IE51030B1

IE51030B1 - Recombinant dna technique for the preparation of a protein resembling human interferon

Info

Publication number: IE51030B1
Application number: IE121/81A
Authority: IE
Original assignee: Searle & Co
Priority date: 1980-02-06
Filing date: 1981-01-20
Publication date: 1986-09-17
Also published as: BE887397A; IE810121L

Description

This invention relates to a recombinant DNA technique for the preparation of a protein resembling human interferon.

Interferon is a natural molecule, which is 5 produced by cells in response to virus infection and certain other stimuli, 'see, for example, Isaacs, A., and Lindenmann, J., ?roc. Roy. Soc. 3., (1957), 147, 258-267).

This molecule, when exposed to cells, renders the cells resistant to virus infection. Bence, interferon is a well recognised anti-viral agent, (see, for example, Isaacs, A. et al, (1957), Proc. Roy. Soc. Β., 147, 26S-273).

Since the original observation of anti-viral activity, a number of other properties have been ascribed to interferon, (see, for example, Stewart, W.E. Interferon I, (1979), Academic Press, I. Gresser (Ed.)), including a potential role in inhibiting the growth of cancerous cells in vivo, (see, for example, Paucker, K., 20 al, (1962), Virology, 7, 324-334; Gresser, I., et al, (1970), Ann. N.Y. Acad. Sci., 173, 694-699; Mellstedt, H., et al, (1979), Lancet, 1, 245-247; Blomgren, K, et al, (1976), Acta. Med. Scand., 199, 527-532; Merigan, T.C., et al, (1978), New Engl. J. Med. 298, 981-987; and Merican, T.C. et =1, (1978), New Engl.

II, J. Med., 299, 1449-1453).

Interferon also exhibits a marked degree of species specificity, (see, for example, Tyrell, D.A.J., (1959), Nature, 154 , 452-453, and Gresser, I· ret al, (1974), Nature, 251, 543-545), which means that, for treatment of human disease, interferon derived from human cells is preferred.

For a detailed review relating to interferon, reference may be made to Texas Reports on Biology and Medicine, 35, 1979, published at the University of Texas Medical 3ranch, Galveston, Texas, U.S.A., (S. Baron and F. Diazani, Editors).

As regards conventional sources, tissue culture cells are the only practicable source of material. A process based on tissue culture cells is regarded as labour-intensive, unreliable, expensive and generally results in low yields.

An object of the present invention is to provide means for the preparation of a protein having properties resembling those of human interferon by recombinant DNA techniques in bacteria, which are cheaper and more reliable and which also have the potential to produce modified forms of interferon. Furthermore, higher yields may be obtained.

Interferon is a glycoprotein having a molecular weight of about 20,000. The amino-termir.al polypeptide sequences of human and mouse interferons are known, (see, for example, Knight, Ξ., Jr., et al, (1980), Science, 207, 525-526; Zoon, K.C., et al, (1980), Science, 207, 527; and Taira, H., et al, (1980), Science, 207, 528-529).

Figure 1 of the accompanying drawings shows selected regions of these sequences, together with the corresponding codon possibilities. (In accordance with common practice, N indicates that any of the four nucleotides may he present.) Out of the thirteen amino acids known for human fibroblast FS-4 interferon, three are common (underlined) with the mouse interferons A and B obtained from Ehrlich ascites tumour cells, while only one amino acid is common between human fibroblast and lymphoblastoid interferons. Indeed, at the DNA level, there seems more likelihood of similarity between human fibroblast interferon and mouse interferons A and B (dotted) than between the two human proteins.

Clearly, the region corresponding to the five amino terminal residues of human fibroblast interferon displays a minimum number of coding oligonucleotide permutations over a sequence of sufficient length to be reasonably specific for an individual mRNA in a higher eukaryote cell, (ses, for example, Montgomery, Di...,et al (1978), Cell, 14 , 673680). The codons for the serine and leucine residues were deduced by first choosing those triplets most likely to shew maximum homology with the analogous mouse condons. The ambiguities now remaining in these and the other triplets were then further reduced by acting on known frequencies of codon usage in human genes, (see, for example, Grantham, F.. , et al, (1980), Nucl. Acids Res., 3., r47-r62). Thus, it was possible to reduce the number of potential coding sequences to two, differing in only the third residue of the serine codon, which does not exhibit a clear discrimination in this position. On this basis, the sequences of two oligocecxyribonucleotides (IFIA and IB) were deduced, which were expected to prime specifically the synthesis of interferon cDNA when incubated with reverse transcriptase and pOlyA-mSNA (3'terminally polyadenylated' mSNA) extracted from induced cells.

An additional factor involved in the prediction of the structure of interferon mRNA - specific primers was the belief that a G residue in DNA may form a moderately stable base pair with a U residue in RNA (or a T residue in DNA). Hence, if in certain positions the primers contained a G residue instead of the correct A residue, a degree of specific priming might still be expected. (See, for example, Powers, G.J., et al, (1975), J.A.C.S., 97, 875-684.) The oligonucleotides (IFIA and IB) were 32 synthesised and radioactively-labelled with . P-phosphate at the 5’ termini thereof, hybridised to mRNA preparations from human fibroblasts, either mock induced or induced to produce interferon by priming with interferon and subsequent superinduction using poly(I):poly(C) and cycloheximide,and incubated with the enzyme reverse transcriptase with the conventional decxynucleoside triphosphate substrates. Only 1FIA cave a transcript, of about 150 nucleotides, specific to mRNA from induced fibroblasts. The nucleotide sequence of this negative strand transcript was determined by known methods and is shown in Figure 2 of the accompanying drawings.

The top strand in accompanying Figure 2 is the sequence of the DNA transcript. This DNA sequence defines the sequence of the 5’-end of human fibroblast interferon mRNA, shown as the bottom strand in accompanying Figure 2. The mRNA sequence in turn defines the protein sequence, beginning from the first ADG, of the amino -terminal region of the presumed interferon precursor molecule.

A second oligocecxynucleotice (IFII! was synthesised which was complementary tc part of the determined sequence as indicated in Figure 3 of the 32 accompanying drawings. [ P] IFII was hybridised as above to cONA prepared by known procedures from total mRNA from induced and mock induced fibroblast cells and a transcript produced as above. When the products of this reaction were fractionated on a size basis using denaturing gel electrophoresis, s particular radioactive transcript of about 700 nucleotides was obtained from cDNA from induced, but not mock induced, cells.

When electrophoresed through native gels, the interferon-specific product was about 850 base pairs in length, corresponding approximately to the size of the complete interferon mRNA molecule. This discrepancy in size may be explained by assuming that, as well as acting as template for IFII, the single strand interferon cDNA also self-primes by virtue of the 3'-end looping back on itself. This transcript is then elongated up to the position of the IFII-primed transcript where it is 1030 halted, thus giving rise to a full-length doublestranded interferon gene, (i.e. a gene coding for interferon), in which the 3’-end of the self-primed transcript and the 51-end of the 1FI1-primed transcript are not covalently joined. Hence, the size of the interfercr.-specif ic product will vary depending on 'the type of gel system.

The interferon gene was eluted from native gels and sequenced by known methods resulting in the elucidation of the gene sequence coding for the aminoterminus of the mature fibroblast interferon. An extended version of XFII, IFIII, (shown ir. Figure 4 of the accompanying drawings), was also made and the above process repeated. The same results were obtained.

This sequence largely verifies the predictions used in the synthesis of IFIA. It may be seen from a comparison of accompanying Figures 1 and 5 that the actual mRNA sequence specifying the amino-terminus of the mature interferon polypeptide is identical to that on which the structure of IFIA was based, except for only one nucleotide difference in the codon for the fifth amino acid from the amino-terminus of the mature polypeptide. Thus out of a total of sever, ambiguous nucleotide positions, six were anticipated correctly in the case of IFIA.

(See, for example, Houghton, M., et al, (1980), Nucl. Acids Res., 8, 1913-1931.) An additional oligoceoxynucleotide primer, IFIV, (shown in Figure 4 of the accompanying drawings), was then made which corresponded to a region near to the end of the newly-determined sequence. Sy repeating the above process, further sequence information was? obtained from which another primer, IFV, (shown in Figure 4 of the accompanying drawings), was deduced and synthesised. The process was then repeated yet again. In this way, the entire gene sequence .coding for the mature fibro10 blast interferon polypeptide, (shown in Figure 5 of the accompanying drawings), was determined step-wise. (See, for example, Houghton, M., et al, (1980), Nucl. Acids Res., 8., 2885-2894.) Figure 6 of the accompanying drawings shows the double strand gene sequence that codes for the 5’ untranslated mRNA sequence and the protein-coding mRNA sequence.

An illustrative scheme of the basic procedures used to clone the interferon gene into bacterial plasmids is shown in Figure 7 of the accompanying drawings.

The end-products of these procedures were recombinants containing interferon genes coding for the mature interferon polypeptide and the 3' untranslated interferon mRNA sequence. In addition, these recombinants contained one molecule of IFIII primer preceding the mature interferon gene sequence and also a synthetic Hind III linker molecule at both ends of the gene.

A partial DNA nucleotide sequence of one of these interferon gene recombinants is shown in Figure 8 of the accompanying drawings, which shews that during the cloning procedures, (presumably as a result of the SI nuclease treatment), the 5' terminal nucleotide Of IFIII was lost. Also, it was observed that the mRNA triplet coding for the tyrosine residue at amino acid position 30 in the mature protein was UAU, rather than the UAC triplet which had previously been determined by sequencing reverse transcripts directly (see accompanying Figure 5.). This silent nucleotide change is presumably a reflection of gene polymorphism and means that both types of gene will result in the expression of identical interferons.

In order to express the interferon gene in bacteria, it was transferred from pBR 322, (see, for example, Bolivar, F., et al, (1977), Gene, 2, 95-113) into the Kind III site of the so called pWT 2x1 expression plasmids. The latter series of plasmids contain the promoter sequence responsible for the transcription of the tryptophan opercn and cater for all three possible translation phases, (see, for example, published U.K Patent Specification No. 2,052,516). Hence, if the interferon gene is inserted into these plasmids in the correct orientation with respect to transcription, only one will result in the faithful expression of the gene. From the sequence shown in Figure 8 of the accompanying drawings, it was predicted that pWT 221 would provide the open translation phase necessary for the faithful expression of the mature interferon polypeptide, although the latter would be fused to an amino-terminal peptide of sixteen residues resulting from expression of the short trp E gene sequence and the Hind III linker molecules present in the pWT 2x1 plasmids and also from the expression of the Hind III linker and the IFIII primer used in the original cloning of the interferon gene.

