WO1984001153A1

WO1984001153A1 - Production of interferon in yeast by use of invertase promoter

Info

Publication number: WO1984001153A1
Application number: PCT/US1983/001370
Authority: WO
Inventors: David Botstein; Donald W Bowden; Ronald W Davis; Gerald R Fink; Robert G Knowlton; Jen-I Mao; Alison Taunton-Rigby; Gerald F Vovis
Original assignee: Collaborative Res Inc
Priority date: 1982-09-15
Filing date: 1983-09-09
Publication date: 1984-03-29
Also published as: EP0118551A1

Abstract

A recombinant DNA segment which contains a SUC2 promoter linked to an interferon gene for directing the expression of the gene within a yeast cell. The expression of the gene for human leukocyte interferon is obtained in yeast and controlled by the SUC2 promoter of the yeast strain Saccharomyces cerevisiae. The SUC2 promoter is a DNA segment that contains the transcription start signal for the structural gene for invertase. The modified yeast strain of the invention displays a method of passive regulation of the expression of the interferon gene in yeast. Synthesis of the interferon is repressed when the yeast are grown in glucose medium, and derepressed when glucose is depleted.

Description

PRODUCTION OF INTERFERON IN YEAST

BY USE OF INVΕRTASE PROMOTER

BACKGROUND OF THE INVENTION

Recombinant DNA techniques have been previously described in which a foreign protein is cloned and expressed in yeast. Evidence for gene expression in yeast came from studies on the in vivo transcription of a rabbit globin gene introduced into Saccharomyces cerevisiae on a yeast plasmid vector. [See Beggs, J. D. , van den Berg, J. , van Obyen, A., and Weissmann, C. , Nature 283, 835-840 (1980) .]

To maximize expression of interferon in yeast, the 5'-promoter region, translation start and signal peptide sequence of the human leukocyte interferon gene were replaced with those from the yeast alcohol dehydrogenase (ADH1) gene. Full length, biologically active interferon molecules were produced in yeast, although the levels of interferon expression varied. [See Hitzeman, R. A., Hagie, F. E. , Levine, H. L. , Goeddel, D. V., Ammerer, G. , and Hall, B. D., Nature 295, 717-722 (1981) .]

The ability to control the activity of the promoter is highly useful to permit the attainment of high levels of interferon expression. It has now been discovered tha t the promoter for the S UC2 invertase gene is controllable by a mechanism of passive regulation in the production of interferon in a yeast cel l.

SUMMARY OF THE INVENTION

It is an object of the present invention to obtain living cells which are capable of producing interferon in culture for high-volume production.

It is a further object of the present invention to provide interferon derived from living cells which contain genetic material derived from recombinant DNA material.

It is a still further object of the present invention to obtain living cells which are capable of producing interferon wherein the synthesis of the interferon is regulated over a broad range by changes in the culture medium.

It is an additioπai object of the present invention to provide a modified strain of Saccharomyces cerevisiae which produces human leukocyte interferon under the control of the promoter of the yeast SUC2 invertase gene.

A still further object of the present invention is to obtain living cells which are capable of producing a polypeptide product by a method wherein the synthesis of the polypeptide is passively regulated by a level of material in culture medium for the cells. A still fu rther object of the present inven tion i s to provide genetic recombinant mater ial carry ing a pr omoter of the yeast SUC2 invertase gene .

It is desirable to have yeast cells capable of producing polypeptide products such as interferon obtained therefrom at a preselected stage of cell growth whereby the cells carry a passive promoter. The cells each contain the promoter in operative relationship to a gene, the relationship of which is not found in nature. The promoter is introduced into the cells or ancestors of the cells for the purpose of permitting a passive signal to turn on synthesis of a desired polypeptide product by expression of the gene upon activation of the promoter after a period of promoter inactivity during cell growth.

According to the present invention, the expression of the gene for human leukocyte interferon is controlled by the SUC2 promoter of the yeast strain Saccharomyces cerevisiae. The SUC2 promoter is a DNA segment that contains the transcription start signal for the structural gene for invertase.

