage: The medium contains 15 g/l Na 2
HPO
4 12H 2 0; g/l NaCI; 1.0 g/l NH 4 Cl; 3.0 g/l KH 2
PO
4 0?.5 g/l MgSO 4 7H 2 0; 0.011 g/l CaC1 2 5 g/l casamino acid (Merck 2238); 6.6 g/l glucose-monohydrate; 0.1 g/l ampicillin; 20 mg/l cysteine and 1 mg/l thiamine- 20 hydrochloride. Each of 4 1000 ml Erlenmeyer flasks o containing 200 ml of this medium is inoculated with 1 ml of a thawed culture of HB101/pRHW12 and incubated by shaking at 250 RPM at 37 0 C for 16 to 18 hours.
Production stage: 00 0 The medium consists of 10 g/l (NH 4 2 HP0 4 4.6 g/1 K 2
HPO
4 .3H 2 0; 0.5 g/1 NaCl; 0.25 g/1 MgSO 4 .7H 2 0; 0.011 g/l CaCI2; 11 g/l glucose-monohydrate; 21 g/l casamino acids (Merck 2238); 20 mg/1 cysteine, S1 mg/l thiamine-hydrochloride and 20 mg/1 0 acid. 8 litres of a sterile medium in a 14 1 fermentnr 0. (height:radius 3:1) are inoculated with 800 ml of said cultures. The fermentation runs at 28 0
C,
1000 RPM of agitation (effigas-turbine), an aeration rate of 1 vvm (volume/volume/minute) and an initial pH of 6.9. During fermentation, the pH decreases to 6.0 and is then regulated automatically at this
IF
61 level by 3N NaOH. After the optical density at 546 nm has reached 18 to 20 (usually after to 9.5 hours of fermentation) the culture is cooled to 20'C under aeration and agitation and then brought pH =2.2 by addition of 6N H2SO 4 without aeration.
After one hour of agitation at 800 RPM and the resulting biomass is centrifuged in a CEPA laboratory centrifuge type GLE at 30,000 RPM.
The cell paste is frozen and stored at 1i Purification of omega (Gly)-interferon a) Partial purification All steps are performed at 4'C.
140 g of biomass coli HB101 transformed ":'.with the expression plasmid pRHWI2) are resuspended in 1150 ml of precooled 1% acetic acid and stirred oooo for 30 minutes. The pH of the suspension is shifted aol •to 10.0 by the addition of 5 M NaOH. The suspension 20 is stirred for another two hours. The pH is then readjusted to 7.5 using 5 M HCI and the stirring continued for a further 15 minutes. The suspension is centrifuged at 4°C for 1 hour at 10,000 rpm (J21 centrifuge (Beckman) 25 The clear supernatant is applied to a 150 ml controlled pore glass-column (CPG 10-350, mesh size 120/200) at a flow rate of 50 ml/hour. The column is washed using 1 1 25 mM Tris pH 7.5/1M NaCI and the bo~und material is •-luted with a solution containing 25 mM Tris pH 7.5/1M KCNS/50% ethylenei~ii •glycol at a flow rate of 50 ml/h,)ur.
:The interferon activity containing fractions are pooled and dialysed over night against about i100 vol. of 0.1 M Na-phosphate/10% polyethyleneglycol 40,000. The resulting precipitate is removed by centrifugation at 40C for 1 hour at 10,000 rpm (J21 centrifuge, JA20 rotor) [See Table 1 below].
I
62 b) Further purification The dialysed and concentrated CPG-eluate is diluted with 5 vol of buffer A (0.1M Na-phosphate pH 6.25/25% 1, 2 -propyleneglycol). The solution is applied to a MonoS 5/5 (Pharmacia, cation exchange) column equilibrated with buffer A using a "superloop" (Pharmacia). Elution is carried out using a linear gradient from 0 to 1M NaCl in buffer A in a total of 20 ml at a flow rate of 0.5 ml/hour. The flow through and the 1 ml fractions are collected and tested for interferon activity by means of a plaque reduction assay using human A549 cells and EMCvirus. The active fractions are pooled.
Table 1 0.0 0
S.
055
S
555
S
S volume Biological test protein total u/mg yield (ml) (u/ml U total (mg/ml) (mg) start 1150 15000 17.3x10 6 3.6 4140 4180 100% ft 2200 4 600 4 1.3x10 6 0.74 1628 e 600 4 eluate 124.3 170000 21.0x10 6 16.8 2088 10000 121% after dia- 41 300000 12.3xl0 6 12.6 516.6 23800 71% lysis 1- Plaque-reduction assay: A549 cells, EMC-virus flow through units using interferon-a2 as a standard -63 Example 12 Characterisation of HuIFN-omegal A. Antiviral activity on human cells B. Antiviral activity on monkey cells C. Antiproliferative activity on human Burkitt's lymphoma cells (Cell line: Daudi) D. Antiproliferative activity on human cervical carcinoma cells (cell line: Hela) and synergism with HuIFN-gamma and human tumour necrosis factor E. Stability at low pH F. Serological characterisation.
A. Antiviral activity on human cells Assay cell line: Human lung carcinoma cells A549 (ATCC CCL 185) I Challenge virus: Murine encephalomyocarditis virus (EMCV) Assay method: Inhibition of cytopathic s e effect 20 A partially purified preparation of HuIFNomegal with a protein content of 9.4 mg/ml was diluted in cell culture medium and applied to the assay plates. The preparation showed an antiviral effect with a specific activity of 8300 lu/mg relative 25 to the reference standard preparation Go-23-901-527 (National Institutes of Health, Bethesda, Maryland,
USA).
f B. Antiviral activity on monkey cells 30 Assay cell line: GL-V3 vervet monkey kidney cells (Christofinis G.J., J. Med. Microbiol. 3, 351-258; S" 1970).
Challenge virus: vesicular stomatitis virus
(VSV)
Assay method: Plaque reduction A partially purified preparation of HuIFNomegal (see Example 12A) was diluted in culture 64 medium and applied to the assay cells. The preparation showed a specific activity of 580 units/mg.
C. Antiproliferative activity on human Burkitt's lympnoma cells (Cell line: Daudi) The human Burkitt's lymphoma cell line Daudi was grown in the presence of various concentrations of HuIFN-omegal (see Example 12A). Cultures were started at 100,000 cells/ml; cell densities were determined after 2, 4 and 6 days in culture (37 0
C)
Untreated cultures served as controls. All cultures were run in triplicate. Figure 19 shows the results of the experiment. Cell proliferation was partially and transiently inhibited at 10 lU/ml, and was strongly inhibited at 100 1U/ml.
The following symobols are used in Figure 19: O control, lIu/ml, 0 10 Iu/ml, i 100 IU/ml.
D. Antiproliferative activity on human cervical 20 carcinoma cells (cell line: Hela) The human cervical carcinoma cell line HeLa was grown in the presence of one of the following proteins or a mixture of two of the following proteins: HuIFN-omegal (see Example 12A) at 100 IU/ml.
25 HuIFN-gamma (see Example 12A) at 100 IU/ml.
Human tumour necrosis factor (HuTNF), *98% pure, prepared by Genentech Inc., San Francisco, USA (see Pennica D. et al., Nature 312, 712-729, 1984) at 100 ng/ml.
All binary combinations of the mentioned S proteins had the same concentrations as above.
In each case, 2 cultures were started at 50,000 cells/3 cm petri dish and incubated for 6 days at 37 0 C; thereafter cell densities were determined.
HuIFN-omegal and HuTNF had only a weak influence on the growth of the cells, whereas HuIFN-gamma showed a clear cytostatic activity. A combination of IFN-gamma with IFN-omegal showed a synergistic cytostatic/cytotoxic activity. The results of this experiment are shown in Figure In this figure, the following symbols are used: C untreated control, T HuTNF, 0 HuIFN-omegal, G HuIFN-gamma.
E. Stability at low pH A preparation of HuIFN-omegal (see Example 12A) was diluted in cell culture medium RPMI 1640 medium containing 10% fetal calf serum) and adjusted to pH 2 with hydrochloric acid. Following incubation at 4 0 C for 24 hours, the preparation was neutralized using sodium hydroxide and its antiviral activity titrated as in Example 12A. The preparation showed 75% of the activity of a control incubated at neutral pH; HuIFN-omegal therefore can be regarded as stable S. at low pH.
F. Serological characterisation To compare the serological properties of HuIFN-omegal and Hu-lFN-a2, samples of both proteins (see Example 12A) were diluted to contain 100 iu/ml, mixed with equal volumes of solutions of various antisera or monoclonal antibodies and incubated for 90 minutes at 37 0 C. The antiviral activity of these samples was then compared with those of untreated controls. Table 2 shows the results of the experiment. The antiviral activity of HuIFNj omegal was neutralized only by an antiserum to human leukocyte-derived IFN at relatively high concentration, but not by a polyclonal antiserum to HuIFN-, HuIFN-a2 or various monoclonal antibodies that neutralise HuIFN-a2. HuIFN-omegal is thus serologically unrelated to HuIFN-a2 as well as to HuIFN-B. Symbols used in Table 2: not tested, 0, no neutralization, partial neutralization, complete neutralization.
66 Table 2 C C S. C
C
C
C.
C C
C
CO
C S
C
CCCO
C
eC 0O C
S
C CO 0 6 0@
C.
C eeC e.g.
C. 6 OC 0 06
S.
Antiserum Dilution Neutralization of monoclonal Pg/mi antibody HuIFN-cz2 HuIFN-omegal EBI-l 1 1 100..
1000 0 EBI-3 1 1 100..
1000 0 L3B7 2 100 0 1000 0 sheep n-3 leukocyte TEN 1: 50 000..
1: 5 000 0 1: 500 1: rabbit anti HuIFN-ca2 1: 1 000..
1: 100 0 1: 10 0 sheep anti 4 HuIFN-3 1: 50 -0 1) EP-A 0.119.476 2) Drug Research 35 364-369 (1985) 3) Research reference reagent catalog no. G-026-502-568 4) Research reference reagent catalog no. G-028-501-568 Research Resources Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland,
USA.
J~ I ~1

Claims (32)