Indeed, when the interferon gene was transferred to the pWT 2x1 plasmids and the trp-promoter de-repressed by incubating in the absence of tryptophan and in the presence of 3 6-indole acrylic acid, anti-viral activity was only demonstrated in the case of the pWT 221 derivative, as expected.

Figure 9 of the accompanying drawings, (wherein Veros = control cells uninfected with EMC virus (Encephalcmyocarditis virus); Std. A = interferon standard A; Std. B = interferon standard B; 221 = extract of recombinant containing the interferon gene inserted into pWT 221; 231 = extract cf recombinant containing the interferon gene inserted into pWT 231; 231 + Std.B = extract of recombinant containing the interferon gene inserted into pWT 231 to which interferon Standard B has been added prior to extraction; and EMC = control cells infected with EMC virus )f shows a typical result Obtained when the bacterial extracts were assayed for anti-viral activity in an in vitro system. In this system, the samples were diluted in 0.5 log1Q steps and applied to monolayers of African green monkey cells (Verog. After an overnight incubation, the cells were challenged with EMC virus prior to cell-staining, whereby those samples containing interferon activity were seen to protect the vero cells against the cytopathic effect (cpe) of the virus. By comparing the results obtained with known interferon standards, the level of activity could be quantitated.

No activity was detected in extracts of the pWT 211 or pWT 231 interferon recombinants bnly the result obtained with the pWT 231 recombinant is shown in accompanying Figure 9), while the observed activity from extracts of pWT 221 recombinants is equivalent to an approximate yield of 106 international reference units of interferon per litre of bacteria, (i.e. equivalent to 106 units of an international standard preparation of interferon, 1 unit being defined in terms of that amount which results in a 50% reduction of viral replication in tissue culture). It is likely that considerable interferon activity is lost during the extraction procedure since, when a native fibroblast interferon standard was added to the ?WT 231 recombinant prior to extraction, a recovery of only about 30% was achieved.

(It should be noted that the observed toxic effect of the recombinant extracts in the anti-viral assays is due to the presence of Triton (Registered Trade Mark) X-100 which was used in this particular extraction process.) It was now necessary to remove the DNA coding for the non-interferon amino acids at the amino-terminus of the fused interferon polypeptide. This may be conveniently carried out by making use of the plasmid pWT 501, (see, for example, U.K. Patent Application No. SO 36080, which has subsequently been published as EP 0 052 002A).

The plasmid pWT 501 has a molecular length of 3805 bp (base pairs) and the restriction map thereof is illustrated in Figure 10 of the accompanying drawings.

This plasmid may be produced by a process which comprises restricting pWT 221 using Hha I, isolating the trp-promoter-containing fragment, incubating this fragment with DNA polymerase I, ligating the resulting blunt-ended fragment to Hind III linkers, restricting the ligated material using Hind III, isolating the trppromoter-containinc fragment, ligating this fragment to Kind Ill-cut, bacterial alkaline phosphatase-treated Μ, , see, for example, Boyer, H.W., pAT 153, (see, for example, Twigg, A.J., and Sherratt.

D., (1980), Nature, 283, 218-218), transforming E. coli K-12 HB101 (genotype gal , lac", ara , pro , arg , strr, rec A , r^ , and Roullard - Dussoix, D., J. Mol. Biol., 41, 459-472) using the resulting plasmid and selecting for ampicillinresistance and tetracycline-resistance.

A small Hind III/Taq I restriction fragment which contains the trp-promoter may be isolated from this plasmid. The Taq I cleavage site in question is located in the DNA sequence corresponding to the ribosome binding site in the mRNA region that codes for the trp leader polypeptide, occurring just before the initiating methionine triplet. This fragment may be joined to a Sac I/Hind III restriction fragment of the cloned interferon gene that contains all of the mature protein-coding sequence preceded by only six base pairs as shown in Figure 11 of the accompanying drawings. Since the aminoterminal residue of the mature interferon happens to be methionine, the joining process may be carried out in such a way as to produce a chimeric DNA which, when transcribed, contains an artificial ribosome binding site that utilises this methionine codon as the initiating codon for translation. The conjoint molecule was then cloned into Esherichia coli (E. coli) K-12 HB101 using pAT 153 as the plasmid vector. This process is illustrated in Figure 11 of the accompanying drawings.

Another important consequence of the above process is that the resulting expression plasmid will lack the trp attenuator region which plays an important role in regulating transcription of the trp-operon, (see, for example, Miozzari, G.F., and Yanofsky, C., (1978), J. Bacteriol., 133, 1457-1466).

Recombinants containing the requisite conjoint molecule in either orientation were then analysed regarding the ability thereof to produce interferon. They were found to produce significantly higher levels, of roughly 6-fold (in both orientations), of extractable anti-viral activity when compared with the recombinants containing the interferon gene inserted into pWT 221. As expected, the size of the interferon product (about 19,000 daltons) is smaller than that obtained using pWT 221 as the expression plasmid (about 24,000 daltons). This is illustrated in Figure 12 of the accompanying drawings and is reasonably consistent with the expected nature of the interferon polypeptides produced in both cases. (Xn accompanying Figure 12: a = recombinant containing conjoint molecule in right-ward orientation (i.e. transcription proceeds from the trp-promoter towards the tetracyclineresistance gene in the plasmid); b = recombinant containing conjoint molecule in left-ward orientation (i.e. transcription proceeds from the trp-promoter away from the tetracycline-resistance gene); c = transformant containing pAT 153 only; d = recombinant containing the interferon gene, but lacking the trppromoter; f = expression recombinant consisting of the interferon gene inserted into pWT 221; and g = expression recombinant consisting of the interferon gene inserted into pWT' 211) . Accompanying Figure 12 also illustrates the abundance of the 19,000 dalton interferon polypeptide relative to the 24,000 dalton product synthesised in the pWT 221 recombinant. This increase in expression of the interferon gene may be attributed to the removal of the trp-attenuator region.

The artificial ribosome binding sequence contains six nucleotides between the remains of the Taq I cleavage site and the ATG initiator codon. The original plasmid (pWT 50l) contains only five nucleotides (of different sequence) in this analogous region (see accompanying Figure 11). Therefore, further changes in the size and sequence of this particular region in the interferon recombinant may increase the efficiency of ribosome binding and consequently improve the yield of the interferon polypeptide.

It is to be expected that the yield of interferon-like polypeptide may be further improved by increasing the number of interferon genes and/or trppromoters in each plasmid. This may be done by known methods .

Also, the use of bacteria fermentors in which high cell densities may be obtained, will obviously increase the yield of interferon activity per litre of bacteria.

Apart from being able to protect Verc cells against the ope of SMC virus, the interferon polypeptide produced in bacteria also protects V3 African green monkey cells against infection by vesicular stomatitis virus (VSV) in vitro. In addition, like the native fibroblast interferon, the bacterial interferon polypeptide has also been found capable of enhancing the natural killing activity of human lymphocytes in vitro.

The genetically-engineered interfercn-like protein is primarily intended for use in the treatment of disease and it will be used in effective amounts, optionally with a pharmaceutically-acceptable carrier. The dosages used in studies using native human interferons have varied considerably, for example from 10$ to 107 international reference units per day for various periods, (see, for example, Scott, G.M., and Tyrell, D.A.J., (1980), Brit. Med. J., 280, 1558-1562). The preferred treatment regime would be expected to vary according to the type and severity of the disease. The interferon polypeptide produced from bacteria may exhibit different ac tivities and pharmacokinetic properties relative to native fibroblast interferon in some instances.

The interferon recombinant DNA has also been used to screen human gene banks for the presence of clones containing the chromosomal interferon gene.

This was achieved by first extracting the Hind III restriction fragment containing the interferon gene from the pWT 221 recombinant plasmid, radio-labelling and then hybridising to plaques produced by transfection 1030 of bacteria with a human chromosomal DNA/χphagehybrid library. Those plaques that specifically bound the interferon gene probe were purified and >. recombinant DNA prepared therefrom. The fibroblast (or 6) interferon gene contained in these molecules was then sequenced and the sequence is shown in Figure 13 of the accompanying drawings.

An uninterrupted gene sequence identical to the one illustrated in accompanying Figure 6 was observed immediately followed by a sequence corresponding to the 3' untranslated interferon mRNA sequence that has previously been determined, (see, for example, Taniguchi, T. , et al, (1980), Gene, 10, 11-15; and Derynck, R., et al, (1980), Nature, 285, 542-547).

This observation indicates that there are no intervening sequences (introns) within this particular fibroblast interferon gene in the genome.

In additicn, short gene sequences upstream of and downstream from the interferon gene were also determined. The upstream flanking region may constitute part of the RNA polymerase initiation/promoter sequence that controls the transcription of the interferon gene in human fibroblasts. (See, for example, Houghton, Μ., et al, (1981), Nucl. Acids Res., 9, No. 2).

It appears that bacteria cannot faithfully express eukaryote genes containing introns and so an implication Of the above is that it should be possible to induce bacteria to produce human interferons by transforming them with chromosomal genes coding for interferon. These chromosomal genes might be isolated from readily-available human gene banks and such an approach offers advantages over first isolating the interferon mRNA from induced eukaryote cells, synthesising the cDNA gene in vitro, transforming bacteria and so on.

As it appears that interferon genes in general do not co.ntain introns, since a leukocyte interferon gene also lacks them, (see, for example, Nagata, S., et al, (1980), Nature, 287, 401-408) , such an approach might be applied to various interferon genes.

By using the cloned interferon gene, i.e. cDNA gene, as a probe for homologous sequences in total genomic DNA restricted with a variety of restriction enzymes, the presence of one predominant type of fibroblast interferon gene could be observed, (see, for example, Houghton, M., et al, loc cit). However, there was some evidence for the presence of other genes that were partially homologous to the probe (ibid). The possibility remains therefore that other interferon genes could be isolated (from restriction digests of total genomic DNA or directly from human chromosomal gene banks) using the present particular cloned interferon gene. Furthermore, having successfully employed specific oligonucleotides tc detect bacterial recombinants containing interferon cenes (see (11) below), it is now feasible to use selected oligonucleotides to detect and isolate different interferon cenes contained in human chromosomal gene banks or present in a restrictic: digest of total genomic DNA. For example, the present interferon gene sequence shows certain similarities with human leukocyte genes, (see, for example, Taniguchi, T., et al, (1980), Nature, 2S5, 547; and Streuli, M., et al, (1980), Science, 209, 1343-1347). By using an oligonucleotide probe that is common between the fibroblast and leukocyte interferon gene sequences, it is possible to isclaze both gene types from a human gene bank, for example.