The modified yeast strain of the present invention displays an unprecedented mode of regulation of the expression of the interferon gene in yeast. Synthesis of the interferon is repressed when the yeast are grown in glucose medium, and derepressed when glucose is depleted. No yeast strain previously described shows regulation of interferon synthesis over a broad range by changes in the culture medium.

A DNA segment is provided which contains a SUC2 promoter linked to an interferon gene for directing the expression of the gene within a yeast cell. The segment is preferably a

0.9 kilobase DNA sequence from the yeast genome that contains the promoter for the major regulated messenger RNA for invertase, whose protein is secreted and glycosylated.

In a method of obtaining expression of interferon in yeast, a SUC2 promoter of invertase in yeast is inserted before the interferon gene in a chromosome or plasmid in such a fashion that the plasmid or chromosome is replicated and carried by the cell as part of its genetic information.

Synthesis of interferon (IFN) from the SUC2 promoter is advantageous for several reasons:

-the SUC2 promoter is strong, leading to abundant synthesis of IFN.

-the regulation of the SUC2 promoter by glucose permits propagation of the yeast without the potentially deleterious effects of IFN production, since overly high levels of IFN may be toxic to cells. The glucose regulation of the SUC2 promoter affords maximal synthesis of IFN at low glucose concentration. -construction of a yeast strain with these properties is particularly desirable for commercial production of IFN because of existing large-scale yeast fermentation technology and also because of the low toxicity of S. cerevisiae.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Microorganisms prepared by the genetic engineering processes described herein are exemplified by cultures now on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. These cultures are identified by Accession Number 20644, Strain Designation CGY144 and were deposited by Collaborative Research, Inc.

As more fully described below, a particular DNA segment is linked to an interferon gene and incorporated in a modified strain of Saccharomyces cerevisiae so that it produces human leukocyte interferon (LelFN) under the control of the SUC2 promoter of the yeast invertase gene. The S. cerevisiae is genetically transformed with a novel recombinant DNA plasmid. The plasmid was constructed by ligation of DNA segments from the E. coli plasmid pBR322, yeast genomic and plasmid DNA's and synthetic DNA linkers. The construction of plasmid pBR322, sequenced by J. G. Sutcliffe, Cold Spring Harbor Symposium 43, 77-90 (1979) , is shown diagrammatically in Table 1.

Generally, in preparing the plasmid for joining with the exogenous gene, a wide variety of techniques can be used, including the formation of or introduction of cohesive termini. DNA molecules with blunt ends can also be joined together. Cohesive termini may be produced in a variety of ways; the plasmid and gene may be cleaved in such a manner that the two chains are cleaved at different sites to leave extensions at each end which serve as cohesive termini.

Cohesive termini may also be introduced by removing nucleic acids from the opposite ends of the two chains or alternatively, introducing nucleic acids at opposite ends of the two chains. Methods which may be employed in joining cleaved DNA segments depend on the nature of the termini, as described below.

"Blunt-ended" refers to DNA molecules with duplex base-paired termini. (See Sgaramella, V., van de Sande, J. H. , and Khorana, H. G. , Proc. Nat. Acad. Sci. USA 67, 1468-1475 (1970).) The DNA blunt-end termini may be joined by T4 DNA ligase with an apparent K_m of about 50 μM DNA 5'-ends. (Sugino, A., Goodman, H. M. , Heyneker, H. L. , Shine, I., Boyer, H. W., and Cozzarelli, N. R. , J. Biol. Chem. 252, 3987-3994 (1977).)

Blunt-ended DNA's are produced as for example, by cleavage with any of a number of restriction endonucleases, such as Haelll. Alternatively, random shear breakage or a restriction enzyme making staggered cuts, such as EcoRI, Hindlll, or BamHI, may be used, but the DNA termini must then be made blunt by biochemical methods. Such biochemical methods include incubation with single-strand-specific nuclease S1, as described in the following articles:

Ulbrick, A., Chine, J. , Chirgwin, J. , Pictet, R. , Tischer, E. , Rutter, W. J. , and Goodman, H. M., Science 196, 1313

(1977); Maniatis, T. , Hardison, R. C. , Lacy, E. , Lauer, J. ,

O'Connell, C. , Guon, D., Sim, G. G. , and Efstratiadis, A. ,

Cell 15, 687 (1978); Scheller, R. H. , Thomas, T. L. , Lee, A.