1. -He omega interferon polypeptides of Type 1 coded for by the cDNA inserts of plasmids P9A2 and E76E9 (deposited at the DSM with E. coli HB 101 under the numbers DSM 3004 and 3003 respectively), the polypeptides coded for by pseudo omega interferon genes 2, 3 or 4, further interferon polypeptides having omega interferon properties comprising 168 to 174 amino acid residues which have an amino acid divergence of at least to 50% compared to alpha-interferons and a divergence of at least compared to beta-interferon, and coded for by sequences which will hybridise with the sequence corresponding to the omega-interferon-coding sequences of plasmids P9A2 and E76E9 or degenerate variations thereof under stringent hybridisation conditions as hereinbefore defined for I detecting about 85% or higher homology, optionally further comprising a SLeader peptide, and N-glycosylated derivates thereof with omega 15 interferon activity. I 2. Omega interferon polypJeptides as claimed in claim 1 which have a divergence of 40 to 48% compared to a-interferons and N-glycosylated I derivatives thereof with interferon activity. o 20 3. Omega interferon polypeptides as claimed in claim 1 or claim 2 of 172 amino acids, and N-glycosylated derivatives thereof with interferon activity.
4. The interferon polypeptide of claim 3 designated omega (Gly)-interferon having the amino acid sequence: S.. p.~LI' 68 J U Cys Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr Leu 1 ii I U '1 Val Lys Ser Leu Ala Gin Glu Arg 20 Tyr Ser Leu Asp Gin Gin Trp Gin Gly Arg Ser Le u Leu Arg Leu Gin Asn Leu Giu Tyr Asp Phe His Arg Gin Ilie Met Gin Ser P he Cys Le u Gin Met Asp Phe 50 Lys Ala Phe Ser Thr Leu His Leu 110 Ala Gly 125 Gin Giy 140 Al 1a Tr p i155 S er Th r Arg Ar g His Leu Le u Giu Ala ile Giu A sn Arg phe Val1 Ph e Asp Th r Ile Arg Val M, t Ile Pro Met His Gin Cys Ser Vai Vai Gi1n Ser 40 Gin -55 Ser 70 Thr 85 Leu 100 Leu 115 Ser 130 Ty r 145 Arg i160 Giu Pro G I U Val1 Glu His Leu Pro Leu Mlet Arg Phe Mi t Leu Arg Thr Gin Ala Lys Giu Leu Leu Val His Ser Gly Val Leu Glu Ile Arg Cys Leu Lys Gly Giu Met Ser Ala Leu His 105 Val Gly 120 Thr Leu 135 Lys Lys 150 Met Lys 165 Ser Lys Does a. 000 a 00 000 *0 60 170 25 Asp Arg Asp Leu Giy Ser Ser and derivatives thereof N-cjiycosyiated at amino acid position 78, O~.th interferon activity. The interferon polypeptide of claim 3 designated omega (Glu)-interferon, having the same amino acid sequence as the interferon polypeptide claimed in claim 4 except that amino acid residue Ill is glutamic acid rather than glycine, and derivativ-s thereof N-glycosylated at amino acid position 78 with interferon activity. 69
6. An omega interferon polypeptide as claimed in any one of claims 1 to 5 which is fused to the leader peptide: Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly.
7. An omega interferon substantially as hereinbefore described.
8. -Apolydeoxyribonucleotide comprising a coding sequence for an omega-interferon as claimed in claim 1 or comprising a pseudo gene sequence capable of hybridising with a sequence corresponding to such an omega interferon-coding sequence under 15 stringent hybridisation conditions as defined herein, S. suitable for detecting about 85% or higher homology with the chosen interferon coding sequence. I 9. A polydeoxyribonucleotide as claimed in claim 8 comprising the omega (G]y)-interferon coding sequence: 2 0 TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 135 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 180 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 225 S 2 5 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 270 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 315 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 36C SAGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 405 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA S 3 0 TCC TTG TTC TTA TCA A AA AAC ATG CAA GAA AGA-CTG AGA AGT AAA 495 GAT AGA GAC CTG GGC TCA TCT 516 ,or a degenerate variation thereof. 70 A polydeoxyribonucleotide as claimed in claim 8 comprising the coding sequence given in claim 9 except that codon 111 is GAG (coding for glutamic acid) rather than GGG (coding for glycine), or a degenerate variation thereof.
11. A polydeoxyribonucleotide as claimed in any one of claims 8, 9 and 10 wherein the omega-interferon coding sequence or pseudo gene sequence is fused with a coding sequence for a leader peptide.
12. A polydeoxyribonucleotide as claimed in claim 11 Scomprising an omega (Gly)-interferon coding sequence or an omega (Glu)-interferon coding sequence fused S. 15 with the nucleotide sequence: oATG GCC CTC CTG TTC CCT CTA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC s**o coding for the leader peptide defined in claim 6.
13. A polydeoxyribonucleotide as claimed in claim 12 i 30 comprising the omega (Gly)-interferon gene of sequence: ee 0 *g I' W ^LI 0SS4 #9 9* 4 Sc,- S S'S. Iv 4 55 0 S 6 St.. S** *O59 S PS S .5 5 @4 4. 59 I S *@05 5* 0 #9 #4 71 GATCTGGTAAACCTGAA 17 GGAAATATAGAAACCTATAGGGCCTGAGTTCCTACATAAAGTAAGGAGGGTAAAAATGG 76 AGGCTAGAATAAGGGTTAAAATTITTGCTTCTAGAACAGAGAAAATGATTTTTTTCATAT 135 ATATATGAATATATATTATATATACACATATATACATATATTGACTATAGTGTCJTATAC 194 ATAAATATATAATAnPTATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT 253 AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCAT'TTGATTAAGCATTCATTT 312 GTATTGACACAGCTGAAGTTTAGTGGAGTTTAGCTGAAGTGTAATGCAAAATTAATAGA 371 TTGTTGTCATCCTCTTAAGGTCATAGGGAGAACACACAAATGAAAACAGTAAAAGAAAC 430 TGAALAGTACAGAGAAATGTTCAGAAAATGAAAACCATGTGTTTCCTATTAAAAGCCATG 489 GATACAAGGAATGTCTTCAGAAAACCTAGGGTGCAAGGTTAAGGCATATCCCAGGTCAG 548 TAAAGCCAGGAGC-ATCGTCATTTCCCA ATG GC( CTCGTG TTC CCT CTA CTG 599 GGA GCG GTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC 644 TGT GAT CTG CCT GAG AAC CAT GGC GTA CTT AGG AGG AAC ACC TTG 689 15GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 734 AAG GAC AGA AGA GAC TTC AGG TTC CCC GAG GAG ATG GTA AAA GGG 779 AGC CAG TTG GAG AAG GCC CAT GTC ATG TCT GTG CTC CAT GAG A'TG 824 GTG GAG GAG ATG TTC AGC CTC TTC GAG ACA GAG CG TCG TCT GCT 869 GCC TGG AAG ATG ACC CTC CTA GAG CAA CTG GAG ACT GGA GTT CAT 914 2 0CkG CAA GTG CAA GAG GTG GAG AGC TGG TTG GTG GAG GTA GTG GGA 959 GAA GGA GAA TGT GGT G GG GCA ATT AGC AGG GCT GCA CTG ACG TTG 1004 AGG AGG TAG TTC GAG GGA ATG GGT GTG TAG GTG AAA GAG AAG AAA 1045 TAG AGG GAG TGT GGC TGG GAA GTT GTG AGA ATG GAA ATG ATG AAA 1094 TCC TTG TTC TTA TCA ACA AAG ATG CAA GAA AGA GTG AGA AGT AAA 1139 2 5GAT AGA GAG CTG GGG TGA TGT TGA AATGATTCTCATTGATTAATTTGGCAT 1190 ATAAGAGTTGCAGATGTGACTGTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCA 1249 CAGAATTGACTGAATTAGTTCTGGAAATAGTTTGTCGGTATATTAAGCGAGTATATGTT 1308 AAAAAGACTTAGGTTGAGGGGGATCAGTCCGTAAGATGTTATTTATTTTTACTGATTTA 1367 TTTATTGTTACATTTTATCATATTTATAGTATTTATATTCTTATATAACAAATGTTTGC 1426 30CGTTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA 1485 TTTTGTTATTTATTAAATTATTTTAACAAAAGTTGTTGAAGTTATTTATTGAAAAG 1544 CAAAATCGAAAGAGTAGTTTTGTGAACGAAATCAAGGAATGGACGGTAATATAGACTTA 1603 CCTATTGATTGATTGGATTTAGATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT 1662 TGGAATTGATGTGAAGGAATGGAGGTGTAGTGATTGGATGACTGTATGAAAATATGTGA 1721 3 5TGTAAGGAATAAATATATACAGTTAGTATGTATGGGACAAAAATTAAAAAGTTATTTTA 1780 AAAAAGAAATAGAGGTGAATAAAGAGAGTTTCTTTGCGTGTTGAAGAGCTTTGATTGTT 1839 ACAGGAAAAGAAAGAGTAAAGATGTAGCAATTTGGTTATATGAA .AGTAGAAAGATA 1898 \AGTAAAAGAALAATGATGTTGTCATAGTAGAAGGTT 1933 ram 2 72