Similarly, other partially homologous interferon genes could be obtained by the use of other oligonucleotides homologous to different regions cf the present cloned interferon gene. Such an approach may be more effective than simply using the entire cloned fibroblast interferon gene as a probe for partially-homologous genes, owing to a diminution of the background in such experiments by the use of specific oligonucleotides.

Common sequences between different interferon genes indicate that they may code for a common function displayed by the various interferon polypeptides.

Hence, the gene sequence coding for the 50-60 amino acids approximately at the carboxyl terminus of the molecule may be of particular significance and it is ’ to be understood that the product of a recombinant containing such a sub-gene or part thereof may be of particular value. Similarly, other sub-genes (whether displaying some homology with other interferon gene sequences or not) may also be of particular value.

It is also to be appreciated that the fibroblast interferon gene sequence may be limited with regard to expression in bacteria due to the use of codons that may be infrequently used in bacterial mRNA molecules. Hence, it is clear that by changing certain interferon gene codons, such that they will still encode the same amino acid, but will be more efficiently translated by the bacterial translational apparatus, the overall yield of interferon may be improved. The logic involved in deciding which codons, if any, would benefit from adjustment may be based on observed codon usage in prokaryote mRNA molecules, (see, for example, Grantham, R., et al, loc cit).

Also, it is to be understood that cloning the interferon gene additionally containing the DNA for the pre-peptide signal sequence may result in some advantage.

For example, the interferon product, possibly after cleavage of the signal sequence, may be sequestered into the periplasmic space. In addition, the conformation of the interferon polypeptide may be more similar to the natural state as compared to that obtained by direct expression of the gene sequence coding for the mature interferon only. Consequently, the activity of the interferon product may be greater.

Alternative approaches to the production of interferon by genetic engineering include introducing the required interferon gene into eukaryote cells, for example mouse L cells or yeast cells.

Mouse L cells may be transformed with exogenous DNA and this may be carried out with human interferon genes in a number of ways. For example, a chromosomal interferon gene containing all of the DNA sequence required for correct RNA polymerase initiation could be cloned into the mouse cells thus allowing transcription and subsequent expression. Alternatively, an interferon gene lacking this promoter sequence might be suitably linked to another piece of eukaryote DNA containing a promoter, e.g. the thymidine kinase gene that is used as a basis for transformant selection, (see, for example, Wigler, M., et al, (1979) , Cell, 16 , 777-785). In this way, transcription of the interferon gene would proceed via the linked gene sequence.

It is also possible that transformation of L cells with an interferon gene lacking a promoter sequence may still result in expression of the DNA due to fortuitous insertion into particular regions of the genome.

Alternatively, certain plasmids inhabit yeast cells and interferon genes could be introduced into such plasmids and suitably modified versions thereof so as to obtain expression of the heterologous DNA and yield interferon polypeptide.

It is possible that interferon produced by suck methods may be more biologically active and effective than that produced in prokaryotes where certain important modifications may not be made to the eukaryote protein. The yield of interferon may also be higher in these eukaryote systems.

Accordingly, in a first embodiment, the 10 present invention provides a gene for the expression of a protein having properties resembling those of human interferon which comprises a coding strand and a complementary strand, the coding strand comprising the sequence.15 5' GGC.CAT.ACC.CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG.GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT. GTT.CGT,GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC. TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.20 TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT. GAG.GAG.ATT. AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC. ACT.GGC.TGG. AAT. GAG. ACT. ATT.GTT.GAG.AaC.25 CTC.CTG.GCT.AAT.GTC.TAT.CAT,CAG.ATA.AAC.CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA. AAA.CTG. GAG. AAA.GAA. GAT.TTC.ACC.AGG.GGA.AAA. CTC. ATG. AGC. AGT. CTG. C AC. CTG. AAA. AGA. TAT. TAT. GGC-. AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC. AAG. GAG.TAC.AGT.CAC.TGT,GCC.TGG. ACC.ATA.GTC. AGA.GTG,GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.51030 ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA. AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC. ATT.CTT.CAA. CCA. GCA. GAT.GCT.GTT.TAA.GTG.ACT. GAT.GGC. TAA.TGT.ACT. GCA.TAT.GAA.AGG.ACA.CTA.GAA.GAT.CTT.GAA. 5 ATT.TTT. ATT. AAA.TTA.TGA.GTT.ATT.TTT. ATT. TAT.TTA.AAT. TTT. ATT. TTG. GAA.. AA.T. AAA. TTA. TTT. TTG. GTG. CAA. AAG. TCA. ACA.TGG.CA 3’ or ε sub-unit thereof or an equivalent thereof.

(It is to be understood that the so-called coding strand has the same sense as the mRNA.) The above corresponds to the entire mRNA-coding sequence plus the upstream and downstream gene sequences obtained by isolating the gene from a chromosomal gene bank and sequencing.

In a further embodiment, the present invention provides such a gene, the ceding strand of which comprises the sequence: ' GGC.CAT.ACC.CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC,GAC.ACT.20 GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC. AGG.ATG.AAC.TTT.GAC.ATC.CCT. GAG. GAG.ATT.2 5 AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG. ACC.ATC.TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC. ACT.GGC.TGG.AAT. GAG. ACT.ATT.GTT. GAG.AAC.51030 CTC.CTG.GCT.AAT.GTC. TAT.CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA. CTC. ATG. AGC. AGT. CTG. CAC. CTG. AAA. AC-A. TAT. TAT. GGG. AGG. ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC.5 TGG. ACC.ATA.GTC. AGA. GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT,CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC.ATT.CTT. CAA. CCA. GCA. C-AT. GCT. C-TT. TAA. GTG. ACT. GAT. GGC. TAA. TGT. 10 ACT. GCA. TAT.GAA.. AG G.ACA.CTA.GAA. GAT. TTT.GAA. ATT.TTT.ATT.AAA.TTA.TGA.GTT. ATT.TTT.ATT. TAT.TTA.AAT.TTT. ATT.TTG.GAA.AAT. AAA.TTA.TTT.TTG.GTG.CAA.AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.CGA.TTT.GAT.TTA.TAT.AAC.CA..3' or a sub-unit thereof or an equivalent thereof.

This corresponds to the above with some additional downstream sequence.

The present invention also provides such a cene, the coding strand of which comprises the sequence: ' GGC.CAT.ACC.CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.20 CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC. ACT.GTT.CGT.GTT.GTC.AAC.ATG. ACC.AAC.AAG.TGT.CTC.CTC.CAA. ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT. CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA. AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.2 5 AAT.GGG.AGG.CTT.GAA. TAT.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT. GAG. GAG. ATT.AAG.CAG.CTG.CAG.CAG.TTC. CAG. AAG. GAG. GAC. GCC. GCA. TTG. ACC. ATC. TAT. GAC-. ATG. CTC. CAG.AAC.ATC.TTT.GCT. ATT.TTC. AGA.CAA. GAT.TCA.TCT.AGC 030 ACT. GGC. TGG. AAT. GAG. ACT. ATT. GTT. GAG. AAC. CTC. CTG. GCT. AAT. GTC. TAT. CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG. GAA. GAA. AAA. CTG. GAG. AAA. G AA. GAT. TTC. ACC. AGG. GGA. AAA. CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT. TAT. GGG. AGG. ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC.TGG. ACC.ATA.GTC. AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT .TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT. TCA. AGC. ATT. CTT.CAA. CCA. GCA. GAT. GCT. GTT. TAA.GTG. ACT.GAT.GGC.TAA.TGT. ACT. GCA. TAT.GAA.AGG.ACA.CTA.GAA. GAT.TTT.GAA.ATT.TTT.ATT. AAA.TTA.TGA.GTT.ATT.TTT.ATT. TAT. TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.TTA.TTT.TTG·GTG.CAA." AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.CGA.TTT-GAT.TTA. TAT.AAC.CA.. 3' or a sub-unit thereof.

This constitutes a polymorphic form of the above. Certain particular portions of such genes also constitute embodiments of the present invention.

Accordingly, in one particular further embodiment, the present invention provides such a gene, the coding strand of which comprises the sequence; ' TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC. ACT.GTT.CGT.GTT.GTC.AAC.ATGACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG. CTC.CTG.TGG,CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG,AAC.TTT.GAC.ATC.CCT.GAG.GAG.ATT.AAG.51030 CAG. CTG. CAG. CAG. TTC. CAG. AAG. GAG. GAC. GCC. GCA. TTG. ACC. ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA. CAA. GAT.TCA.TCT.AGC. ACT.GGC.TGG.AAT. GAG.ACT.ATT.GTT. GAG. AAC. CTC. CTG. GCT. AAT. GTC. TA.T. CAT. CAG. ATA. AAC. CAT. 5 CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG. GAG.AAA.GAA. GAT. TTC. ACC.AGG.GGA. AAA.CTC.ATG,AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT. TAT.GGG.AGG. ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC.TGG. ACC.ATA.GTC.AGA.GTG. GAA. ATC.CTA.AGG.AAC.TTT.TAC.TTC. ATT.AAC. AGA.CTT.ACA.GGT. 10 TAC. CTC. CGA. AAC. TGA. AGA. TCT, CCT. AGC. CTG. TGC. CTC. TGG. GAC. TC-G. A-'A. ATT. GCT. TCA. AGC. ATT. CTT. CAA. CCA. GCA. GAT. GCT.GTT.TAA.GTG.ACT.GAT.GGC.TAA.TGT.ACT. GCA.TAT.GAA. AGG. ACA.CTA.GAA. GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA. GTT.ATT.TTT. ATT. TAT.TTA.AAT.TTT. ATT.TTG.GAA.AAT. AAA. 15 TTA.TTT.TTG.GTG.CAA.AAG.TC 3' or an equivalent thereof.

Possibly with the addition of one or two A residues at the 3'-end, this constitutes the gene sequence corresponding to the entire mRNA sequence.