S., Klein, W. H. , Niles, W. D., Britten, R. J. , and Davidson,

H. , Science 196, 197 (1977); and Charnay, P., Perricaudet,

M., Galibert, F. , and Tiollais, P. , Nucleic Acids Res. 5,

4479 (1978) . Alternatively, blunt termini can be created by incubation with T4 DNA polymerase [see Stakura, K., Hirose,

T. , Crea, R. , Riggs, A. D. , Heyneker, K. L. , Bolivar, F. , and

Boyer, H. W., Science 198, 1056 (1977) ; and Fraser, T. H. , and Bruce, B. J. , Proc. Nat. Acad. Sci. USA 75, 5936 (1978)],

E. coli DNA polymerase [see Seeburg, P. H. , Shine, J. ,

Martial, J. A., Baxter, J. D., and Goodman, H. M. , Nature

270, 486 (1977) ; Heffron, F. , Sa, M., and McCarthy, B. J. ,

Proc. Nat. Acad. Sci USA 75, 60612 (1978); and Backman, K. ,

Ptashne, M. and Gilbert, W., Proc. Nat. Acad. Sci. USA 73,

4174 (1976)], and reverse transcriptase [see Ulbrick, A.,

Chine, J. , Chirgwin, J. , Pictet, R., Tischer, E. , Rutter, W.

J. , and Goodman, H. M. , Science 196, 1313 (1977)] with added deoxynucleotide triphosphates. "Cohesive-ended" refer s to DNA molecules wi th s ingle-s tr anded termini . The s ingle-s tranded extensions are complementa ry and antiparalle l. ( See Mertz , J. E. , and

Davis, R. W., Proc. Nat. Acad. Sci. USA 69, 3379-3374 (1972).)

Joining of base-paired duplexes occurs when the nucleoside at a 5'-end carries a phosphate group and the complementary nucleoside opposite to it carries a free 3'-hydroxyl group. Two phosphodiester bonds would be made essentially simultaneously and the joined duplexes would have their nucleotide sequence inverted with respect to one another.

There are three general approaches to creating cohesive-ends on DNA:

1. digest DNA with type II restriction endonucleases that introduce staggered scissions at unique sequences;

2. treat linear DNA molecules with terminal deoxynucleotidyl transferase to generate single- stranded tails of either poly(dA) and poly(dT) or poly(dC) and poly(dG) at the 3'-hydroxyl terminus of different populations of DNA molecules; and

3. add to blunt-ended molecules linkers, which are short duplexes containing a restricton endonuclease cleavage site. Such linkers are joined to DNA by T4 DNA-ligase catalyzed blunt-end joining. After digesting the product with the restriction enzyme that cleaves within the linker, the DNA is terminated with cohesive ends. These methods are well known, as exemplified in the following articles: Sadler, J. R. , Betz, J. L. , Teiklenburg, M., Goeddel, D. V., Yansura, D. G. , and Caruthuers, M. H. , Gene3, 211 (1978); Bahn, C. P., Marians, K. J. , Wu, R. , Stawinsky, J. , and Narang, S. A., Gene 1, 81 (1976); and Scheller, R. H. , Dickerson, R. E. , Boyer, H. W., Riggs, A. D. , and Stakura, K. , Science 196, 177 (1977).

"Linker" refers to a duplex, blunt-ended DNA molecule from 6-14 base pairs in length, containing the recognition site for a restriction endonuclease that produces cohesive or blunt termini.

In the preferred embodiment of the present invention, the plasmid serves as the vehicle for introduction of the foreign gene into the yeast cell. However, it is not necessary to use a plasmid, since any molecule capable of replication in yeast can be employed. The DNA molecule can be attached to a vector other than a plasmid, which can be a virus or cosmid as is known in the art; or it can be integrated into the chromosome.