14. A polydeoxyribonucleotide as claimed in claim 12 comprising the same gene sequence as given in claim 13 except tha codon 111 of the omega-interferon coding sequence is GAG rather than GGG. A polydeoxyribonucleotide as claimed in claim 11 which comprises the IFN-pseudo-omega2-gene of sequence: AAGCTTGAGCCCCCAGOAAGCATAACCACATGAACCTGAATGAATATATT CTAGAAGGAGGGAAGCACCAGAGAAGTTCTTTCACTAATAACCATCAACGTCTTCTGTG AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAG GGTAAAACTGGAGGCT AGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG ATGTTTCTCATCTGATrTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGG AATATTGAACATCCA'TTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCA TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA GGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC TCTGCCTCCTGTCAGATCACCAGTGG CATTGGAT TCTCAAAAGAGCAGGAACCCTATTG TGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGAGAATCTAATGCCTCTG AAAGCATTCCCTCC CTGAC C C CATTTTT CGTGGA AAAA TTA TCTTCCACC AAAC TGG TG GCCAAAAG GTTG TG GATG CTGATA'A GAAG ACATG TAAATGAAAACAA TAAATGGA-ATT AAAAATTTAGAGAAATGCTCA GA AAAA TG AAAA CTA TTTGTG CTCCA TTAAAAG CCATG C ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAA soe *0 9, ON 0 $to GoofI 51 169 228 287 346 405 464 523 582 641 700 759 818 877 928 973 1018 1063 1108 1153 1198 1243 1288 1333 1380 1439 1498 1557 CCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC GCA GCC CTA GAG GTG TGC AGC TGT GGC TCT TCT GOA TAT AAC CTG CCT CAG AAC CAT GGC CTG CTA GOC AGG GTG CTT TTG GGC CAA ATG AGO AGA ATC TCT CCC TTC AAG GAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG AOC CAG TTG GAG AAG GCC CAG OCT ATG TCT TTC CTC TTACAGCAGGTCTTCAACTTCTCA CAC AAA GCG CTC ATG GAA CAT GAC CTT CCT OGA CCA ACT CCA CAC TTT GCA GCT GGA ACA CCT OGA GAC CTG CTT GOT GCA GGA AGG AGA AGC TGG 000 CAG TOG GTG ATT GAG GOC TCT TTG AGG AGO TAT TTC CAG OAA TCC ATC TCT ACC TOA CCT CTA CTO TCT CTA OGA AAC ACC TTO TTO TOT CTA OTO OA-A OTC TAT OAT OTO CTC TOG TG AG TGA TCA OAT GO AGA ACA CTG 0CC AAGAGAAGAAA TAAAGATTOTOCCTGOOAAGTTOTCAGAOTOOAAATCATGAGATCCTTTTCATCCACAA GTTTGCAAGAAAGATT'CAGAAGTAAGOATGAAGACCTOOOCTCATCATGAAATOATTCT CATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCA -73 TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA 1619 j AGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC 1675 CCTGATGCTGTTTATTTATCTTCCTTCTT ITTTTTCATGCCTTGTATTTATGATATTTA 1734 TATATTTTA TATTTTCATCTTCACATCGTATPTAAAATTTATAAAACATTCACTTTTTCA 1793 TATTAAGTT'TGCATTTTGTTTTATTAAATTCATAT'CAAAGAAAACTCTGTAAATGTTTC 1852 Ii TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTGAAAATATGGTAAAA 1911 TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG 1970 TGGGGTTTTCTTAAkAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG 2029 AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT 2088 ACAAAGAGAAGTAAGAkAAGTGATGTTCTCACATCAGAAGCTT 2132 *16. A polydeoxyribonucleotide as claimed in claim 11 which comprises the IFN-pseudo-omega3-gele of sequence; CCATG CATAGCAGGAATGCCTTCAGAGA- ACCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG £4 0::CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTC CTG CTT CCT CTA CTC 115 *GTG GCC CTG CCG CTT TG GAC TGT GGC CCT GTT GGA TCT CTG AGC 160 P .TOG GAC CTG CCT CAG AAC CAT GGC CTA CTT AGG AGG AAC ACC TTG 205 GCA CTT CTG GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TOT CTC AAG GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 295 TG AGT TGC AGA AGG CCC COG CCC TOT CTC TGC TGG ATG AGA TG 340 .:TTG AGG AGA TGT TGA 0CC TGT TGG CCA GAG AOT OCT GGT GTG GTG 385 GGT OGA ACA TGA CGGTGGTGGACGAACTGGACACTOOAGTTCATCTGCAGCTGGA 440 ATGCCGGATGCTTGCTTAGGOCAGACAAAAAGAGAGOAAGAATCTGTGGGGGTGATTG 499 GGCCTACACTGGCCTTGAGGACOTACTTTGAGGOAATGCATGGGAATCCAGGGAATG 558 TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG 617 AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA 676 CCTGGGTTCATCCTOAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG 735 IGTCTTTGGTCATTTCAATAGGTTCTTATTTCTGCAG 772 74
17. A polydeoxyribonucleotide as claimed in claim 1lwhich comprises the IFN-pseudo-omega4-gele of sequence: AA'.CTTTGGGCATGACCTAGTAGGTGACTCTT 32 AGTTGGAGTGGTCAGTTGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCC2CC 91 ACGTTTGCAGCCGTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCCACTGGA 150 GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA 209 TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG 268 jTAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT 327 GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC 386 AGGGGTTCCCTGCCTCGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTGG 445 4CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT 504 j CTCCTTCATTAGTTCCAACGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA 563 GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA 622 see 0000 GTGTTTAGCCAGAGGAGTGGGGTCGCTGCATGCTGGTGTAAGTATGGGACTTCACTTCT 681 :00e: TGGGAAACAGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC 740 CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAAACAAAGAGC 799 TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 858 GCAGTGGCAGAGGTAGGATGGTTGTGGGAGCCTCTCCCCAGGCAAACTCTAGTCAACTA 917 CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGCAGGGGGCC 976 000 TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGPAAGGGGCTGGACTCCTCTTCATA 1035 0**e TAGGGCATGAGTGTGGCGTGCAGGTATGAAAGGCTTGTTCTTCCCA1094 GCCAGAGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 O ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTGAGGAGACGAGGCAAG 1212 CACCAGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330 8TGGGGAT'CTGGAGCAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGG 1389 CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 OGCGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1507 of0 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566 CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625 CATTTfTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684 CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG 1802 GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1861 -GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920 CAGCACAATGGCAA~CCTACCATTCCCTTTGGAGCCCATCCCAGGGATATTCGAACT 1979 GCTGTCCACTAGAAAACAGTGGTGGAGGTGACTGGAGACCCCAGTGGAGAGT'TTCACCT 2038 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAJACCAATCTGACTGCTTCCCCGTA 2097 GAGCTGCTGGACTGTGCTGGGTGGCTGCTCCAGTCCCTAGCTGCCTTGGACTCCCGAGA 2156 ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC 2215 CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGCCTGGATTCCC 2274 AACTCGAGTGGGTCTTACCCTGAGACAGGCCATCCPJAGGTGCCCCTGTCATTGTCACTG 2333 CCCAGCCCCCTGCATGA.AACCCCTTTCCTAGGGGTATGTATAGGGGTCTAGCGTCCTCC 2392 TTGGCTGGAGTTATAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAACCCTCCAGGGTAC 2451 CCTCTCAACGTCCGTAACTCTGCqGAGCGCA 2510 ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 2569 jTCCCAGAAGCGTGGGTCCCCAGGCCTGCTCACTTACTCACCACTTCCCTGGGCAGGAGA 2628 GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT 2687 TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG 2746 AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGACAGTCGCACACACTAGCA 2805 GGTTCCAGTCGGCCATCTTGCAACCCCTGAAJACTATTTGTTTCCAGCTATAAGCCATT 2864 ::GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2923 CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 *GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT OGA TCT CTG CCC 3019 :-OTTT GAC CTG CCT CAG AAC CGT GCC CTA CTT ACC AGC AAC ACC TTG 3064 GCA TTC TGG GCC AAA TGC ACA ATC TCQ ACT TTC TTC TGT CTC AAG 3109 GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 3154 ATT TGC AGA ACG CCC AGG CTG TGT CTG TCC TCC ATC A GA TG C 'IT.C 3199 AGC AGA TCT TCA GCC TCT TCC CCA CAG AGC GCT CCT CTG CTG CCT 3244 2 5GGA~ ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG 3300 CCTGGAGTCTTGCTTAGGGCAGGCAACACCAGACCAJ\CAATCTGTCCCCGTGATTCCCA 3359 CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAA TCCAGAGA-ATCTAC 3418 CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAJ.A 3477 sees: TCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGACCTGGGGTC 3536 .00: 3 0TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC 3595 TAGTTC 2'TTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAJJATA 3654 AGCTT 3659 I J 1 III I- S S S S S S5 *5 S *555 SO S S 76
18. A polydeoxyribonucleotide as claimed in claim 8 substantially as hereinbefore described.
19. A recombinant DNA molecule which contains a polydeoxyribonucleotide as claimed in any one of claims 8 to 18. A recombinant DNA molecule as claimed in claim 19 which is a plasmid vector, phaqe vector or cosmid vector.
21. The plasmid of claim 20 designated P9A2 (deposited in E. coli HB101 at the DSM under number DSM 3004).
22. The plasmid of claim20 designated E76E9, (deposited in E. coli HB101 at the DSM under number DSM 3003).
23. A cosmid vector as claimed in 20 selected from the cosmid vectors designated Cos9, CoslO and CosE as hereinbefore defined.