In another particular embodiment, the present invention provides such a gene, the coding strand of which comprises the sequence: ' ATG. ACC. AA C. AAG. TGT. CTC. CTC. CAA. ATT. GCT. CTC. CTG. TTG. TGC.TTC.TCC. ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG. CTT.GGA.TTC.CTA.CAA. AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG. AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC. 25 CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG.GAG. ATT. AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC. GCA.TTG.ACC.ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC. 51030 AGA.CAA. GAT.TCA.TCT.AGC. ACT.GGC.TGG.AAT. GAG.ACT.ATT. GTT,GAG.AAC.CTC.CTG.GCT.AAT.GTC. TAT. CAT.CAG. ATA. AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC. ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.5 AAA. AGA. TAT. TAT.GGG.AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC. AAG. GAG.TAC.AGT.CAC.TGT.GCC.TGG. ACC.ATA.GTC.AGA.GTG. GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA. 3' or an equivalent thereof This corresponds to the gene sequence coding for the pre-peptide and the mature protein, Also, the present invention provides such a gene, the coding strand of which comprises the sequence: ' ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA. AGC. AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG. AAT. GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG. GAG.ATT,AAG.CAG.CTG.CAG,CAG.TTC.CAG.AAG.GAG.GAC.GCC. GCA.TTG. ACC.ATC. TAT. GAG.ATG.CTC.CAG. 20 AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.AAT.GTC. TAT. CAT. CAG. ATA. AAC. CAT. CTG. AAC-. ACA. GTC. CTC-. GAA. GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGC-.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.ATT.25 CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT. GCC. TGG.ACC.ATA.GTC. AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC. ATT. AAC. AGA. CTT. ACA.GGT.TAC.CTC.CGA. AAC. TGA. 3' S103Q or an equivalent thereof.

This corresponds to the gene sequence coding for the mature protein.

Sub-units of such genes also const!tute.embociments of the present invention.

The present invention also provides a process for the production of a gene or an equivalent thereof or a sub-unit thereof according to any of the first three of the above-mentioned embodiments which comprises using as a probe a molecule having a sequence of a gene or an equivalent thereof or a subunit thereof according to any of the other of the abovementioned embodiments to isolate from human chromosomal DNA a human chromosomal interferon gene.

It is to be understood that, although single or double strand DNA may initially be used as the probe, at the hybridisation stage a single strand form will be present.

The present invention further provides a process for the production of a gene or an equivalent thereof or a sub-unit thereof according to any one of the above-mentioned embodiments, other than the first three thereof, which comprises isolating poly A-inSNA from induced fibroblast cells, synthesising single strand cDNA using oligo dT primer and reverse transcriptase and synthesising double strand DNA therefrom using reverse transcriptase or Ξ. coli DNA polymerase I and optionally a primer.

It is preferred to use a primer in the third step of this process. Preferably, the reaction mixture is fractionated after the second step and/or after the third step. Self-priming is preferably inhibited after the second step. The product may be identified, after cloning, by using a primer to screen colonies.

The present invention also provides a process for the production of a gene or an equivalent thereof or a sub-unit thereof according to any one of the abovementioned embodiments, other than the first three thereof, which comprises isolating mRNA from induced fibroblast cells, synthesising single strand cDNA using a specific primer and reverse transcriptase and synthesising double strand DNA therefrom using reverse transciptase or E. coli DNA polymerase I and optionally a primer.

This may be considered to constitute a modification of the above process and the above-mentioned preferred procedures may be applied thereto.

Also, the present invention provides a process for the production of a gene or an equivalent thereof or a subunit thereof which has a portion common with or related to a portion of a gene or an equivalent thereof or a sub-unit thereof according to any of the first three of the abovementioned embodiments which comprises using as a probe a molecule having a sequence corresponding to at least part of a common or related portion to isolate from human chromosomal DNA a human chromosomal gene.

The present invention also provides a plasmid recombinant which comprises a plasmid vector having inserted therein at an insertion site such a gene or a sub-unit thereof or an equivalent thereof, the plasmid recombinant enabling translation in the correct phase for the mRNA corresponding to the inserted gene or subunit thereof or equivalent thereof and having a bacterial promoter upstream of and adjacent to the insertion site such that the inserted gene or sub-unit thereof or equivalent thereof is under bacterial promoter control.

It may be considered advantageous to insert a plurality of DNA fragments. Similarly, it may be thought desirable to use a plurality of bacterial promoters. One preferred case uses at least one trppromoter. It is particularly advantageous to use a plasmid recombinant which lacks at least part of the DNA sequence responsible for attenuation of tranoription.

A particular example of a preferred plasmid recombinant comprises a modified derivative of pWT 501. In some instances, advantage may be gained from the fact that the inserted DNA also codes for an initiation codon and/or at least part of a ribosome binding site.

The production of such a plasmid recombinant by a process which comprises inserting the DNA at the insertion site of an appropriate plasmid vector also constitutes an embodiment of the present invention.

In one particular case, such a process may comprise: isolating a trp-promoter-containing —150 bp Hind III fragment from pWT 501; self-ligating; partially digesting with Taq I; treating with Si nuclease; treating with E. coli DNA polymerase I; restricting with Hind III; isolating a trp-promotercontaining -ΊΟΟ bp fragment; ligating to a ~700 bp fragment obtained by treating a recombinant plasmid of pWT 231 containing the gene or a sub-unit thereof or an equivalent thereof with Sac I, SI nuclease, E. coli DNA polymerase I and Hind III; restricting with Hind III; and ligating to Hind Ill-restricted, bacterial alkaline phosphatase-treated pAT 153.

The present invention further provides a cell which comprises inserted therein such a gene or a sub-unit thereof or an equivalent thereof or such a plasmid recombinant.

In a preferred case, the cell is an E. coli K-12 HB 101 cell.

The production of such a cell by a process which comprises inserting the DNA or the plasmid recombinant into a cell also constitutes an embodiment of the present . invention.

In a further embodiment, the present invention provides a process for the production of a protein having properties resembling those of human interferon which comprises culturing such a cell and recovering expressed protein.

The following illustrates the present invention: (1) Synthesis of the oligodeoxynucleotides The six oligonucleotides, CpApGpGpTpTpGpTpApGpCp TpCpApT (IFIA), CpApGpGpTpTpGpTpApApCpTpCpApT (IFIB), CpTpCpTpTpTpCpCpApTpG (IFII), CpApGpCpTpCpTpTpTpCpCpApTpG (IFIII),GpApGpGpApGpApTpTpApApG (IFIV) and CpCpTpGpGpApAp GpApApA (IFV) were synthesised by conventional triester methodology, (see, for example Hsiung, E.M., et al, (1979), Nucl. Acids Res., 6, 1371-1385), and in accordance with the reaction scheme illustrated in Figure 14 of the accompanying drawings wherein N represents G^so^v, T, C^z or A^z. The fully protected oligonucleotides were deblocked with 2% (w/v) benzene sulphonic acid, 0.1 M tetraethyl ammonium fluoride in tetrahydrofuran (THF)/pyridine/ HjO (8/1/1 by volume), followed by treatment with ammonia. The deblocked oligonucleotides were purified by high pressure liquid chromatography (HPLC) on 'Partisil (Registered Trade Mark) 10 SAX', microparticulate silica which has been derivatised with quaternary ammonium groups (Whatman). (2) Phosphorylation of the oligonucleotides Each synthetic oligonucleotide (120 pmcles) _ oo was incubated with 203 pmoles of 5'-(γ- ‘R) ATP (Radiochemical Centre, Amersham, Buckinghamshire, England; specific activity >5000 Ci/mmol) and 3 units of polynucleotide kinase (EC 2,7.1.78), (1 unit is the amount that catalyses the production of 1 n 32 mole of acid-insoluble P after incubation for 30 minutes at 37°C according to Richardson, C.C., (1972), Progress in Nucleic Acids Research, 2, 815), in a total volume of 20 jil containing 50 mM Tris-HCl (tris (hydroxymethyl) aminomethane hydrochloride), pH 9.0, 10 mM Mg Cl^, 0.1 mM EDTA (ethylenediaminetetra-acetic acid disodium salt), 0.1 mM spermidine and 1 mM dithiothreitol (DTT). After 60 minutes at 37°C, when most of the primer molecules were labelled, the mixture was either diluted to 200 yil and adjusted to 1% (w/v) sodium dodecyl sulphate (SDS) and 10 mM EDTA and, since little oligonucleotide was present, 5 pg/ml yeast tRNA, shaken with an equal volume of water-saturated phenol and shaken again after addition of the same volume of chloroform, centrifuged (10,000 x g; 2 minutes) and the resulting aqueous phase extracted as above, which was then extracted twice with an equal volume of chloroform and then concentrated by ethanol precipitation 'involving adjusting the NaCl concentration to 200 mM, adding 2.5 volumes of absolute ethanol and standing overnight at -20°C) , (IFIA and IE); or simply heated at 100°C for 5 minutes (IFII, III, IV and V). (3) Isolation of poly A - mRNA 1 roller bottles of human fibroblasts(FS-4 (derived from human foreskin cells) or 17/1 (derived from human embryo lung cells)) were primed with homologous interferon overnight, (60 international reference units/ml), before being superinduced for 5 hours with poly(I):poly(C), (30 pg/ml), and cycloheximide, (2 pg/ml). Mock induced cells received no poly(I):poly(C). Cells were removed from the glass surface by briefly treating with a phosphatebuffered saline (PBS) solution containing 1.25 mg/ml trypsin (BC 3.4.4.4), 0.5 mg/ml EDTA, 2 pg/ml cyclo5 heximide and harvested into ice-cold R.P.M.l. 1640 (Searle modification) medium (Flow Laboratories) containing 2 pg/ml cycloheximide and 10% (v/v) calf serum. After cuickly washing the cell pellets (1,000 x g; minutes) with R.P.M.l. 1640 containing 2 pg/ml cyclo10 heximide, the cells were lysed by homogenising in 0.2 M TriS-HCL, pE 9, 50 mM NaCl, 10 mM EDTA, 0.5% (w/v) SDS, 1 mg/ml heparin and then deproteinised with an equal volume of phenol equilibrated in the above buffer, (without heparin), followed by three extractions with an equal volume of buffered-phenol/ CHClj mixture (1:1 v/v). Following ethanol precipitation and dissolution, the RNA was selectively precipitated with 3M NaCl and polyadenylated mRNA isolated by oligo (dT) cellulose chromatography, (see, for example, Aviv, K., and Leder, P., (1972) Proc. Nat. Acad. Sci. USA., 69, 1408-1412). (4) Reverse transcription of fibroblast mRNA using IFIA and IB pmoles of 5'[ P]-labelled oligonucleotide (IFIA or IB' were incubated with 22.5 pg of poly(A)containing fibroblast mRNA at 25°C for 1 hour in 36 pi of 0.4M KCl ro allow the primer to hybridise to the complementary sequence in the mRNA.