The recombinant plasmid or plasmid chimera is constructed in vitro. Since the annealing and ligation process not only results in the formation of the recombinant plasmid, but also in the recircularization of the plasmid vehicle, a mixture of ligation products i s obtained involvi ng the orig inal plasmid and the foreign DNA. Only the or iginal plasmid and the DNA chimera cons isting of the plasmid vehicle and linked foreign

DNA will normally be capable o f replication. When the mixture is employed for transfo rmation of bacteria , replication of both the plasmid vehicle genotype and the foreign genotype will occur.

The transformation of the bacterial cells will result in a mixture of bacterial cells, the dominant proportion of which will not be transformed. Of the fraction of cells which are transformed, some significant proportion, but in some cases a minor proportion, will have been transformed by the recombinant plasmid . In any event , only a very small fraction o f the total number of cells which are present will have the desired phenotypic characteristics.

In order to isolate only the bacteria containing the DNA chimera or the original plasmid, a selectable genetic marker is included on the original plasmid, such as resistance to an antibiotic or heavy metal. The cells can then be grown on an agar medium containing the growth inhibiting substance. Since E. coli is used as the bacteria for transformation in the present invention, ampicillin is used as the growth inhibiting material to afford selection in E. coli. Only available cells having the resistant genotype will survive. If the foreign gene does not provide a phenotypical property, which allows for distinction between the cells transformed by the plasmid vehicle and the cells transformed by the plasmid chimera, a further step is necessary to isolate the replicated plasmid chimera from the replicated plasmid vehicle. The steps include lysing of the cells and isolation and separation of the DNA by conventional means or random selection of transformed bacteria and characterization of DNA from such transformants to determine which cells contain molecular chimeras. This is accomplished by physically characterizing the DNA by electrophoresis, gradient centrifugation, sequence analysis or electron microscopy.

Cells from various clones may be harvested and the plasmid DNA isolated from these transformants. The plasmid

DNA may then be analyzed in a variety of ways. One way is to treat the plasmid with an appropriate restriction enzyme and analyze the resulting fragments for the presence of the foreign gene. Other techniques have been indicated above. Once the recombinant plasmid has been replicated in E. coli and isolated, the E. coli may be grown and multiplied and the recombinant plasmid employed for transformation of the S. cerevisiae strain.

The term SUC2 promoter as employed in the present invention, also designated P_SUC2, is a 0.9 kilobase DNA sequence from the yeast genome that contains the promoter for the major, regulated messenger RNA (mRNA) transcript of the invertase gene whose protein is secreted and glysoylated as opposed to the intracellular, nonglycosylated invertase. In yeast, RNA transcription initiates within 100 base pairs of the right end of the sequence and proceeds rightward. See M.

Carlson and D. Botstein, Cell 28, 145-154 (1982). The sequencing information for the 151 base series at the 3' end of SUC2 is shown in Table 3.

The interferon gene referred to, which can be promoted by the promoter used in this invention, is any one of the three classes of interferon genes described below:

(a) leukocyte - derived from leukocyte or lymphoblastoid cells, designated LelFN or IFN-α;

(b) fibroblast - derived from fibroblast cells, designated FIFN or IFN-β; and

(c) immune - derived from mitogen- or antigen-stimulated lymphoid cells, designated IFN-γ.

Such interferon genes are described in:

-Goeddel, D. V., Leung, D. W. , Drell, T. J. , Gross, M., Lawn, R. M., McCandliss, R. , Seeburg, P. H. , Ullrich, A., Yelverton, E. , and Gray, P. W. , Nature 290, 20-26 (1981) .

-Allen, G. and Fantes, K. H. , Nature 287, 408-411 (1980) and preceding reference.

-Zoon, K. C. , Science 207, 527-528 (1980) . -Mantei, N. , Schwartzstein, M. , Streuli, M. , Panam, S., Nagata, S., and Weissman, C. , Gene 10, 1-10 (1980) .

-Streuli, M. , Nagata, S., and Weissman, C. , Science 209, 1343-1347 (1980) .

In the preferred embodiment, expression of human leukocyte interferon is enhanced compared to levels of production in prior methods, and with the use of the S. cerevisiae strain a new human leukocyte interferon is produced. The yeast strain employed as the host cell in the preferred embodiment of the present invention is

Saccharomyces cerevisiae , a common laboratory strain of yeast used for its low toxicity and well-known genetic characteristics. This strain is readily cultivatable on a large scale. However, the recombinant DNA material of the present invention containing the SUC2 promoter can be used to express interferon in any yeast cells capable of transformation.