24. A plasmid as claimed in claim 20 selected from the plasmids designated pRH57, pRHW22, pRH51 and PRH52 as hereinbefore defined. A recombinant DNA molecule as claimed in claim 20 which is an expression vector nuitable for transformation of a microorganism or cells derived from a multicellular organism, having a 25 coding sequence for an omega interferon or a pseudo gene sequence capable of hybridising with a sequence corresponding to such an omega interferon-coding sequence under stringent hybridisation conditions as defined herein (suitable for detecting about 85% or higher homology with the chosen interferon coding sequence) at an appropriate site for expression within a desired host.
26. A recombinant DNA molecule as claimed in claim 25which is an expression vector suitable for transformation of E. coli. ~Z O h, 77
27. A recombinant DNA molecule as claimed i claim 25 which is a yeast expression vector.
28. A recombinant DNA molecule as claimed in claim 27wherein an omega-interferon coding sequence is connected to the portion of the leader sequence of the MF-alpha-1 yeast gene beginning at position 256 from the initiation codon.
29. An expression vector as claimed in claim derived from plasmid pBR322 wherein the shorter EcoRl/BamH1 fragment belonging to plasmid pBR322 is replaced by a polydeoxyribonucleotide comprising the sequence: EcoRI Sau3A gaattcacgctGATCGCTAAAACATTGTGCAAAAGAGGGTTGACTTTGCCTTCGCGA 59 mRNA-Start Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAACCTTAAAG ATG '11 I RBS H!'n TT Cys Asp TGT GAT C IFN-omega-coding sequence-- Sau3A The expression vector of claim 29 desionated pRHW12 as hereinbefore defined.
31. The expression vector of claim 29 designated pRHW11 as hereinbefore defined. h 30 30 32. A recombinant DNA molecule as claimed in claim 19 or as hereinbefore described.
33. A recombinant DNA molecule as claimed in claim 19 or substantially as hereinbefore described with reference to any one of Examples 1, 5 and 8. lis- 78
34. A process for the preparation of the expression vector of claim 30 which comprises isolating the NcoI-AluI fragment from the cDNA insert of plasmid P9A2 arnd ligating it with the larger fragment obtained by cutting the plasmid pRHWlO with BamHl, filling the cutting sites to obtain a linearized blunt- ended form using the Klenow fragment of DNA polymerase 1 and the 4 deoxynucleoside triphosphates and subsequently cutting with NcoI.
35. A process for the preparation of the expression vector of claim 31 which comprises isolating the NcoI-AluI fragment from the cDNA insert of plasmid E76E9 and ligating it with the larger fragment obtained by cutting the plasmid pRHWlO with BamHl, 15 filling the cutting sites to obtain a linearized *g blunt-ended form using the Kienow fragment of DNA polytnerase 1 and the 4 deoxynucleoside triphosphates foes and subsequently cutting withi NcoT.
36. A process as claimed in claim 34 or claim 35 wherein plasinid iRJW1U cor.-,ti licted by inserting the DNA fragment: *ag Ii nd I II Sau3A NCOI1 aAGCTTAAAG ATGTGTGAPC, TGCCrCAGAA CCJVT'GGCCTA CTTAGCAGGA GCTTICTCAC CAAATG'AGGCA GAATCTCCCC TTTCTTGTGT 100 to C ICAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG .150 so~* :3 CCAGTTGCAG AAGGCCCATG TC-ATG ICTGT CCTCCATGAG ATGCTGCAGc 200 AGATCACACA TCTTTA- ctt Sau3A Ii ndil at the HindIII site of plasmid pERlO3. 79 C 4C *6~S 9* C C C. S C S S heS@ C C C C *5 S C. a C C@ 0* C S. C S S S IC
37- A process as claimed in claim 36 wherein the DNA fragment inserted at the HindIII site of plasmid pER1O3 is constructed by ligating the following 189bp Sau3A fragment obtainable from the cDNA insert of plasmid P9A2 or plasinid E76E9 Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr Leu GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 42 Sa-u3A NcoI 20 25 Vai Leu Leu His Gin Met Arg Arg Ile Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTIC 87 40 15 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Giu Met Val Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 132 50 5 Ser Gin Leu Gin Lys Ala His, Val tSeVlLuHsGiHe 20 AGC GAG TTG CAG AAG GCC CTJYAK'Y QiVA GAG ArC-I 177 Leu Gin Gin Ile CTG GAG CA atc 185 Sau3A 25 with the following 108 bp fragmenrt obtainable by digesting the 389 bp EcoRi-PvuII fragment of plasmid pER33 with Sau 3A EcoRr Iau3A aa t tcacgc UGATICGCTAAAAC ATT'GTIGC AAAAAAGGGT'TGACI TTTGCCTTCGCGA 59 JfmRNA-Start Met ACCAGTTAACTAGTACACAkAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 Cys Asp B idI TGTjgat c 123 Sau3A Al: T,~ ~4I I and cutting the resulting fragment with HindIII. 80
38. A process as claimed in claim 36 wherein the DNA fragment inserted at the HindlII site of plasmid pERlO3 is constructed by ligating the following DNA fragment constructed from two synthetic oligo- nucleotides '-AGCTTAAAGATGTGT 31 31- ATTTCTACACACTAGp 51 with the 189bp Sau3A fragment obtainable from the cDNA insert of plasmid P9A2 or plasmid E76E9. 39 A process asi claimed in claim 34 or claim substantially as hereinbefore described with reference to Example A host organism transformed by a vector as claimed in any one of claims 20 to 33. 41 The polypeptide encoded by the IFN -pseudo- 9 4 S 9* 99 I. 9 09A 9 99 £9 9 a 4*~4 S S. 15 omega2 gene claimed in claim 15 having the sequence: Met Ala Leu Leu Phe Pro Leu Leu Aft- *9 0 9 60 0 94 S 9, 4* '9 94 4 9.99 S. 4 0* .9 Ala Ty r Val Liys Ser Leu Met Ala Ala Asn Le u Asp Gln Gln Glu Ala Leu Glu Val Leu Pro Gln 20 Leu Gly Gln 35 Arg Ser Asip Leu Gln Lys 65 Gln Val Phe His Asp Leu Gly Thr Pro 110 -10 Cys Asn Met Phe Ala Asn Pro Gly Se r H is Arg Arg Gl1n Phe Gly Asp Cys Gly Arg Phe Ala Ser Pro Leu Gly Ser 10 Leu Leu 25 Ile Ser 40 Pro Gln 55 Met Ser 70 His Lys 85 Thr Pro 100 Leu Gly 115 Ser Gly Gly Arg Pro Phe Glu Lys Phe Leu Ala Leu His Phe Ala Gly (G1v Ser -1 Ser Leu Gly Asn Thr Leu Leu Cys Leu Val Glu Val Tyr Asp Val Leu Cys Cys Thr Ser Ser 105 Asp Gly Arg 120 Thr Leu Ala DrrT ?xrr, ~r eP,.r~ C1n 'Pm ~Jfl 130 jLeu Arg Arg Tyr Phe Gln Glu Ser Ile Ser Thr 'V 0 0 CS S 005t Sb 0 S 0@ S S @550 0 e.g. OSS* 0 5 9 se S 10 S S 00 S. 9* 0 0*00 S@ C C. S. 81
42. The polypeptide encoded by the TFN-pseudo- omega3 gene clairted in claim 16 having the sequence: Met Val Leu Leu Leu Pro Leu L, u 5-10 -5 -1 Val Ala Leu Pro Leu Cys His Cys Gly Pro Val Gly Ser Leu Ser 10 Trp Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr Leu 25 Ala Leu Leu Gly Gin Met Cys Arg Ile Ser Thr Phe Leu Cys Leu 40 Lys Asp Arg Arg Asp Phe Arg Phe Pro Leu Giu Met Trp Met Ala 55 Val Ser Cys Arg Arg Pro Arg Pro Cys Leu Ser Ser Met Arg Cys 15 65 70 Phe Ser Arg Ser Ser Ala Ser Ser Pro Gin Ser Ala Pro Leu Leu Pro Gly Thr 20 43. The polypeptide encoded by the IFN-pseudo- omega4 gene claimed in claim 17 having the sequence: Met Val Leu Leu Leu Val Leu Leu 10 -5 -1 25Vdi*L Ala Leu Leu Leu Cys Gin Cys Gly Pro Val Gly Ser Lou Gly 5 10 Phe Asp Leu Pro Gin Asn Arg Gly Lou Lou Ser Arg Asn Thr Leu 25 31)0 Ala Phe Trp Ala Lys Cys Arq Ile Ser Thr Phe Lcu Cys Lou Lys 30 35 40 Asp Arg Arg Asp Phe Arg Phe Pro Leu Giu Met Trp Met Ala Vai 55 Ile Cys Arg Arg Pro Arg Leu Cys Leu Ser 5cr Met Arg Cys Phe 70 Ser Arg Ser Ser Ala Ser Ser Pro Gin Ser Ala Pro Lou Leu Pro Gly Thr K, L -82
44. A process for preparing a polypeptide as claimed in any one of claims 1 to 7or 41 to 43 which includes the step of transforming a suitable host organism with an expression vector as claimed in claim containing a coding sequence for the desired polypeptide at an appropriate site for expression and isolating the desired polypeptide from the resulting transformants. A process as claimed in claim 44 substantially as hereinbefore described.
46. A process as claimed in claim 44 substantially as hereinbefore described with reference to Examples and 11.
47. A pharmaceutical composition comprising at least one interferon polypeptide as claimed in any e one of claims 1 to 7 and/or at least one N-glyccsylated derivative of such an interferon polypeptide with interferon activity, in association with a pharmaceutically ee acceptable carrier or excipient.