This was then diluted with transcription mix to give a final solution containing 0.5 mM of each of dATP, dCTP, dGTP and dTTP, 50 mM Tris-HCl, pH 8, 4 mM MgCl2, 60 mM KC1, 5 mM DTT, <101% (v/v) Triton X-100 (non-ionic detergent, isooctylphenoxypolyethoxyethanol), 0.22 pM IFIA or IB, 50 pg/ml actinomycin D, pg/ml polvA-mRNA, 20 units/ml of rat liver ribonuclease inhibitor, (1 unit is the amount that gives \o a 50% inhibition of 5 ng pancreatic ribonuclease according to Shcrtman, K., (1961), Biochim. Biophys. Acta., 51, 37), (see, for example, Gribnau/ A.A.M., et al, (1969), Arch. Biochem. Biophys., 130, 48-52), and 100 units/ml of AMV (aviar. myeloblastosis virus) reverse transcriptase, (1 unit is the amount that incorporates 1 n mole of dTMP into acid-precipitable product after incubation for 10 minutes at 37°C according to Houts, G.E., et al, (1979), J. Virol., 29, 517). The incubation I was continued for 60 minutes at 37°C, after which the 2-° mixture was extracted with phenol and chloroform and concentrated by ethanol precipitation (see (2) above). (5) Analysis of the reverse transcription products ^obtained with IFIA and IB The ethanol-precipitated products were - dissolved in water and incubated in the presence of O.lM NaOH and 1 mM EDTA at 37°C for 30 minutes. An equal volume of 10M urea, 25% (w/v) sucrose, 0.05% (w/v) each of xylene cyanol and bromophenol blue was then added and the mixture heated to 90°c fcr 90 seconds before applying to a 12.54 (w/v) polyacrylamide gel containing 7M urea, (see, for example, Maxam,A., and Gilbert, W., (1977), Proc. Nat. Acad. Sci. USA, 74, 560-564). After electrophoresis, the gel was autoradiographed to locate the position of labelled molecules. The labelled product of about 150 nucleotides and produced only by mRNA from induced cells was eluted from the gel slice and its sequence determined, (see, for example, Maxam and Gilbert, loc cit). The sequence is illustrated in Figure 2 of the accompanying drawings. (6) Preparation of cDNA from fibroblast polyA-mRNA cDNA was prepared by incubating the following mixture at 375^ for 2 hours: 0.5 mM dCTP, dGTP, dTTP, 0.15 mM [3H] dATP (167 mCi/mmole), 5 mM DTT, 50 mM Tris-HCl, pH 8, 10 mM MgClj, 0.01% (v/v) Triton X-100, pg/ml actinomycin D, 20 units/ml rat liver ribonuclease inhibitor, 50 mM KCl, 20 pg/ml oligo20 (dT)12-18' 60 pg/ml polyA-mRNA and 100 units/ml AMV reverse transcriptase. Following incubation, the mixture was extracted with phenol and chloroform and ethanol precipitated (see (2) above). The DNA was recovered by centrifugation, dried and dissolved in • 25 200 pi of 0.3N NaOH. After incubating for 1 hour at 50°C, it was neutralised and eluted as the excluded peak from a G50 (M) Sephadex (Registered Trade Mark), particulate cross-linked modified dextran polymer (Pharmacia), column (25 x 0.5 cm) equilibrated in 50 mM NaCl/0.1% (w/v) SDS. The peak fractions were pooled and concentrated by ethanol precipitation. (7) Transcription of cDNA with oligonucleotides IFII, III, IV and V_ The 5' (32P)-labelled oligonucleotide (3pK) was pre-incubated with total single-stranded cDNA (440 pg/ml), derived from total polyA-mRNA, for 2 hours at 25°C in the presence of 0.4 M KC1. This was then incubated at 37 °C for 3 hours in a final solution now containing 0.01% (v/v) Triton X-100, 50 mM TrisHCl, pH 8, 20 mM DTT, 10 mM magnesium acetate, 0.5 mM each of dATP, dCTP, dGTP and dTTP, 80 mM KC1, 1,000 3’ units/ml AMV reverse transcriptase, 5' ( P)-labelled oligonucleotide (0.12 pM) and singletstranded cDNA (17.6 pg/ml). Following extraction with phenol and chloroform, the aqueous phase was run through a G50 (M) Sephadex column in 50 mM NaCl, 0.1% (w/v) SDS and the excluded fraction was precipitated with ethanol, dissolved and electrophoresed. (8) Analysis of transcripts produced by IFII, III, IV and V_.

The transcripts were either treated as above, i.e. for transcripts produced by IFIA and IB, except for the use of a 5% (w/v) polyacrylamide gel in 7M urea, or 030 they were prepared and eiectrophoresed through native 1.4* (w/v) agarose gels, (see, for example, Eharp, P.A., et al, (1973) , Biochemistry, 12, 3055-3063), which were then autoradiographed. The interferon genes were eluted from selected agarose gel slices by first passing through a 19G, then a 21G syringe needle (Gillett^ and stirring the gel fragments for 3 hours at 25 °C in 10 mM Hepes, N-2-hycroxy-.ethylpiperazine-N'-2-ethanesuiphonic acid (Oltrol), pH 7, 0.1 mM EDTA and 0.02% (v/v) Triton X-100. Following centrifugation (700 x g; 5 minutes) through a glass wool plug overlaid on a GF/C and a GF/B filter paper .(Whatman), the filtrate was extracted with phenol and chloroform and then precipitated with ethanol.

The DNA was then sequenced as mentioned above. (9) Preparation of interferon genes for cloning (a) Production of single-stranded cDNA from total polyA-mRNA isolated from induced fibroblasts: cDNA was synthesised in a 20 ml incubation containing the constituents described above (6). Following extraction with phenol and chloroform, the aqueous phase was chromatographed on a Sephadex G50 (M) column (28 x 4 cm) from which the cDNA was eluted in the excluded fraction in 50 mM NaCl, 0.1% (w/v) SDS. The cDNA was precipitated with ethanol and then recovered, dissolved in 0.4 ml 0.2N NaOH and incubated at 50°C for minutes before being centrifuged through an alkaline sucrose gradient as described below. (b) Purification of interferon single strand cDNA: The above cDNA was divided into two equal portions and each centrifuged at 36,000 rpm at 2°C for 24 hours in a Beckman (Registered Trade Mark) SW 41 rotor through a 5-20% (w/w)isokinetic sucrose gradient (11 ml) containing 0.2N NaOH, 100 mM NaCl, 10 mM EDTA. 0.5 ml fractions were collected by upward displacement using 50% (w/v) sucrose and corresponding fractions from both gradients were pooled, neutralised and precipitated with absolute ethanol. The precipitates were collected by centrifugation, washed in absolute ethanol and then dissolved in 100 pi Hj0· Those fractions containing long interferon cDNA transcripts were identified by analysing the products obtained after priming small aliquots of each fraction with 32 ( P) IFIII anc reverse transcriptase as described above (7) and (8). The main fractions which gave rise to the synthesis of an interferon DNA molecule of about 700 nucleotides in length were pooled in preparation for a large scale synthesis of double strand interferon genes. (c) Inhibition of the self-priming activity of the purified interferon single strand cDNA: Before carrying out a large scale preparation of double strand interferon genes, the self-priming 1030 activity of the purified interferon single strand cDNA (when subsequently incubated with reverse transcriptase) was reduced by incubating the cDNA at 37°C for 3 hours in 1.5 ml containing 15C mM sodium c&cocylate, pH 7.2, 1 mM 2-mercaptoethanol, mM CoClji 100 pg/ml gelatin, 20 pg/ml cDNA, 1.2 mM rATP and 600 units/ml terminal transferase (EC 2.7.7.31), (1 unit is the amount that incorporates 1 n mole dATP intc acid-precipitable product in I hour at 37°C using d(pA)5Q as primer according to Bollum, R.J., et all (1974), Methods in Enzymology, 29, 70).

Following extraction with phenol and chloroform as described above, the aqueous phase was brought to 0.3N NaOH and incubated for 1 hour at 50’C. The mixture was then neutralised and chromatographed on a Sephadex G50 (medium) column (20 x 2 cm) and the cDNA eluted in the excluded fraction in 50 mM NaCl, 0.1% (w/v) SDS. It was subsequently precipitated with ethanol and re'rtissoved in HjO.

The result of this process is the addition of a single AMP residue to the original 3'-hydroxyl terminus of the cDNA. The alkali treatment also produces a mixture of 2' and 3' phosphate residues at this terminus which may no longer take part in a self-priming reaction with reverse transcriptase. Therefore, when double strand genes are subsequently made using an oligonucleotide to prime specific transcription on the single strand cDNA, the level of contaminating self-primed products is reduced and the remaining single strand cDNA may be subsequently degraded by SI nuclease (EC 3.1.4.21).

The self-priming activity may also be severely inhibited by pre-incubating the cDNA in appropriate buffer mixtures with either reverse transcriptase or terminal transferase in the presence of the four cideoxynucleoside triphosphates.

Alternatively, the cDNA may be incubated with terminal transferase and deoxyadenosine triphosphate, thus producing a poly(dA) tail at the 3'-hydroxyl terminus which also effectively inhibits the self-priming activity. (d) Syntheses of double strand interferon genes using IFIII as primer: Interferon single strand cDNA was purified by centrifugation through an alkaline sucrose gradient and its self-priming activity reduced as described above. 20 yg of this material was then pre-incubated at 25°C for 1 hours in 0.36 ml containing 0.4M XCI and 575 pmoles of IFIII that had been previously incubated with polynucleotide kinase (EC 2.7.1.78) and (γ- P) ATP also as described above. Double strand genes were then produced by incubating further at 37°C for 3 hours in 1.8 ml now containing 0.01% (v/v) Triton X-100, 50 mM Tris-HCl, pH 8 , 20 mM DTT, mM magnesium acetate, 0.5 mM dATP, dCTP, dGTP and dTTP, 1,000 units/ml reverse transcriptase, 80 mM KCl, 11.1 pg/ml cDNA and 0.32 jiM (32P) IFIII.

Following extraction with phenol and chloroform, the genes were eluted from a Sephadex G50 (medium) column (20 x 2 cm) in 50 mM NaCl, 0.1% (w/v) SDS.