Saccharomyces cerevisiae is a yeast whose vegetative reproduction occurs by multilateral budding cells. Such cells are usually found in pairs or small clusters. The species is usually diploid where spores are produced directly in vegetative cells, but the species can also grow as haploid. In addition, S. cerevisiae forms an ascus with one to four spheroidal spores in each ascus. The ascus for this species does not rupture at maturity. The yeast has a strongly fermentative as well as respiratory metabolism. Selected strains are referred to as distillers' yeasts and baker's yeast.

The vast majority of yeasts can be cultivated under relatively uniform conditions on common laboratory media. The usual growth requirements of yeast include:

(a) organic carbon compound for carbon and energy;

(b) organic or inorganic nitrogen for the synthesis of proteins and nucleic acids; (c) various minerals (including compounds furnishing trace elements); and

(d) frequently a mixture of vitamins.

Such growth requirements are met by yeast nitrogen base (YNB, obtained from Difco) , a chemically defined medium which contains a number of tr ace elements, 9 vitamins, trace amounts of amino acids to stimulate growth of certain fastidious yeasts and the pr incipal minerals, potassium phosphate , magnesium sulfate , sodium chloride, and calcium chloride. The nitrogen source is ammonium sulfate. The desired carbon source must be added and is normally at a concentration of 0.5 - 3% . Additions are made to this medium to fit particular strain requirements. The pH range of the medium is usually from pH 3 - 8. The preferred range is pH 4.5 - 6.5.

In the present invention, the arrangement of the DNA segments in the plasmid construction is shown diagramatically in Table 4.

This construction consists of several components generally used in "shuttle" vectors, i.e., plasmids that can be maintained either in E. coli or yeast. The plasmid described in Table 4 is a modified construction of plasmid YIp5, as described by K. Struhl, D. T. Stinchcomb, S. Scherer and R. W. Davis, Proc. Nat. Acad. Sci. USA 76, 1035-1039 (1979) [see Table 2]. Segment (1) is a 2.4 kilobase fragment of plasmid pBR322, and contains a DNA replication origin and the β-lactamase gene, allowing propagation of the DNA in E. coli and continuous selection for its presence by ampicillin resistance. Segment (2) is a 1.6 kb Hpal to Hindlll fragment of the yeast 2μ plasmid containing an initiation site for replication in yeast. [The 2μ plasmid is described by J. L. Hartley and J. E. Donelson, Nature 286, 860-865 (1980) .] Segment (3) is a 1.1 kb DNA containing the yeast URA3 gene which encodes the enzyme orotidine-5' monophosphate decarboxylase that catalyzes an essential step in uracil biosynthesis in yeast. [The URA3 gene is described by M. Bach, F. Lacroute, and D. Botstein, Proc. Nat. Acad. Sci. USA 76, 386-390 (1979) .] Presence of this gene on the vector allows the continuous selection for yeast harboring the plasmid by its complementation of a ura3- mutation in the host genome. Segment (3) is inserted at the Aval site of plasmid pBR322, thus dividing this region of pBR322 into Segments (6) and (7) . The unique aspect of this construction is the inclusion of DNA segments (4) and (5). Segment (4) is a 0.9 kb EcoRI to Hindlll DNA fragment of the yeast genome that contains the regulated promoter for the major messenger RNA transcript of the invertase gene. In yeast, RNA transcription initiates within 100 base pairs of the right end of segment (4) and proceeds rightward (as shown in Table 4). The actual coding sequence for invertase has been separated from its promoter by cleavage of the DNA with restriction endonuclease Hindlll, leaving cohesive ends. In this new construction, segment (5) is ligated to segment (4) at this HindIII cleavage site.