48. A pharmaceutical composition as claimed in claim 47 comprising a synergistic mixture of at least one interferon polypeptide as claimed in any one of claims 1 to 7 and i-interferon, in association with a pharmaceutically acceptable carrier or excipient. I 49. A pharmaceutical composition as claimed in claim 47 comprising a synergistic mixture of at least one interferon polypeptide as claimed in any one of claims 1 to 7 and human tumour necrosis factor in association with a pharmaceutically acceptable .4 carrier or excipient. I 50. Use of an interferon polypeptide as claimed in claim 1 in the preparation of a pharmaceutical composition suitable for anti-viral or anti-tumour treatment. !j tI 4' 83 51,. A pharmaceutical compos~tion as claimed in claim 47 substantially as hereinbefore described. DATED this 26th day of February, 1990 Boehringer Ingeiheim. International GmibH by its Patent tttorneys DAVIES COLLISON S S 5* S S SOS S SO S S S *5*1* S 5.5. S *S 0 S. S S S S S Sc.. S 55 ii J2~ 'I K 4 o~ S S E76 E9 100 200 300 400 500 600 700 800 900 bp I I I I I I Alu I Hinf I Sau3A-1 Pst I I I I I I I I II FIG. 1. a.. as. a a a a a a a a a a a a a P9 A2 100 200 300 400 500 600 700 800 900 bp I IIIII I I AlulI Hinf I Sau 3A I Pst I I I III I I I I 4-oi 4- 4- F IG. 2. T Cys TGT Asp GAT Le u C TG P r cc~ F I G. 3a. PA TCT( 10 o Gin Asn His Gly Leu Leu Ser Arg Asn Thr T' CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC 25 s Gin Met Arg P g Ile Ser Pro Phe Leu Cys CCAA ATG AGG AG3A ATC TCC CCT TTC TTG TGT Leu TTG Leu CTC GGGC 7 Val Leu Leu Hi: GTG CTT CTG CA 0 000 0 0 0 0* Lys Asp Arg Arg AAG GAC AGA AGA Ser Gin Leu Gin AGC GAG TTG CAC Leu Gin Gin Ile CTG CAG CAG ATC Asp Phe Ary GAC TTC AGG 40 Phe Pro Gin TTC CCC CAC Ciu GAG Val C TC Met Val ATG GTA Lys AAG Aia His Vai CCC CAT GTC 55 Met Scr ATG TCT Lys Gly AAA CCC Leu His Giu CTC CAT GAG Met ATG 187 65 Phe Ser TTC AGC 70 Leu Phe His Thr Giu CTC TTC CAC ACA GAG Arg CC Ser Ser TCC TCT Ala GCT 232 Ala Trp CCC TG Gin Gin CAG CAA Asn Met AAC ATG Leu Gin CTG CAA Thr ACC His CAC Giu GAA Gly GGA 110 Giu 5cr Ala GAA TCT GCT Leu Leu CTC CTA Leu Giu CTG GAG Giy Ala CCC GCA Gly Ile GGA ATC Trp Giu TGG GAA Asp Gi.n GAC CAA 100 Thr Cys Leu ACC TGC TTG 85 Leu CTC Ile ATT 115 Ser Ser AGC AC His Thr CAC ACT Leu Gin CTG CAG Gly Leu His GGA CTT CAT 277 105 Val Val Cly GTA GTC GGA 322 S S SS S S5 5 5S Arg Arg Tyr AGG AGG TAC 125 Phe Gin TTC CAG 140 Cys Ala TGT GCC Arg Val CCT GTC 130 Ty r TAG Pro CCT Leu CTG Met ATG Ala GCA Ly s AAA Giu GAA Leu Thr CTG ACC 120 Leu TTG 367 135 Giu Lys Lys GAG AAG AAA 412 150 lle Met Lys ATC ATG AAA 457 Ty r TA C Ser AGC Asp GAG 145 Val Val Arg GTT GTC AGA I p F IG. 3b. CONTINUATION OF FIG.3a. 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Ara Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 502 170 rAsp Arg Asp Leu Gly Ser Ser GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 554 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 613 se1 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 672 ego AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 731 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 790 TTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTAT 849 TTTGTTATTTATTAAATTATTTTCAAAC-poly-A 877 1 '4 F IG. 4a. E7 6 E9) CTCTGGGC 7, Cys Asp TCT GAT Val ILeu GTC CTT Lys Asp AAG GAC Ser Gin AGC GAG Leu Gin CTG GAG Ala Trp GCG TGG Leu Pro CTG CGT Leu His CTG CAC Gin CAG Gin CAA Asn AAC His CAT Gly Leu GGC CTA Arg Ile AGA ATC 10 Leu CTT 25 Ser TCC Ser Arg Asn Thr AGG AGG AAC ACC 0S esee S 5* S. 0 0 56 S S*S S S 0eSO *5 5 OSS *5 S Arg Arg Asp AGA AGA GAC Leu Gin Lys TTG CAG AAG 65 Gin Ile Phe CAG ATC TTC 80 Asn Met Thr AAC ATG ACC Leu Gin His CTG CAA GAG 110 Giu Ser Ala GAA TGT GGT 125 Tyr Phe Gin TAG TTC GAG Met Arg ATG AGG Phe Arcj TTC AGG Ala His CCC CAT Ser Leu AGC CTC Leu TTG Leu CTC Pro Phe CCT TTC Giu Met GAG ATG 40 Phe Pro Gin TTC CCC GAG Vai Lys Giy GTA AAA CCC 55 Val Met Ser Vai Leu GTG ATG TCT GTC GTG His CAT Giu Met GAG ATG Leu Gys TTG TGT 98 143 118 233 278 Gin GAG Glu GAA Gin CAA Gly GGA Arg AGG Leu GTC Leu CTG Glu GAG Gly GGA Leu CTA Giu GAG Ala CA Ile ATG 70 Phe His Thr TTG GAG ACA 85 Asp Gin Leu GAG CAA CTC 100 Thr Cys Leu ACC TGC TTG 115 Ile Ser Ser ATT AGC AGC His CAC Thr Gly ACT GGA Leu Gin Val Val CTG GAG GTA GTG Pro Ala Leu Thr CGT GCA GTG ACC Leu Lys Giu Lys GTG AAA GAG AAC Met Ciu Ile Met 105 Gly GGA 323 120 Leu TTG 368 135 Lys AAA 413 150 Ly s Leu His CTT CAT Giu Arg Ser Ser GAG CCC TCG TGT Aia GCT Arg AGG Arg Vai CGT GTG S S @0 S OS S OS *5 130 Ty r TAG 145 Arg 140 Ala Tyr Ser Asp Gys Trp Giu Val Vai TAG AGC GAG TGT CCC TGG GAA GTT GTC AGA ATC GAA ATG ATG AAA 458 FIG. 4b. CONTINUATION OF FIG. a. 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Arg Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 503 170 Asp Arg Asp Leu Gly Ser Ser GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 555 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 614 *fee AGAATTGACTGAATTAGTmCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 673 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 732 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 791 TTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTAT 65 0 TTTGTTAT 858 0 44 .v F IG. a) I I I E H B SPC M EHB SPC kbp -21,2 5,1 4,3 -1,9 -1,58 -1,38 -0,95 -0,83 -0,56 S S 6eSO 5 5 S OS 5 S S S b) P9A2 pP 3' S S A i I Probe I-I 100bp It L I /S~u3A pER 33 txSau3A M"l C;i AAGCTTAEAA ATG TGTL TTC2AATETC IAC ACA CTAG SIuJA ",nOIISJ3A F IG. 6. S,.3A Alul xN xA AMC S P9A2 coaI 1, //1 X S" 3A A: II I P% G" al LouLtGd CCI Cr10 EEC CAI GC/C JAC GGA r I c1 CT C Ligase r. 0 0 *Oe 0 S0 0 *00 *000 0 0000 OS *00 0 0* 0 9 0O 00 0 *00 Ili.-Il I-'CA S..3A A -1A ,InE Jx Hind IIl ,Sau3A lnd Ini If."dill ll-n~ll Ligase S.u3A S..3A Li -s I xHind Ill An pER1O3 T En~ x BarnH I, Klenow f Ill In xNco I PclG Eau3A ATIc V111111111111 Ligase 0000 0S S. S S. S S S *5 a a a IFN-*A IFN-oB IF1-C IFH-oD IFN-OF XFN-oG IFN-oH IRI-eLK IFN-iL IN-B UH-a IFH-aA IFN-a8 IFN-aC XrN-oD UNH-ar ITN-etH ITN-.C IFN--w XTH-rzK RIF-o1, XFH-w 31 (18,7) 31 32 (13,1) (12,3) (10,6) (10,2) (11,8) (52,S) (28,5) (12,5) (13,9) (12,7) (12,5) (12,7) (12,9) (12,9) (5513) X,1) (18,7) (19,2) (12,1) 1 ,1) (10,0) 8,1) ,5) 1 0,8) (55,9) (29.3) (17,5) (22,9) (19,3) (10,8) (11,2) (12,9) (12,5 (16, 3) (29,7) (18,1) (18,7) (10,8) (16,9) (10,2) (11,0) 8,3) 1 8,5) (54,7) (3,8) (15,1) 29 (17,5) 33 (19,9) 34 (20,5) (17,5) 3 (18,1) 33 (19,9) 35 (21,1) (16,3) 24 (14,S) 11 6,6) 3 1,8) (14,5) 32 (19,J) 35 (21,1) 34 (20,S) (12,0) 27 (16,3) 20 (12,0) 21 (12,7) 24 (14,5) 28 (16,9) 29 (17,5) 1 8,9) 24 (14,5) 26 (15.7) (10.0) 41 7,9) 12 1 7,2) 109+4 (68,1) 116+4 (72,3) 113]+4 (70,5) 115+4 (71,7) 115+, (71,7) 111+4 (69,3) 110 4- (68,7) 112+4 (69,9) 1134 (20,5) 270 (52,0) 63 +6 (41,6) 69 +6 (45,2) 67 +6 (44,0) 70 46 72 +6 (47,0) 68 +6 (44,6) 71 +6 (46,4) 69 +6 (45,2) 68 +6 (44,6) 112+10 (73,5) 46 52 53 (10.2) 281 (54,1) 147 (28,3) 812) (54,3) (29,9) (2,5) (55,) (29 .7) 2S0 (55,9) 153 (29,5) Nucleotide differences F 16.7. 549/85 F IG. 8a. 130 140 150 3IFN cxA gaggagtttgGCaaccagttccaAaaggct IFN c(B qaggagtttgatgataaacagttccagaag IFN axC gaggaqtttgatggcaaccagttccagaag TFN uD gaggagtttgatggcaaccagttccagaag TFN aF gaggagtttgatggcaaccagttccagaag IFN cxG gaqgagtttgatggcaaccagttccagaag IFN aH gaggaatttgatggcaaccagttccagaaa IFN aI( gaggagtttgatggccaccagttccagaag IFN uL gaggagtttgatggcaaccagttccagaag TFN omeqal gagatggtaaaagggagccagttqcagaag IFN beta gagattaagcagctgcagcagttccagaag EFN FIG. 8b.3536 3403536 INomegal GgggCaaTtAGcaGcCctGCActgaccTtg I F x t a g a g g g c c a t t g t t IFN aA ctgatgtagaggactccattctggctgtg IFN aC ctgatgatgaggactccatcctggctgtg IFN uC ctgatgaatgaggactccatcttggctgtg *IFN cxF ctgatgaatgtqactccatcttggctgtg IFN aF ctgatgaatgtgqactccatcctggctgtg *IFN (xG ctgatgaatgtggactctatcctgactgtg IFN aH ctgatgaatgaggactccatcctggctgtg SIFN aLf ctgatgaatgaggactccatcctggctgtg IFN beta aggggaaaactcatgagcagtctgcacctg Y F IG. 8c. I FN I FN I FN IFN I FN I FN IFN I FN I FN I F N I FN a D aC aF aL omega 1 beta 200 210 220 tcttcaacctct tTaCcacaaaAgaTtcat t ottcaa totc t tca gca c aaa g gact cat ccttcaacctcttcagcacaaaggactcat cc tto aa totct tcagc acag agqac tca t ccttcaato to t tagc ac aaagg actca t ccttcaa to tottcagcacaa aggac tca t ccttcaatctcttcagcacaaagaactcat cc ttcaato tot toagcacag agg actoa t cc ttcaa to tottcagcac ag agg ac tcat tot toagcto t t ccacacagagcgc tcct tctttgctattttcaqacaagattcatcta F IG. 9. 