Yeast tRNA was added as carrier (3 pg/ml) and the concentration of NaCl adjusted to 0.2 M before precipitating with 2.5 volumes Of absolute ethanol. After standing at -70°C for 30 minutes, the precipitate was collected by centrifugation and the dried pellet redissolved in 100 pi Ι^θ. (e) Purification of double strand interferon genes: The DNA was sedimented through a 5-20% (w/w) linear sucrose gradient containing 10 mM Tris-HCl, pH 7.5, 0.8M NaCl, 8 mM EDTA. A 5.2 ml gradient was centrifuged in a Beckman SW 65 rotor at 42,000 rpm for 18 hours at 2°C, after which 0.2 ml fractions were collected and-precipitated with ethanol. The DNA was collected by centrifugation, redissolved and then small aliquots were electrophoresed through a 5% (w/v) polyacrylamide gel containing 7M urea which was then autoradiographed. Those fractions displaying the 700 nucleotide interferon DNA molecule were pooled . (f) Preparation of interferon genes containing Hind III linkers at the termini thereof: 5103 The above pooled gradient fractions were first treated with Si nuclease by incubating at 37°C for 30 minutes in 50 pi containing 0.1 mM ZnSO^, 150 mM NaCl, 25 mM sodium acetate, pH 4.6, and 120 units/ml Si nuclease, il unit is the amount that solubilises 10 pg nucleic acid in 10 minutes at 45°C according to Vogt. V.M. , (1973), Eur. J. Biochem., 33, 192).

After addition of 0.5 pi 10% (w/v) SDS, 1 pi 0.5 M EDTA and 1 pi 0.5 M Tris-HCl, pH 8.3, the mixture was extracted with phenol and chloroform and the aqueous phase was precipitated with ethanol. At this stage there was a total of 0.24 pg of double strand DNA.

In order to ensure the presence of blunt ends for the subsequent ligation with Hind III linker molecules , the DNA was recovered from ethanol and incubated at 14 °C for 20 minutes in 50 pi containing 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 1 mM 2mercaptoethanol, 0.2 mM dATP, dCTP, dGTP and dTTP and 40 units/ml of E coli DNA polymerase I (EC 2.7.7.7), (1 unit is the amount that incorporates 10 n moles of total nucleotides into an acid-precipitable fraction in 30 minutes at 37°C using poly-d(A-T) as primer according to Richardson, C.C., et al, (1964), J. Biol. Chem., 239, 222). The DNA was then extracted in the usual manner, precipitated with ethanol and the pellet obtained by centrifugation was then dried . 2.5 pg of Hind III linker (Collaborative Research) were phosphorylated by first incubating - · 4a at 37°C for 30 minutes in 50. pi containing 50 mM Tris-HCl, pH 7.8, 10 mM MgClj, 10 mM 2-mercaptoethanol, 0.25 mM (γ-32Ρ) ATP (20 mCi/pmole), 50 pg/ml BSA (bovine serum albumin. ·) and 100 units/ml of polynucleotide kinase. To ensure complete phosphorylation, the mixture was incubated for a further 30 minutes at 37°C after the addition of 10 pi containing 50 mM Tris-HCl, pH 7.8, 10 mM MgClj, 3.7imM rATP and 500 units/ml of polynucleotide kinase. The mixture was then extracted with phenol and chloroform and precipitated with ethanol after the addition of yeast tRNA to 10 pg/ml.

Ligation of the phosphorylated Hind III linkers to the interferon genes was achieved by dissolving the interferon gene pellet in 40 pi containing 5C mM Tris-HCl, pH 7.8, 10 mM MgCl^, mM rATP, 20 mM DTT, 20 pg/ml of phosphorylated linker and v480 units/ml of T4 DNA ligase (EC 6.5.1.1), (1 unit is the amount that catalyses the conversion of l n mole of 32PP^ into (32P)-ATP in 20 minutes at 37°C according to Weiss, B., et al, (1968), J. Biol. Chem., 243, 4543). This was then incubated at 25°C for hours.

These conditions provided a molar excess of linker over DNA of about 100-fold.

After heating the mixture at 65°C for 5 minutes, the DNA was restricted with Kind III by adding 60 pi Of a mixture containing 400 units/ml Hind III (EC 3.1.23.21), (1 unit is the amount that digests 1 pg ?\-DNA in 15 minutes at 37°C in 50 pi) , mM DTT, 167 pg/ml gelatin, S3 mM Tris-HCl, pH 7.5, S3 mM NaCl, 17 mM MgCl2 and 8 mM 2-mercaptoethanol. Following incubation at 37°C for 2 hours, the mixture was brought to 02.% (w/v) SDS and 20 mM EDTA and the DNA was extracred with phenol and chloroform and yeast tRNA was added to the final aqueous phase to give a concentration of 5 pg/ml. This was then chromatographed on a Sephadex G150 superfine column (50 x 0.7 cm) where the double strand genes were eluted in the excluded fraction using 50 mM NaCl, 0.1% (w/v) SDS and 5 pg/ml yeast tRNA. The latter were then precipitated with ethanol after bringing the tRNA concentrations to 10 pg/ml. The recovered DNA was estimated to contain about 0.125 pg of double strand genes. (g) Final fractionation of interferon genes: The DNA was then dissolved in 50 pi KjO and 20 pi were electrophoresed through a native 1.4% (w/v) agarose gel (20 x 14 x 0 5 cm) for 2 hours at 120 volts. DNA of between 600 and 1000 bp in length was then eluted from the gel as follows: The appropriate slice 'was excised and passed through a Gillette (Registered Trade Mark) 21 G needle. The latter was washed with 4 ml 10 mM Hepes, pH 7.5, 0.1 mM EDTA, 0.02% (v/v) Triton X-100, pg/ml yeast tRNA and the slice frozen at -70eC. It was then thawed and agitated on a rotary mixer overnight at room temperature (about 25°C) before being filtered through a Whatman (Registered Trade Mark) 52 paper. The filtrate was adjusted to 0.1 M ammonium acetate, mM magnesium acetate, 0.02% (w/v) SDS and bound to a 1 ml column of diethylaminoethyl (DEAE) 52 cellulose. This was then washed 3 times with 1.5 ml aliquots cf the above buffer before eluting the DNA with 0.5 ml aliquots of 1.1 M NaCl, 0.1M ammonium acetate, 2 mM magnesium acetate, 0.02% (w/v) SDS, 0.02 mM EDTA. The DNA eluted with the second and third aliquots, which were then pooled, supplemented with 5 pg yeast tRNA and then precipitated with ethanol. The precipitate was recovered by centrifugation, washed with absolute ethanol, dried and dissolved in 20 pi HjO. 4 pi were taken for liquid scintillation counting and the remainder (about 8 ng) was dried by vacuum dessication. (10) Cloning the interferon genes into X1776 using pBR 322 as the plasmid vector The vector was prepared as follows: 10 pg Of the plasmid pBR 322 was restricted with Kind III, extracted with phenol and chloroform and precipitated with ethanol. The recovered linear plasmid was then incubated at 65°C for 30 minutes in 25 pi containing 20 mM Tris-HCl, pH 7.5, 0.1% (w/v) SDS and 0.7 mg/ml bacterial alkaline phosphatase (EC 3.1.3.1), before being extracted three times with phenol and chloroform, twice with diethyl ether and again precipitated with ethanol. The recovered precipitate was then dissolved in 100 pi K2O.

The dried gene pellet (see (9) (g) above) was then dissolved in 10 pi containing 50 mM TrisHCl, pH 7.8, 10 mM MgCl2, 1 mM rATP, 20 mM DTT, pg/ml of the treated vector DNA and ~16 units/ml of T4 DNA ligase. This mixture was then incubated at 15°C overnight before being diluted to 100 pi with 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 M NaCl and used to transform E. coli K-12X1776 according to known methods, (see, for example, Curtiss, R., III, et al, (1976) , Recombinant Molecules: Impact on Science and Society (R.F. Beers, Jr., and E.G.

Bassett (Eds.), 45-56). In this way a total of 370 ampicillin (luO pg/ml)-resistant transformants was obtained which were then tested for sensitivity towards 10 pg/ml tetracycline. 62% were found to be cetracycline-sensitive and were thus identified as recombinants. 160 of the latter were then grown on millipore filters overlaid on culture plates in order to identify interferon gene recombinants by colony hybridisation with (γ-32Ρ) IFIA and (γ-32Ρ) IFIV. '(11) Screening of recombinants Recombinants were grown on nitrocellulose filters (9 cm diameter) until the colonies reached a diameter of 2 or 3 mm. After lifting the filters from the plates, they were placed for 7 minutes on a pad of Whatman No. I filter papers soaked in 0.5N NaOH, then for 2 minutes on a pad soaked in 1M Tris-HCl, pH 7.5, and then for 7 minutes on a pad soaked in 0.5M Tris-HCl, pH 7.5, 1.5M NaCl. The filters were then dried by placing on a vacuum manifold, for about 5 minutes and finally washed with 100 ml of absolute ethanol before being heated for 2 hours at from 80 to 85°C.

The filters were pre-incubated at 25°C for 2 hours in 3 x SSC (1 x SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.6) containing 0.02% (w/v) BSA, 0.02% (w/v) Ficoll (Registered Trade Mark), (copolymer of sucrose and epichlorohydrin) and 0.02% (w/v) polyvinvlpyrolidone before being drained of liquid and placed on a filter paper to air-dry for a few minutes. The oligonucleotides 32 IFIA and IFIV were radioactively labelled using (γ- P) ATP and polynucleotide kinase as described above and then suspended in 3 x SSC. 300 pi (containing 0.85 pmole of each (γ- P) oligonucleotide (about 1 pCi/ pmole)) were applied uniformly over each filter (new standing on a non-absorbent surface)· and left for about 5 minutes to soak in. The filters were submerged in light paraffin and incubated for 2 or 3 days at 25°C. Subsequently, they were drained of oil, rinsed in chloroform, air-dried and washed four times in 3 x SSC at 25°C (20 minutes each wash), before washing twice in 2 x SSC at the same temperature. After gently blotting dry, the filters were sealed in a polythene bag and autoradiographed at -7C°C in a Kodak (Registered Trade Mark) regular intensifier cassette.

The autoradiograph indicated the presence of five colonies that were significantly darker than the rest. Plasmid DNA was prepared from these according to known methods, (see, for example, Birnboim, H.C., and Doly, J, (1979), Nucl. Acids Res., 1_, 1513-1523) and subsequent restriction and nucleotide sequence analysis confirmed that each of these recombinants contained a fibroblast interferon gene coding for the mature interferon polypeptide and the 3'-untranslated mRNA sequence. (12) Transfer of the interferon gene into pWT 2x1 expression plasmids Plasmid DNA was prepared from one of the above interferon recombinants by centrifuging a cleared lysate through density gradients containing ethidium bromide, ;see, for example, Katz, L., et al, (1973), J. Bacteriol, 114, 577-591? and Wensink, P.C., et al, (1974), Cell, 315-325). 3 pg were then restricted with Hind III and the extracted DNA was electrophoresed through a native 5 % (w/v) polyacrylamide gel. The interferon gene (about 730 bp) was visualised by ’ 54 staining with 1 pg/ml ethidium bromide and viewing under UV light (254 nm) , thus enabling the excision of a small gel slice containing the gene. This slice was S1030 dispersed by passing through the orifice of a 1 ml plastic syringe, the latter then being washed with 4 volumes, relative to the gel slice, of AGEB (0.5M ammonium acetate, 10 mM magnesium acetate, 0.1% (w/v) SDS and 0.1 mM EDTA), which was then mixed with gel slurry and gently shaken for 16 hours at 37°C.