Segment (5) contains the gene for the new human leukocyte IFN in its mature form, oriented such that transcripts originating from the SUC2 promoter copy the gene into mRNA coding for interferon. The interferon coding sequence is not the full sequence of human LelFN mRNA, but one in which the sequence coding for the signal peptide of the interferon precursor has been replaced by a translation initiation codon directly preceding the sequence coding for the mature secreted protein for the new human leukocyte interferon. The sequence of the DNA between the HindIII site joining segment

(4) and (5) and the beginning of the modified IFN gene is:

Codon (a) is the initiating methionine codon and (b) is the initial cysteine codon of the new human LelFN. As shown, the construction also contains the cleavage site of restriction endonύclease Clal. The synthetically produced sequence shown above may exist in nature in random fashion.

Following the initiation codon is the 498 base pair coding sequence of the new HuLelFN, its translation stop codon, and approximately 600 base pairs of untranslated sequence from the mRNA of the new HuLelFN. Segment (5) is joined to Segment (6) of pBR322 DNA by a HindIII linker.

This plasmid vector is maintained in multiple copies per cell in a strain of Saccharomyces cerevisiae (CGY 123) that has the genotype MATa leu2-3 leu2-112 ura3-50. Since this host is a uracil auxotroph, only those cells containing the plasmid with the URA3+ gene can grow in minimal medium without uracil. This selection reduces the accumulation of yeast cells that have lost the plasmid by segregation during cell division.

Different DNA sequences exist between the SUC2 promoter and the start of the IFN coding sequence. For example, by cutting the plasmid DNA described in (I) with endonuclease

Clal (creating cohesive ends), followed by nuclease S1

(creating blunt ends), and re-closing the structure with DNA ligase, the DNA sequence at the junction of segments (4) and

(5) has been changed to:

The synthetically produced sequence shown above may exis t in nature in r andom f ash ion.

Additional DNA sequences have been added to the

C-terminal end of the IFN gene. In one case a 1.4 kb segment from the yeast genome containing the transcription termination site of the invertase gene has been inserted between segment (5) and segment (7).

The essential feature in the plasmid that must be maintained is the orientation of the SUC2 promoter relative to the IFN gene to be transcribed. Permissible modifications in the plasmid DNA would include:

--changes in the length or sequence of DNA between the SUC2 promoter and the initiation of the IFN gene. These sequences must not contain transcription stop signals or out-of-frame translation start codons; --substitution of other IFN genes; —DNA sequence changes (probably in segment (4)) that affect the level of transcription from the SUC2 promoter or its mode of regulation;

--any arrangement of DNA with the IFN gene transcribed from the SUC2 promoter. This constructon could differ from that in (I) in having the SUC2 promoter - LelFN combination integrated in a yeast chromosome rather than in an extrachromosomal plasmid; --translation initiation at a signal peptide coding sequence rather than the methionine codon initiating the mature IFN sequence. This signal peptide (that encoded by a portion of the invertase gene, for example) could lead to secretion of IFN from the yeast cell.

The yeast strain described herein produces a new human

LelFN when cultured in a medium with low glucose levels. The yeast are propagated in a medium containing 6.7 g/l yeast nitrogen base, 50 mg/l L-leucine and 2% glucose. To induce interferon synthesis, the yeast cells are removed from the high glucose medium by centrifugation, and resuspended in minimal medium containing 0.05% glucose. In 2 to 3 hours, the SUC2 promoter is fully active and the yeast accumulate

IFN. The cells are harvested by centrifugation. The cell-free extract containing LelFN is obtained by breaking open the yeast cells by vigorous vortexing with glass beads in a solution of 0.1 M Tris-HCl, pH8, 20% glycerol, and ImM

PMSF.