0* 0S 9.. SS *0 S0 Hind III1I Bamnf I fragment of pRHW12 Ml3mp9 Primer extension by Kienow Polymerase ~72 FIG. k I I I 1 4. H ~1 4 ~e I I 9 *6 S. I SI S. I F IG. 11k. IFN-omegal) GATCTCGTAAACCTGAA GCAAATATAGAAACCTATAGGGCCTGACTTCCTACATAAAGTAAGGAGGGTAAAAATGG AG GCTAGAATAAGGCTTAAAATTTTGCTTCTAGAACAGAGAAAATGATTTTTTTCA TAT ATATATGAATATATATTATATATACACATATATACATATATTCACTATAGTGTGTATAC ATAAATATATAATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCATTTGATTAAGCATTCA-TT GTATTGACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAAATTAATAGA TTGTTGTCATCCTCTTAAGGTCATAGGGAGAACACACAAATGAAAACAGTAAAAGAAAC TGAAAGTACAGAGAAATGTTCAGAAAATGAAAACCATGTGTTTCCTATTAPAAGCCATG CATACAAGCAATGTCTTCAGAAAACCTAGGGTCCAAGGTTAAGCCATATCCCAGCTCAG 17 76 135 194 253 31.2 371 430 489 548 TAAAGCCACGAGCATCCTCATTTCCCA Met Ala Leu ATG GCC CTC Tyr Ser Pro TAT AGC CCT S 0* S S *5 S Ala Ala CCA GCC Cys Asp TGT GAT Val Leu GTG CTT Leu Val Met CTA GTG ATG 5 Leu Pro Gin CTG CCT GAG 20 Leu His Gin CTG CAC CAA Arg Arg Asp AGA AGA GAG Leu Gin Lys TTG GAG AAG Gin Ile Phe GAG ATC TTC Thr ACC Ser AGC Leu CTG -5 Val1 C TT Ser AGC Gly Ser GGA TCT Arg Asn AGG AAC Phe TTC 1 Leu Cly CTG GCC Thr Leu ACC TTG -16 Pro Leu Leu CCT CTA CTG Asn His Gly Leu AAC CAT CCC CTA Met Arg Arg Ile ATG AGG AGA ATC Phe Arg Phe Pro TTC AGG TTC CCC Air% His Val Met GCC CAT GTC ATG I 0 of 0 0 0. 0 Ly s AAG Ser AGC Asp GAG Gin GAG Gin GAG 10 Leu CTT 25 Ser TCC 40 Gin GAG 55 Ser TCT 70 Thr ACA 85 Leu 599 644 689 734 Pro Phe CCT TTC Glu Met GAG ATG Vai Leu GTG CTC Glu Arg GAG CG Leu Cys TTG TGT Val Lys Gly GTA AAA GCC 779 His Glu CAT GAG Ser Ser TCG TCT Le u CTC Oses S s S 00S Leu CTG Ser Leu AGG GTC Phe His TTC GAG Met ATG Aia GCT His 824 869 80 Thr Ala Trp Asn Met Leo Leu Asp Gin His Thr Gly Leo GCC TGG AAC ATC ACC CTC CTA GAG CAA CTG GAG ACT GGA GTT CAT F IG. 11b. CONTINUATION OF FIG. Ila. Gin Gin CAG CAA Glu Gly GAA GGA Leu Gin CTG CAA Giu Ser GAA TCT His Leu Giu CAC CTG GAG Arg Arg Tyr Phe AGG AGG TAC TTC Tyr Ser Asp Cys TAC AGC GAC TGT Ser Leu Phe Leu TCC TTG TTC TTA Asp Arq Asp Leu GAT AGA GAC CTG 110 Ala GCT 125 Gin CAG 140 Ala GCC 155 Se r TCA Gly Ala GGG GCA Gly Ile GGA ATC 100 Thr Cys Leu ACC TGC TTG 115 Ile Ser Ser ATT AGC AGC 130 Arg Val Tyr CGT GTC TAC Leu Gin CTG CAG Val1 GTA ?ro CC 7, Leu CTG Met ATG Val1 GTG Ala Leu GCA CTG Lys (flu AAA GAG 120 Thr Leu ACC TTG 1004 13% Lys Lys AAG AAA 1045 10 Gly GGA 959 Trp Glu Val Val TGG GAA GTT GTC 145 Arg AGA Glu Ile Met GAA ATC ATG Leu Arg Ser CTG AGA AGT 150 Ly s AAA 165 Ly s AAA 160 Thr Asn Met Gin Glu Arg ACA AAC ATG CAA GAA AGA 1094 1139 0 0*0 00 0 0 000 0 00 0 0 170 Gly Ser Ser GGC TCA TCT TGA AATGATTCTCATTGATTAATTTGCCAT 1190 i~1 ATAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCA CAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTT AAAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTA TTTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGC CTTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA **TTTTGTTATTTATTAAATTATTTTCAAACAAAACTTCTTGAAGTTATTTATTCGAAAAC CA AAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA CCTATTCATTCATTCCATTTACATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT 0 TGGAATTGATGTCAAGCAATGCAGGTGTACTCATTGCATGACTGTATCAAAATATCTCA TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA AAAAAGAAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA AGTAAAAGAAAATGATGTTCTCATACTAGAAGCTT 1249 1308 1367 1426 1485 1544 1603 1662 1721 1780 1839 1898 1933 IG.I12a. (IFN-pseudo-omega2) ISAAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATGAATATATT 51 ICTAGAAGGAGGGAAGCACCAGAGAAGTTCTTTCACTAATAACCATCAACGTCTTCTGTG 110 AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC 169 CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAG 228 GGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG 287 ATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATIATATCTTAAGTTTACTGGG 34 6 AATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCA TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA 464 ~jiGGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC 523 TCTGCCTCCTGTCAGATCAGCAGTGGCATTGGATTCTCAAAAGAGCAGGAACCCTATTG 582 TGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGAGAATCTAATGCCTCTG 641 AAAGCATTCCCTCCCTGACCCCATTTTTCGTGGAAAAATTATCTTCCACCAAACTGGTG 700 GCCAAAAGGTTGTGGATGCTGATATAGAAGACATGTAAATGAAAACAATAAATGGAATT 759 AAAAATTTAGAGAAA'GCTCAGAAAAATGAAAACTATTTGTGCTCCATTAAAGCCATGC 818 ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAA 877 I-20 -16 0Met Ala Leu Leu Phe Pro Leu Leu CCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC CCT CTA CTG 928 -5 -1 1Ala Ala Leu Giu Val Cys Ser Cys Gly Ser Ser Gly Ser Leu Gly GCA GCC CTA GAG GTG TGC AGC TGT GGC TCT TCT GGA TCT CTA GGA 973 10 Tyr Asn Leu Pro Gin Asn His Gly Leu Leu Gly Arg Asn Thr Leu TAT AAC CTG CCT CAG AAC CAT GGC CTG CTA GGC AGG AAC ACC TTG 1018 25 Val Leu Leu Gly Gin Met Arg Arg Ile Ser Pro Phe Leu Cys Leu GTG CTT TTG GGC CAA ATG AGG AGA ATC TCT CCC TTC TTG TGT CTA 1063 40 Lys Asp Arg Ser Asp Phe Arg Phe Pro Gin Giu Lys Vai Giu Val AGGAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG GTG GAA GTC i108 :50 55 Ser Gin Leu Gin Lys Ala Gin Ala Met Ser Phe Leu Tyr Asp Vai AGC CAG TTG CAG AAG GCC CAG GCT ATG TCT TTC CTC TAT GAT GTG 1153 1% 7 F IG. 12 b. CONTINUATION OF FIG. 12a. Phe Leu Gin Gin Val TTA CAG CAG GTC TTC Met Glu His ATG GAA CAT Ala Ala Gly GCA GCT GGA Asp Leu GAC CTT Thr Pro ACA CCT Asn Phe Ser AAC TTC TCA Pro Gly Pro CCT GGA CCA Gly Asp Leu GGA GAC CTG Gin Trp Val GAG TGG GTG 70 His Lys Ala CAC AAA GCG 85 Thr Pro Hlis ACT CCA CAC 100 Leu Gly Ala CTT GGT GCA 115 Ile Glu Gly ATT GAG GGC Phe Thr Ser TTT ACG TCA Gly Asp Gly GGA GAT GGG Ser Thr Leu TCT ACA CTG Leu Leu Cys Cys CTC CTC TGC TGC Ser TCA 105 Arg AGA 120 Ala GCC 1198 1243 1288 1333 S SS S S. 5 SS* 0e S Arg Arg Ser Trp AGG AGA AGC TGG Leu Arg Arg Tyr TTG AGG AGG TAT 110 G ly GGG 125 Phe TTC Gin Giu Ser Ile CAG GAA TCC ATC 130 Ser Thr TCT ACC TGA AAGAGAAGAAA 1380 '~4 TAAAGATTGTGCCTGGGAAGTTGTCAGAGTGGAAATCATGAGATCCTTTTCATCCACAA GTTTGCAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGAAATGATTCT CATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCA TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA AGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC CCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTA *TATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTCACTTTTTCA TATTAAGTTTGCATTTTGTTTTATTAAATTCATATCAAAGAAAACTCTGTAAATGTTTC TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTGAAAATATGGTAAAA TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG :AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT *:ACAAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 1439 1493 1557 1619 1675 1734 1793 i8K 2 1911 1970 2029 2088 2132 F IG. 13. IFN-pseudo-omega 3) CCATG CATAGCAGGAATGCCTTCAGAGAACCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG 64 115 CTAGGCCAGCAGCACCCTCGTTTCCCA Met Val Leu ATG GTC CTC Cys Gly Pro TGT GGC CCT Leu CTG -5 Val GTT Leu CTT Gly GGA -16 Pro Leu Leu CCT CTA CTC Val GTG Trp TGG Ala Leu Pro GCC CTG CCG Leu Cys CTT TGC His CAC Ser TC T 1 Leu Ser CTG AGC 160 Thr Leu ACC TTG 205 Asp GAC Leu Pro Gin CTG CCT CAG Asn His Gly Leu AAC CAT GGC CTA 10 Leu CTT Ser Arg Asn AGC AGG AAC Thr Phe Leu ACT TTC TTG I I 0 0* S S 000* 0* 0 0 0 00* 0000 Ala Leu GCA CTT Leu Gly CTG GGC Lys Asp Arg Arg AAG GAC AGA ACA 20 Gin CAA 35 Asp GAG Arg AGG Ser TCA Met Cys ATG TGC 25 Arg Ile Ser AGA ATC TCC Phe Arg Phe Pro TTC AGG TTC CCC Pro Arg Pro Cys CCC CGG CCC TGT 40 Leu CTG 55 Leu CTG Glu Met Trp Met GAG ATG TGG ATG Ala GCA Cys Leu TGT CTC Val Ser GTC AGT Phe Ser TTC AGC Cys Arg TGC AGA Arg Ser AGA TCT Ser Ser Met TCC TCC ATG Arg Cys AGA TGC 250 295 340 385 Goo0 0 0 0 Ala Ser GCC TCT 70 Ser Pro Gln TCC CCA CAG Ser Ala Pro Leu AGT GCT CCT CTG Leu CTG Pro CCT Gly GGA Thr ACA TGA CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA 440 ATGCCTGGATGCTTGCTTAGGGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA CCTGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG TGTCTTTGGTCATTTCAATAGGTTCTTATTTCTGCAG 499 558 617 676 735 772 F IG. 14a. (IFN-pseudo-omega4) AAGCTTTGGGCATGACCTAGTAGGTGACTCTT 32 AGTTGGAGTGGTCAGTTGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCCCC 91 ACGTTTGCAGCCGTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCCACTGGA 150 GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA 209 TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG 268 TAGTTGTAACTCAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT 327 GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC 386 AGGGGTTCCCTGCCTCGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTIGG 44 CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT 504 GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA 563 GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA 622 GTGTTTAGCCAGAGGAGTGGGGTGGCTGCATGCTGGTGTAAGTATGGGACTTCACTTCT 681 TGGGAAACAGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC 740 CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAA4CAAAGAGC 799 TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 858 GCAGTGGCAGAGGTAGGATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA 917 CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGCAGGGGGCC 976 TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGGACTCCTCTTCATA 1035 TAGTGGCGATGATGTGCTGGAGCTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCCA 1094 GCCAGAGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTGAGGAGACGAGGCAAG 1212 GGAAGAATGCCTGTGCTGGAGTCTCTGGTCAGGTGGCTCTGCCACAGAGGAGAGGTGAC 1271 CACCAGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330 TGGGGATCTGGAGCAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGG 1389 S CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 00 GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1507 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566 CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625 S: CATTTTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684 CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 *-TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGC ATCAG 1802 GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1861 GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920 CAGCAACAATGGCAACCTACCATTCCCTTTGGAGCGCCATCCCAGGGATATTCGAAACT 1979 F I 6. 1 4b. CONTINUATION OF FIG. 14a. GGTGTCCAGTAGAAAACAGTGGTGGAGGTGACTGGAGACGCCAGTGGAGAGTTTCAGCT GGTGAAAAGAAACAGGATTTGGGATGGACATGAATAACCAATCTGACTGCTTCCCCGTA GAGCTGCTGGACTGTGCTGGGTGG CTGCTCCAGTCCCTAGCTGCCTTG'OACTCCCGAGA ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGGCTGGATTCCC AAGTCGAGTGGGTCTTACCCTGAGACAGGCCATGGAAGGTGGGCCTGTCATTGTCAGTG CCCAGCGCCCTGGATGAAACCGCTTTGCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC TTGGCTGGAGTTATAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAAGGGTCCAGGGTAC CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG TGGGAGAAGCGTGGGTCCGCAGGGCTGCTCAGTTACTCACCACTTCCCTGGGCAGGAGA GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGAGAGTGGCACACACTAGCA GGTTCCAGTCGGCCATCTTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAAGCCATT GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2038 2097 2156 2215 2274 2333 2392 2451 2510 2569 2628 2687 2746 2805 2864 2923 es. 0 0 *00 0 0000 -20 Leu -16 Leu Met Val Leu Leu Val Leu CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 Val Ala GTG GCC Phe Asp TTT GAC Leu Leu Leu CTG CTG CTT 5 Leu Pro Gin CTG CCT GAG Cys TGC Gin CAA -5 Cys G:ly Pro Val TGT GGC CCT GTT Gl1y GGA Ser Leu TCT CTG 1 Gly GGC 3019 9e 000 *0 0 S @0 S S.. 10 Gly Leu Leu Leu 7 I @000 S* S 50 S 0* SO Ala GCA Asp GAG Ile ATT Phe Trp Ala TTC TGG GCC 20 Ly s AAA Asn Arg AAC CGT Cys Arg TGG AGA Arg Phe AGG TTC GGC CTA CTT AGC AGG AAC ACC TTG 3064 Ser Arg Asn Thr 25 Ile Ser Thr ATC TCC ACT Phe Leu TTC TTG Arg AGA Cys TGC 35 Arg Asp Phe AGA GAG TTC Arg Arg Pro AGA AGG CCC Ser Ser Ala Cys Leu TGT CTC Pro Leu CCC CTG 40 Glu GAG 55 Ser TCC 70 Ser Arg Leu Cys Leu AGG CTG TGT CTG Ser Ser Pro Gin Met Trp Met Ala ATG TGG ATG GCA Ser Met Arg Cys TCC ATG AGA TGC Ala Pro Leu Leu Ly s AAG 3109 Val1 GTC 3154 Phe TTC 3199 Pro Ser Arg AGC AGA TCT TCA GCC TCT TCC CCA GAG AGC GCT CCT CTG CTG CCT 3244 FIG. 14c. CONTINUATION OFFIG.14b. Gly Thr GGA ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG CC TGGAGTCTTGCTTAGGGCAGGCAACAGGAGAG GAAGAATCTG TGGGGGTGATTGGGA CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAATCCAGAGAATCTAC CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA TC CTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGAC CTGGGGTC TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA AGCTT 3300 3359 3418 3477 3536 3595 3654 3659 II II I *1 F IG. 16. 0* 0 9* 0* S. omega 1 omega2 ornega3 oimega4 omegal 492/577 497/587 493/579 oniega2 85,3 473/577 466/570 ornega3 84,7 82,0 554/592 1 omega4 85,4 81, 8 93,6 Right upper part: Number of identical adjusted bases bases/total Left lower part Percentage homology 45549/85 FI f H A L L F P L L A A L V M T S Y S P V G S L G C 0 L P Q N H 1 ATGGCCCTCCI6TTCCCTCTACTGGCAGCCCTAGI6ATGACCAGCTATAGCCCIGT1GGATCIC 1GG6C E616ATCTCCICAGAACCAT K A L L F P L L A A L E V C S C G S S G S L G Y N L P Q N H 2 AIGGCCCICCICITCCCTACTGGCAGCCCIAGA6GGITGCAGCTGTGGCICTTCIGGATCTCIAGGAlAIAACCTGCCTCAGAACCAT H V L L L P L L V A L P L C H C G P V G S L. S W 0 L P Q 1N H 3 ATGGTCCTCCTGCTTCCTCTACICGTGGCCCTGCCGCTTI6CCACTGTGGCCCTGTTGGATCTCIGAGCTGGGACCIGCCTCAGAACCAT K VL LL V LL VA LL L C Q C GP V G SL GF OLP Q N 4 ATGGTCCIACTGCTTGTTCIACI6GTGGCCCTGCTGCTTTGCCAAI6166CCCTGTTGGATCTCI6GGCIGACC16CClCAGAACCGT 20 30 40 50 60 70 B0 G L L S R N T L V L L ft Q M R Rl 1 S P F L C L K R R 0 F R G L L GR NTI L V L L6GQ MR R 1ISP F L C L KO0R S 0F R 2 GGCCTGCTAGGCAGGAACACCITTGTGCTTTGGCCAALATGAGGAGAATCTCTCCCTICIG6UIAAAGGACAGAAGTACTCAGA G LL SR N IL AL L GQ MC R I S I F LC L K R R DOF R 3 GGCCTACTTAGCAGGA.ACACCTTGGCACTTCTG6CCAAAI6IGCAGA.ATCTCCACTTTCTT6GIGCTCAAGGACAGAAGAGACT ICAGG G LL S RNTILA L GQHM RI SI F LC L K 0R R FR F PQI H V0K6 S Q LQK APALV S VL H EML QQ I F SIL I TTCCCCCAGGAGATG.T6AIGGCAUICAGTTGCA6AAGGCCCGCC16IC6CCTCCATAGAI C I CACAAIC I CACCC F H QTE KR S A QW M LK A 00AK L Y T 61 H L Q H F L 2 TTCCACAAACGICTGGCGCCGAACAGACCTCCA6ACCA.GC TCC CAGAI 1G CTACAACGCAAC TCA.CTG7 F TK LPQ L L MLDGQK .CTTCCACCGAATGTTGCICAGAACA6AACCTCC16CCACICCCCCATA6ICCAGCA1GGACACCIC F P T E C S S A 5 H Q L Q L H S L H L E C LC F P IE RS SA A WNMTIL Q D L H 16 F H Q0 E S HL E o 4 6 1 TCCCCACAGAGCGCTCCTCTGCTGCCTGGAACATGACCCTCCTAGACCAGCTCCACACGATI TCATCAGCAGCC. ACCTGGA 3F 38 39 40 41 NKILL 0 42 43 44 450 QQLE 1 TICAGAAGCGCTT...GTCTACAAAGACAA6TAACAGCTCAC61CC16GAT TITCAUGAAIlGC16A so IH CC W VV V L I CSAGAISSPR 2 CGACCAT.............CTCTGACC1GGAAACGAGA6........ .6AATTGGCCGAIGAICAGAATC1G T LV EM G YE A G IS EGC S L ALR R Y F 2 GCG6GCTGGG6GAATGGGMA IGGAA GCAGAAAFCAGG1GAJGG1GG6CAGTI GCGAG GAGA. CTGA QG so H I RE E E VOCGAPI LRHLE IMYK 3 GC6GCICCAGGGACCAGAGAA1CCI6AGA A6AAAA1AAICGGG GlA16GGCC T AAIGCIGAG16AC. C16 46so30 470 400 40 500 41 40 430 430 450 1 AGGCCTGT ACACA6AAAGTAGAAGAAAAAACC1Li6CICAICAGTTtAIAA CG 2 CAIC.TC CIACAA6TCTACA6AAAIGAGAAGAAAAA AIICCTGGCAATGC1 GATAGA G so so: 3 CAGGCCTCCATCA1AACAGGAACCAGGAGAAAGAAAGIGAA6CIC66G1AGGICGAGGA.CG 4 ACCICICI. .TCA1CACAGAGCTCC. .GAGAGAAAIAAGA6GACCIG6 GGT6CT1TAAGA.CT 60 570 580 590 5 00 61 50 53 54 I t. F IG. 17 omegal omega2 ornega3 omega4 omegal 16/23 13/23 13/23 omega? 69,6 14/23 14/23 omega3 56,6 60,9% 19/23 omega4 56,5 60,9 82,6 S S S. S S SSS Right upper part: Left lower part: Number of identical amino acids in total of 23 amino acids Percentage homology F IG. 18. S* S S S SO S. S 5* .5 S S** I omega 1 omega? omega3 omega4 omega]l 128/166 130/169 131/167 omega2 77,1 Z 118/163 124/ 161 omega3 76,9 72,4 146/166 omega4 78,4 7710 88,0 S S S. S S* S 55 5 S. Right upper part: Lower left part: Number of identical amino acids/ Number of total adjusted amino acids Percentage homology 3 IA F IG.1 9. S. S 0 0 0 0000 S 0000 9S S S 0 S0 5* S. S S. 0 5* 5 S S 55 0.3- LU If1 0.1- LU (3 '1 I> I I I I 0 2 4 6 DAYS I it j~ CU,. U S. C eq.. 0 5@ S S OSU S U. 5 S 0 eq.. S LII7XJH U S 0 OU S q *4 S. S.C T+O 50 Io 0S'O CU 0e C C. S 0* O es 104x celLs/petri dish
AU45549/85A 1984-08-01 1985-07-29 Improvements in or relating to interferons Expired AU600653B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE3428370A DE3428370A1 (en) 1984-08-01 1984-08-01 Interferon, genetic sequences which code therefor, and organisms producing these
DE3428370 1984-08-01
DE19853505060 DE3505060A1 (en) 1985-02-14 1985-02-14 Type I interferons, genetic sequences which code therefor, and organisms producing these
DE3505060 1985-02-14