Yeast tRNA was added to give a final concentration of 25 pg/ml before centrifuging the mixture through a Whatman 52 paper in a 2 ml syringe and then washing the paper with AGEB. The pooled filtrate was then diluted five times with H2O before chromatography on a 1 ml column of DEAE 52-cellulose as described above (9g). The eluate (1 ml total) was extracted twice with 1 ml of iso-amyl alcohol saturated with 1/5 x AGEB containing 1.1 M NaCl (to remove the ethidium bromide) before being precipitated with ethanol after the addition of 5 pg of yeast tRNA.

The DNA was recovered and dissolved in 50 pi HjO (about 0.2 pg DNA). pi was then ligated (in a final volume of 10 pi) with 80 ng Of either pWT 211, pWT 221 or pWT 231, which had been previously restricted with Hind III and then treated with alkaline phosphatase to minimise ligation of the parent plasmid (see (10) . above). Each ligation was then used to transform E. Coli K 12 H3 101 using known methods, (see, for example, Emtage, J.S. , et;aj^, (1980).,. Nature, 283, 171-174).

Transformants were grown on L-agar plates containing 100 pg/ml ampicillin (in fact carbenicillin sodium (obtainable from Beecham) was used, which is a derivative which may be regarded as equivalent; carbenicillin is /-carboxy benzylpenicillin) and plasmid DNA was prepared from 6 to 12 colonies in each case. By carrying out Pst I (EC 3.1.23.31) restrictions and analysing the products by gel electrophoresis, interferon recombinants could be identified as could the orientation of the interferon gene relative to the tryptophan promoter sequence. For each of the three pWT 2x1 plasmids, a recombinant having the interferon gene in the required orientation for expression was selected and investigated with respect to its ability to produce biologically active interferon. (13) Induction of expression plasmids containing the interferon gene 150 ml cultures of each of the three pWT 2x1/ interferon gene clones were grown in L-broth (luria broth: 1% (w/v) bacto tryptone, 0.5% (w/v) bactc yeast extract, 0.5% (w/v NaCl), 0.2% (w/v) glucose, 0.004% (w/v) thymine, pH'7) containing ampicillin to an O.D.,ftn of about 0.3. The bacteria were Delleted by 600 nm centrifugation, washed and then resuspended in inducing medium (42 mM Na2HPO4, 22 mM K^PO^ , 8.6 mM. NaCl, 18.7 mM NH^Cl, 0.1 mM CaCl2, 1.0 mM MgSO^, 1 pg/ml vita min BI, 0.2 % (w/v) glucose, 0.5% (w/v) casamino acids lacking triptophan (obtainable from Difco), 20 pg/ml (5-indole acrylic acid and 100 pg/ml ampicillin).

Following incubation at 37°C for 4 hours (at which time the O.D.,nn __ was about 1.0) the 600 nm cells were centrifuged and extracted as described below. (14) Extraction of interferon from bacteria The following operations were carried out at 2°C: The bacterial pellets were resuspended in 1.2 ml of PBS containing 2% (w/v) HSA (human serum albumin). An equal volume of 50% (w/v) sucrose containing 0.1 M Tris-HCl, pH 8» and 1% (w/v) HSA was then added, followed by 0.8 ml of a fresh lysozyme solution (10 mg/ml in PBS). After 15 minutes, 0.8 ml of 0.5M EDTA, pH 8.5, was added and the cells left for a further 10 minutes. 4 ml of 0.6% (v/v) Triton X-100 were next added and, after mixing for 10 minutes, the extract was sonicated to reduce viscosity and ensure complete lysis, then ultra-centrifuged at 50,000 rpm in a Beckman Ti 50 rotor for 2 hours.

The supernatant was dialysed against PBS, cleared by centrifugation (10/)00 rpm; 10 minutes) before being assayed for interferon activity by monitoring the protection conferred on Vero cells against the cpe of EMC virus in an in vitro micro-plate assay system, (see, for example, Dahl, H., and Degre, M, (1972), Acta. Path. Microbiol. Scan., 1380, 863).

An alternative method of extraction involves simply sonicating the induced bacteria in 10 mM TrisHCl, pH 8.0, followed by centrifugation for 30 minutes at 15,000 x g and collecting the supernatant. (15) Construction of modified expression recombinants The plasmid pWT 501 contains a 150 bp Hind III fragment that contains tbe trp promoter and part of the gene sequence coding for the trp leader polypeptide. This fragment was .recovered from a native 5% (w/v) polyacrylamide gel and self-ligated using T4 DNA ligase (10). The concatemers were then partially digested with Taq I before treating first with SI nuclease, then E. coli DNA polymerase X and finally with Hind III (9 f). In this way, the required Taq I cleavage within the sequence corresponding to the ribosome binding site in the mRNA region coding for the trp leader polypeptide, results in a fragment of about 100 bp following Hind III restriction. This fragment was isolated from an 8% (w/v) native polyacrylamide gel (12) in preparation for ligation to the Sac I/Hind III fragment of the interferon gene. The latter fragment was obtained by first digesting the interferon gene cloned in pWT 231 with Sac 1. Following treatment with SI nuclease, then with E. coli DNA polymerase I, the DNA was restricted with Hind III and the resulting fragment of about 700 bp was recovered from a 5% (w/v) native polyacrylamide gel.

The two fragments were then ligated to each other by incubating at 25°C for 16 hours in a final volume of 20 pi containing 50 mM Tris-HCl, pH 7.8, 10 mM MgClj, 1 mM ATP, 20 mM DTT, —480 units/ml of T4 DNA ligase, 0.5 pg/ml of the Hind III/Taq I trp fragment and 5 pg/ml of the Sac X/Hind III fragment from the interferon gene. Following restriction with Hind III, the conjoint molecule was transformed into E. coli K-12 HB101 using pAT 153 as the vector. The latter had been previously restricted with Hind III prior to treating with bacterial alkaline phosphatase (EC 3.1.3.1) in order to reduce the level of ncn10 reccmbinant transformants (10). Plasmid DNA was prepared from transformants according to known methods, (see, for example, Kauz, et al, and Wensink et al. loc cit), which was then analysed by restriction enzyme mapping and also by nucleotide sequencing. In this way, several recombinants containing the required conjoint molecule were identified. These were then induced tc produce interferon as described above (13), except that the concentration of 3 β-indole acrylic acid was adjusted to 2.5 pg/ml.

In order to prepare radioactive interferon, bacteria were induced as described, except in the absence of casamino acids and in the presence of pg/ml 3 β-indole acrylic acid. At the end of the induction period, 1 ml aliquots were incubated for a 14 further 15 minutes with 5 pCi of a ( C) amino acid mixture. The bacteria were then centrifuged and the pellet lysed by adding 50 pi of a mixture of 10% (v/v) glycerol, 5% (w/v) 2-mercaptoethanol, 3% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.8, 0.01Ϊ (w/v) brcmophenoi blue and heating to 9C°C for 2 minutes. Samples were then electrcphoresed through 12.;% (w/v) poly5 acrylamide gels by known methods, (see, for e?:ample, Laemmli, U.K., (1970), Nature, 227, 680-685). The gel was then dried and autoradiographed in order to visualise the interferon polypeptide, (see Figure 12 of the accompanying drawings).

Claims

Claims:

1. A gene for the expression of a protein having properties resembling those of human interferon which comprises a coding strand and a complementary strand, the coding strand comprising the sequence: 5' GGC. CAT.ACC.CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA,CAG.GTA.GTA.GGC.GAC.ACT. GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG,TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA. AGA.AGC.AGC.AAT.TTT.CAG. TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT. GAG. GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC. GCA.TTG.ACC.ATC.TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT. TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.AAT,GTC.TAT. CAT.CAG.ATA.AAC.CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT. TAT.GGG.AGG.ATT.CTG.CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.GCC.TGG. ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC. ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT. AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC.ATT.CTT.CAA. CCA.GCA.GAT.GCT.GTT.TAA.GTG.ACT. GAT.GGC.TAA.TGT.ACT.GCA. TAT,GAA.AGG. ACA. CTA.GAA.GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.GTT.ATT.TTT.ATT. TAT.TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.TTA.TTT TTG.GTG.CAA.AAG.TCA.ACA.TGG.CA 3' or a subunit thereof or an equivalent thereof.

2. A gene as claimed in claim 1, the coding strand comprising the sequences 5 1 GGC. CAT. ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT. CAG.TGT. CAG. AAG. CTC. CTC-. TGG. CAA. TTG. AAT. GGG. AGG. CTT. GAA. TAC. TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC,CCT.GAG.GAG,ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT. TCA.TCT.AGC. ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT. GAG.AAC.CTC.CTG.GCT.AAT.GTC. TAT. CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA. GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC'.ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT.TAT.GGG.AGG. ATT. CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC.TGG. ACC. ATA. GTC. AGA. GTG. GAA. ATC. CTA. AGG. AAC. TTT. TAC. TTC. ATT.AAC. AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT. AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC.ATT.CTT.CAA.CCA.GCA GAT.GCT.GTT.TAA.GTG.ACT.GAT.GGC.TAA.TGT.ACT.GCA. TAT.GAA.AGG.ACA.CTA.GAA.GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.GTT. ATT.TTT.ATT. TAT.TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.TTA.TTT.TTG.GTG.CAA.AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.CGA.TTT.GAT,TTA.TAT.AAC.CA..3' or a sub-unit thereof or an equivalent thereof.

3. A gene as claimed in claim 1 or claim 2, the coding strand comprising the sequence: 5' GGC. CAT, ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.51030 CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC. GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT. CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.5 AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG. AAT.GGG.AGG.CTT.GAA. TAT.TGC.CTC.AAG.GAC.AGG.ATG.AAC. TTT.GAC.ATC.CCT. GAG.GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT. ATT.TTC. AGA.CAA.GAT.TCA.TCT.AGC.10 ACT.GGC.TGG. AAT. GAG. ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.. AAT.GTC. TAT. CAT.CAG.ATA.AAC.CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.ι ATT.CTG.CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.15 GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT,CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC, ATT.CTT.CAA.CCA.GCA.GAT,GCT.GTT.TAA.GTG.ACT.GAT.GGC.TAA.TGT.ACT. GCA.TAT.GAA.AGG.ACA.CTA.GAA. GAT.2 0 TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.GTT.ATT.TTT.ATT. TAT .~ TTA .AAT.TTT.ATT.TTG .GAA .AAT .AAA .TTA .TTT .TTG-.GTG .CAA .“ AAG .TCA..ACA .TGG .CAG .TTT .TAA..TTT .CGA .TTT GAT TTA TAT.AAC.CA..3' or a sub-unit thereof.