EXAMPLE

1. Isolation of IFN mRNA

3.55 grams of Sendai virus induced lymphocytes were disrupted by means of a Dounce homogenizer into 40 ml of cold buffer (10° C) consisting of 50 mM NaOAc, pH 5.2; 6 M guanidine HCl; and 0.1 M 2-mercaptoethanol. The resulting solution was sonicated at 60W pulsed power for 2x30 seconds and then layered onto 3 ml shelves of 5.8 M CsCl, pH 7.2, containing 0.1 M EDTA. The material was centrifuged at 15° C in a Beckman Type 50 Ti rotor at 40,000 rpm overnight. The pellet was resuspended on ice for 20 minutes in 6.6 ml of the above cold buffer plus 20 mM EDTA, and then treated with 3.3 ml of ice-cold absolute ethanol. After 1 hour at -20° C, the precipitate was pelleted by a centrifugation at 8,000 rpm for 20 minutes at -10° C. The pellet was resuspended two times in 18 ml of the preceding buffer, treated with 9 ml of ice cold absolute ethanol, chilled one hour at -20° C and the pellet collected as decribed previously. The final pellet was resuspended in 8 ml of 0.1 M EDTA with heating at 60° C, and then 0.1 volume of 2M NaOAC, pH 5.0, and 2 volumes of ice-cold absolute ethanol were added and the solution placed at -20° overnight. The RNA precipitate was collected by centrifugation at 8,000 rpm for 20 minutes at -10° C, and was dissolved in 5 ml water. The yield was 396 mg RNA. The RNA solution was diluted with 5 ml of 2x concentrated binding buffer (20 mM Tris-HCL, pH 7.5; 2mM EDTA, pH 7.0; 0.4% SDS; and 0.24 M NaCl). The RNA was applied to a 1 ml oligo-dT-cellulose column, the column was washed with Ix concentrated binding buffer and then the poly A-containing RNA (mRNA) was eluted by washing the column with binding buffer containing no NaCl. About 39 mg of poly A-containing RNA was obtained.

A portion of the poly A-containing RNA was translated in vitro in a rabbit reticulocyte lysate system [Pelham, H.R.B. and Jackson, R.J. , Eur. J. Biochem. 67, 247-256 (1976)] to confirm the isolation of mRNA coding for interferon.

2. Preparation of double-stranded copy DNA (cDNA)

About 2.5 μg of cDNA was synthesized from 25 μg of the lymphocyte poly A-containing RNA by incubation for one hour at 42° C in 50 mM Tris-Hcl, pH 8.3; 100 mM KCl; 8mM MgCl₂; 0.4 mM dithiothreitol; 1.2 mM each dATP, dGTP and dTTP; and

20μg/ml oligo (-dT)₁₂_₁₈, containing 100 units reverse

32 transcriptase and 0.25 mM α-³² P-dCTP(1.8 Ci/mmole). After heating the reaction mixture at 100° C for 3.5 minutes, quick chilling on ice for approximately 3 minutes and removing the precipitated protein by centrifugation, to the supernatant was added Hepes-NaOH, pH 6.9, to 100 mM; MgCl₂ to 5 mM; dithiothreitol to 0.5 mM; and deoxynucleoside triphosphates as above. Incubation of this mixture with 300 units of E. coli DNA polymerase I for 2.5 hours at 15° C produced

1.8μg of double-stranded cDNA. The DNA was phenol extracted, separated from unincorporated triphosphates by chromatography on Sephadex G-100 (13 ml column, 0.68 cm x 37 cm, eluted with 20 mM Tris-HCl, pH 7.5, 3.5 mM EDTA) and ethanol precipitated overnight at -20° C by addition of 1/10 volume 2 M NaOAc, pH 5, and 2.5 volumes cold ethanol. The double-stranded cDNA was then treated with 8,000 units of S1 nuclease at 37° C for one hour in buffer (0.3 M NaCl, 30 mM NaOAc, pH 4.6, 3 mM ZnSO₄) . The reaction was terminated by addition of EDTA to 10 mM, and Tris-HCl, pH 8.3, to 200 mM, and the mixture applied to a Biogel A-150m column (0.7 cm x 35 cm) equilibrated and eluted with 10 mM Tris -HCl, pH 7.5, 250 mM NaCl and 1 mM EDTA. The peak fractions (0.5 ml each) of large molecular weight DNA were pooled and ethanol precipitated by addition of 1/10 volume 2 M NaOAC, pH 5, and 2.5 volumes cold absolute ethanol.