Publications (2)

Publication Number Publication Date
AU4554985A AU4554985A (en) 1986-02-06
AU600653B2 true AU600653B2 (en) 1990-08-23

Family

ID=25823496

Family Applications (1)

Application Number Title Priority Date Filing Date
AU45549/85A Expired AU600653B2 (en) 1984-08-01 1985-07-29 Improvements in or relating to interferons

Country Status (21)

Country Link
EP (1) EP0170204B1 (en)
JP (2) JP2566909B2 (en)
KR (1) KR0136799B1 (en)
AT (1) ATE67786T1 (en)
AU (1) AU600653B2 (en)
BG (1) BG60445B2 (en)
CA (1) CA1340184C (en)
DD (1) DD246318A5 (en)
DE (1) DE3584198D1 (en)
DK (1) DK175194B1 (en)
ES (2) ES8609475A1 (en)
FI (1) FI90667C (en)
GR (1) GR851866B (en)
HK (1) HK187896A (en)
HU (1) HU205779B (en)
IE (1) IE58942B1 (en)
IL (1) IL75963A (en)
MX (1) MX9203645A (en)
NO (1) NO177863C (en)
NZ (1) NZ212937A (en)
PT (1) PT80901B (en)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5231176A (en) * 1984-08-27 1993-07-27 Genentech, Inc. Distinct family DNA encoding of human leukocyte interferons
DE3607835A1 (en) * 1986-03-10 1987-09-24 Boehringer Ingelheim Int HYBRID INTERFERONS, THEIR USE AS MEDICINAL PRODUCTS AND AS INTERMEDIATE PRODUCTS FOR THE PRODUCTION OF ANTIBODIES AND THE USE THEREOF AND METHOD FOR THEIR PRODUCTION
EP0490233A1 (en) * 1986-03-10 1992-06-17 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Monoclonal antibodies against Bgl III-hybrid interferons, their use and process for preparing them
US4863727A (en) * 1986-04-09 1989-09-05 Cetus Corporation Combination therapy using interleukin-2 and tumor necrosis factor
DE3633323A1 (en) * 1986-10-01 1988-04-07 Boehringer Ingelheim Int NEW MONOCLONAL ANTIBODIES AGAINST IFN-OMEGA, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE FOR CLEANING AND DETECTING IFN-OMEGA
DE3635867A1 (en) * 1986-10-22 1988-05-11 Boehringer Ingelheim Int NEW YEAR EXPRESSION VECTORS FOR IFN-OMEGA, METHOD FOR THEIR PRODUCTION AND USE THEREOF
EP1987845B1 (en) 1997-11-20 2012-03-21 Vical Incorporated Treatment of cancer using cytokine-expressing polynucleotides and compositions therefor.
WO2000040273A2 (en) * 1999-01-08 2000-07-13 Vical Incorporated Treatment of viral diseases using an interferon omega expressing polynucleotide
WO2004099231A2 (en) 2003-04-09 2004-11-18 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
KR20090110951A (en) 2002-03-07 2009-10-23 아이드게노쉬쉐 테흐니쉐 호흐슐레 쥬리히 System and method for the production of recombinant glycosylated protein in a prokaryotic host
CA2818688A1 (en) * 2002-03-07 2003-09-12 Eidgenossische Technische Hochschule Zurich System and method for the production of recombinant glycosylated proteins in a prokaryotic host
WO2004096852A1 (en) * 2003-04-25 2004-11-11 The Institute Of Microbiology And Epidemiology, Academy Of Military Medical Sciemces, Pla A RECOMBINANT HUMAN INTERFERON ϖ, THE METHOD FOR EXPRESSING IT AND THE USES OF IT
CA2607595C (en) 2005-05-11 2018-11-27 Eth Zuerich Recombinant n-glycosylated proteins from procaryotic cells
SG10201400320TA (en) 2008-02-20 2014-05-29 Glycovaxyn Ag Bioconjugates made from recombinant n-glycosylated proteins from procaryotic cells
HUE038456T2 (en) 2009-11-19 2018-10-29 Glaxosmithkline Biologicals Sa Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells
HUE037956T2 (en) 2010-05-06 2018-09-28 Glaxosmithkline Biologicals Sa Capsular gram-positive bacteria bioconjugate vaccines
WO2013024158A1 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of protein kinase inhibitors and interferons or of protein kinase inhibitors and direct acting antivirals for the treatment and the prevention of hcv infection
WO2013024156A2 (en) 2011-08-17 2013-02-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Combinations of anti-hcv-entry factor antibodies and interferons for the treatment and the prevention of hcv infection
WO2014033266A1 (en) 2012-08-31 2014-03-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-sr-bi antibodies for the inhibition of hepatitis c virus infection

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000776A1 (en) * 1982-08-18 1984-03-01 Cetus Corp Interferon-alpha 6l
AU1946283A (en) * 1982-08-18 1984-03-07 Cetus Corporation Interferon-alpha 6l
EP0174143A1 (en) * 1984-08-27 1986-03-12 Genentech, Inc. Novel, distinct family of human leukocyte interferons, compositions containing them, methods for their production, and DNA and transfected hosts therefor

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3247922A1 (en) * 1982-12-24 1984-06-28 Boehringer Ingelheim International GmbH, 6507 Ingelheim DNA SEQUENCES, THEIR PRODUCTION, PLASMIDES CONTAINING THESE SEQUENCES AND THE USE THEREOF FOR THE SYNTHESIS OF EUKARYOTIC GENE PRODUCTS IN PROKARYOTS

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000776A1 (en) * 1982-08-18 1984-03-01 Cetus Corp Interferon-alpha 6l
AU1946283A (en) * 1982-08-18 1984-03-07 Cetus Corporation Interferon-alpha 6l
EP0174143A1 (en) * 1984-08-27 1986-03-12 Genentech, Inc. Novel, distinct family of human leukocyte interferons, compositions containing them, methods for their production, and DNA and transfected hosts therefor

Also Published As

Publication number Publication date
GR851866B (en) 1985-12-02
PT80901B (en) 1989-01-17
DK175194B1 (en) 2004-07-05
HU205779B (en) 1992-06-29
ES8609475A1 (en) 1986-08-01
IL75963A0 (en) 1985-12-31
CA1340184C (en) 1998-12-15
DD246318A5 (en) 1987-06-03
ES545725A0 (en) 1986-08-01
IL75963A (en) 1992-05-25
EP0170204A2 (en) 1986-02-05
FI90667C (en) 1994-03-10
KR870001310A (en) 1987-03-13
NZ212937A (en) 1991-08-27
FI852956A0 (en) 1985-07-31
ATE67786T1 (en) 1991-10-15
PT80901A (en) 1985-09-01
HK187896A (en) 1996-10-18
IE58942B1 (en) 1993-12-01
ES8708013A1 (en) 1987-09-01
NO177863C (en) 1995-12-06
DK346385A (en) 1986-02-02
EP0170204A3 (en) 1988-02-17
NO177863B (en) 1995-08-28
JPS61181381A (en) 1986-08-14
FI90667B (en) 1993-11-30
HUT39477A (en) 1986-09-29
JP2567195B2 (en) 1996-12-25
EP0170204B1 (en) 1991-09-25
DK346385D0 (en) 1985-07-30
NO853012L (en) 1986-02-03
AU4554985A (en) 1986-02-06
KR0136799B1 (en) 1998-04-25
FI852956L (en) 1986-02-02
JPH06181771A (en) 1994-07-05
MX9203645A (en) 1992-09-01
IE851906L (en) 1986-02-01
ES552431A0 (en) 1987-09-01
JP2566909B2 (en) 1996-12-25
BG60445B2 (en) 1995-03-31
DE3584198D1 (en) 1991-10-31

Similar Documents

Publication Publication Date Title
AU600653B2 (en) Improvements in or relating to interferons
KR940010864B1 (en) Mathod of making interferons
JP2515308B2 (en) Human immune interferon
CA1341569C (en) Microbial production of human fibroblast interferon
US7635466B1 (en) DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides
JP2642291B2 (en) DNA encoding hybrid human leukocyte interferon
DK170054B1 (en) DNA sequences, transformed host organisms, polypeptide having biological activity as gamma-horse interferon, process for its preparation, and pharmaceutical preparation containing the polypeptide
AU606585B2 (en) Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells
NZ200145A (en) Dna molecules coding for interferon;oligonucleotide intermediates;recombinant dna method for production of interferon;transformed microorganisms
EP0328061A2 (en) Human colony-stimulating factors
CN107286255A (en) It is a kind of by OVA, chicken interferon gamma and chicken interferon α fusion protein constituted and preparation method thereof
EP0174143B1 (en) Novel, distinct family of human leukocyte interferons, compositions containing them, methods for their production, and dna and transfected hosts therefor
JP2501549B2 (en) Interferon manufacturing method
FR2481316A1 (en) Gene for expressing protein similar to human interferon - derived plasmid recombined materials, and modified bacterial cells
GB2143535A (en) Improved plasmid vector and use thereof
WO1985005619A1 (en) Novel polypeptide and process for its preparation