4. A gene as claimed in any of claims 1 to 3, 2b the coding strand comprising the sequence:

5. ' TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG.GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.51030 A CC. AAC. AAG 1 TGT. CTC .CTC. CAA. ATT. GCT. CTC. CTG .TTG.TG'C. * TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.TGT,CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG.GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.AIT.TTC.AGA.CAA. GAT.TCA.TCT.AGC. ACT.GGC.TGG.AAT.GAG. ACT. ATT.GTT.- GAG.AAC, . CTC. CTG. GCT. AAT. GTC. TAT. CAT. CAG. ATA. AAC. CAT. 7 CTG.AAG. .ACA.GTC.CTG.GAA.GAA.AAA.CTG. GAG,AAA.GAA.GAT.- TTC.ACC, .AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.- AGA.TAT. . TAT.GGG.AGG. ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.- GAG,TAC. .AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA. - ATC.CTA. ,AGG.AAC.TTT.TAC.TTC.ATT.AAC.AGA.CTT.ACA. GGT.- TAC.CTC. ,CGA.AAC.TGA.AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.- GAC.TGG, ,ACA.ATT.GCT.TCA.AGC.ATT.CTT.CAA.CCA.GCA.GAT.- GCT.GTT. .TAA.GTG. ACT. GAT.GGC.TAA.TGT. ACT. GCA. TAT.GAA.- AGG.ACA, ,CTA.GAA. GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.- GTT.ATT. .TTT. ATT. TAT. TTA. AAT. TTT. ATT. TTG. GAA. AAT. AAA. - TTA.TTT. ,TTG.GTG,CAA.AAG.TC..3' or an equivalent thereof 5. A ge?e as claimed in any of claims 1 to 3, the coding strand comprising the sequence: 5 1 ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA. ATT.GCT.CTC.CTG.TTG TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT. TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC. AAG. GAC. AGG. ATG. AAC. TTT. GAC. ATC. CCT. GAG. GAG. ATT. 51030 AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC.GCA. TTG.ACC.ATC. TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA. GAT.TCA.TCT.AGC.ACT.GGC.TGG.AAT. GAG.ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.AAT.GTC.TAT.CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG. GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT. TAT.GGG.AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.« GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.ATT.AAC.AGA.CTT.ACA.” GGT.TAC.CTC.CGA.AAC.TGA.. 3’ or an equivalent thereof.

6. A gene as claimed in any of claims 1 to 3, the coding strand comprising the sequence: 5' ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.” ( AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG. AGG. CTT. GAA, TAC. TGC. CTC. AAG. GAC. AGG. ATG. AAC. TTT. ” GAC.ATC.CCT. GAG. GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC.GCA.TTG.ACC.ATC.TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC,AGA.CAA.GAT.TCA.TCT.AGC.ACT. GGC.TGG.AAT. GAG. ACT.ATT.GTT. GAG.AAC.CTC.CTG.GCT.AAT.GTC.TAT.CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA.CTC.ATG.AGC. AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.ATT.CTG. CAT. TAC. CTG. AAG. GCC. AAG. GAG. TAC. AGT. CAC. TGT. GCC. TGG. ACC. ATA. GTC. AGA. GTG. GAA. ATC. CTA ..AGG. AAC. TTT. TAC. TTC.ATT.AAC. AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA. 3' or an equivalent thereof.

7. A sub-unit of a gene as claimed in any of claims 1 to 6.

8. A gene or an equivalent thereof cr a subunit thereof as claimed in any of claims 1 to 7 substantially as herein described.

9. A process for the production of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 4 to 6 which comprises isolating polyA-mRNA from induced fibroblast cells, synthesising single strand cDNA using oligo dT primer and reverse transcriptase and synthesising double strand DNA therefrom using reverse transcriptase or Esherichia coli DNA polymerase I and optionally a primer,

10. A process for the production of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 4 to 6 which comprises isolating mSNA from induced fibroblast cells, synthesising single strand cDNA using a specific primer and reverse transcriptase and synthesising double strand DNA therefrom using reverse transcriptase or E. coli DNA polymerase 1 and optionally a primer. Il» A process as claimed in claim 9 or claim 10 in which a primer is used in the third step.

11. 12. A process as claimed in any of claims 9 to 11 in which the reaction mixture is fractionated after the second step and/or the third step.

12.

13. A process as claimed in any of claims 9 to 12 in which self-priming is inhibited after the second step.

14. A process as claimed in any of claims 9 to 13 in which the product is identified, after cloning, by using a primer to screen colonies. 5

15. A process as claimed in any of claims 9 to 14 for the production of a gene or an equivalent thereof or a sub-unit thereof as' claimed in any of claims 4 to 6 substantially as herein described. 510 30 69

16. A gene or an equivalent thereof or a subunit thereof as claimed in any of claims 4 to 6 when produced by a process as claimed in any of claims 9 to 14. 15.

17. A process for the production of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 1 to 3 which comprises using as a probe a molecule having a sequence of a gene or an equivalent thereof or a sub-unit therecf as claimed in any cf claims 1 to 8 or 16 to isolate from human chromosomal DNA a human chromosomal interferon gene.

18. A process as claimed in claim 17 for the production of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 1 to 3 substantially as herein described.

19. A gene or an equivalent thereof or a subunit thereof as claimed in any of claims 1 to 3 when produced by a process as claimed in claim 17 or claim 18.

20. A process for the production of a gene or an equivalent thereof or a sub-unit thereof which has a portion common with or related to a portion of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 1 to 3 which comprises using as a probe a molecule having a sequence corresponding to at least part of the common or related portion to isolate from human chromosomal DNA a human chromosomal gene.

21. A process for the production of a gene or an equivalent thereof or a sub-unit thereof which has a portion common with or related to a portion of a gene or an equivalent thereof or a sub-unit thereof as claimed 5 in any of claims 1 to 3 substantially as herein described.

22. A gene or an equivalent thereof or a sub-unit thereof which has a portion common with or related to a portion of a gene or an equivalent thereof or a sub-unit thereof as claimed in any of claims 1 to 3 when produced by a process 10 as claimed in claim 20 or claim 21.

23. A plasmid recombinant which comprises a plasmid vector having inserted therein at an insertion site a gene or a sub-unit thereof or an equivalent thereof as claimed in any of claims 1 to 8, 16 or 19, the plasmid 15 recombinant enabling translation in the correct phase for the mRNA corresponding to the inserted gene or subunit thereof or equivalent thereof and having a bacterial promoter upstream of and adjacent to the insertion site such that the inserted gene or sub 20 unit thereof or equivalent thereof is under bacterial promoter control.

24. A plasmid recombinant as claimed in claim 23 comprising inserted therein a plurality of genes or sub-units thereof or equivalents thereof a,s claimed in any of claims 1 to 8, 16 or 19.

25. A plasmid recombinant as claimed in claim 23 or claim 24 comprising a plurality of bacterial promoters.

26. A plasmid recombinant as claimed in any of claims 23 to 25 comprising at least one trp-promoter.

27. A plasmid recombinant as claimed in any of claims 23 to 26 which lacks at least part of the DNA sequence responsible for attenuation of transcription.

28. A plasmid recombinant as claimed in any of claims 23 to 27 comprising a modified derivative of pWT 501.

29. ' A plasmid recombinant as claimed in any of claims 23 to 28 comprising a plasmid vector having inserted therein a gene or a sub-unit thereof or an equivalent thereof as claimed in any of claims 1 to 8, 16 or 19 which also codes for an initiation codon and/or at least part of a ribosome binding site.

30. A plasmid recombinant as claimed in any of claims 23 to 29 substantially as herein described,

31. A process for the production of a plasmid recombinant as claimed in any of claims 23 to 30 which comprises inserting a gene or a sub-unit thereof or an equivalent thereof as claimed in any of claims 1 to 8, 15. 16 or 19 into an insertion site of an appropriate plasmid vector.

32. A process as claimed in claim 31 comprising: isolating a trp-rpromoter-containing -^150 bp Kind III fragment from pWT 501; self-ligating; partially digesting with Taq I; treating with SI nuclease; treating 5 with E. coli DNA polymerase I; restricting with Hind III; isolating a trp-rpromoter-containing ~100 bp fragment; ligating to a ~700 bp fragment obtained by treating a recombinant plasmid of pWT 231 containing a gene or a sub-unit thereof or an equivalent thereof as claimed in 10 any of claims 1 to 8, 16 or 19 with Sac I, SI nuclease, E, coli DNA polymerase I and Hind III; restricting with Hind III; and ligating to Hind Ill-restricted, bacterial alkaline phosphatase-treated pAT 153.

33. A process as claimed in claim 31 or 32 15 substantially as herein described.

34. A plasmid'recombinant as claimed in any of claims 23 to 30 when produced by a process as claimed in any of Claims 31 to 33.

35. A cell which comprises inserted therein a 16. 20 gene or a sub-unit thereof or an equivalent thereof as claimed in any of claims 1 to 8, 16 or 19 or a plasmid recombinant as claimed in any of claims 23 to 30 or 34.

36. A cell as claimed in claim 35 which is an E. 17. 25 coli K-12 HB1O1 cell.

37. A cell as claimed in claim 35 or claim 36 substantially as herein described.

38. A process for the production of a cell as claimed in any ox claims 35 to 37 which comprises inserting a gene or a sub-unit thereof or an equivalent thereof as claimed in any of claims 1 to 8, 15 or 19 or a plasmid recombinant as claimed in any 5 of claims 23 to 30 or 34 into a cell.

39. A process as claimed in claim 38 substantially as herein described.

40. A cell as claimed in any of claims 35 to 37 when produced by a process as claimed in claim 10 38 or claim 39.

41. A process fcr the production of a protein having properties resembling those of human interferon which comprises culturing a cell as claimed in any of claims 35 to 37 or 40 and recovering expressed 15 protein.

42. A process as claimed in claim 41 substantially as herein described.

43. A protein having properties resembling those of human interferon when produced by a process as 20 claimed in claim 41 or claim 42.