3. Addition of Hindlll Linkers

The S1-treated double-stranded cDNA (0.21 μq) was incubated in buffer (60 mM Tris-HCl, pH 7.5; 8 mM MgCl; 5 mM dithiothreitol, 1 mM ATP and 1 mM of each deoxynucleoside triphosphate) with 9 units of E. coli DNA polymerase I at 10° C for 10 minutes and then placed on ice. This blunt-ended double stranded cDNA was next incubated in 65 mM Tris-HCl, pH 7.5; MgCl₂; 5 mM dithiothreitol; 1 mM ATP, with 160 pmoles of 32 P-labelled Hindlll synthetic linker

(100 x excess over cDNA ends) and 4 blunt-end units of T4 DNA ligase at 15° C for 5 minutes, cooled on ice, heat treated to inactivate the ligase, treated with Hindlll restriction endonuclease (New England Biolabs, 9 units) in 5.6 mM

Tris-HCl, pH 7.5, 5.6 mM MgCl₂ at 37° C for 4 hours 45 minutes and then phenol extracted. The reaction was fractionated on a Biogel A-150m column (0.7 cm x 31.5 cm) .

Fractions (0.5 ml each) containing high molecular weight DNA were pooled and ethanol precipitated.

This double stranded cDNA with Hindlll cohesive termini was then ligated to f1 phage CGF4 double-stranded DNA which had been cut open with Hindlll restriction endonuclease and treated with calf intestinal alkaline phosphatase by the method of H. Goodman and R. J. MacDonald [Goodman, H. M. and

MacDonald, R. J. , Methods in Enzymol. 68, 75-91 (1979)] to remove the terminal phosphates (Note: In order to produce phage CGF4, f1 phage R229 [Bo eke, J. D., Mol. Gen. Genet.

181, 288-291 (1981)] was cut with EcoRl endonuclease, rendered blunt ended with T4 DNA polymerase and ligated with

Hindlll synthetic oligonucleotide linkers from Collaborative

Research, Inc. of Lexington, Massachusetts.) The ligation reaction contained 60 mM Tris-HCl, pH 7.5; 6 mM MgCl₂; 7 mM dithiothreitol; 1.2 μg double-stranded cDNA; 1.2 μg CGF4 DNA;

0.5 mM ATP and 450 cohesive end units of T4 DNA ligase.

Ligation was for 19 hours at 15° C. 4. Transfection of E. coli DB4548 with recombinant CGF4 DNA

E. coli strain CGE6 (DB4548; hsdR-, hsdM⁺, sup E, sup F, Bl-, met-) was grown in 150 ml tryptone broth at 37° C with shaking and harvested at OD₇₀₀=0.5 by centrifugation at 7,000 rpm for 10 minutes at 4° C. The cells were resuspended in 70 ml ice cold 50 mM CaCl₂ and allowed to sit at 0° C for 30 minutes. The suspension was then centrifuged at 7,000 rpm for 10 minutes at 4º C and resuspended in 3 ml ice cold 50 mM CaCl₂. After standing at 0° C for 2 hours the cells were used for transfection. Either 1μl or 2 μl of 1:40 dilution of ligation reaction in 50 mM Tris-HCl, pH 7.5, was added to each of 12 tubes containing 50μl sterile 50 mM Tris-HCl, pH 7.5. One-tenth milliliter of the CaCl₂-treated cells was added to each tube and the mixtures set on ice for 30 minutes. After warming to 37° C for 2 minutes, 0.2 ml of CGE5 (JMl01: J. Messing (1979) , F tra D36 pro AB lac lZVM15 in a

lac pro) SupE thi- background) overnight culture and 3 ml of 0.7% soft agar were added, and the mixture poured into tryptone agar plates. Incubation at 37° C overnight produced over 1280 plaques. 5. Identification o f a recombinant-CGF4 car rying the leukocyte interf eron sequence

The plaques were transferred to nitrocelluloses and probed as described by Benton and Davis [Benton, W. D. and Davis, R. W. , Science 196, 180-182 (1977] using a ³²P-labelled synthetic oligonucleotide (with the sequence, CATGATTTCTGCTCTGAC, Collaborative Research, Inc.) which corresponds to a known segment of LelFN. The oligonucleotide (1 μg) was kinased with 0.5 mC γ-³²P-ATP using 6 units of

T4 polynucleotide kinase (P-L Biochemicals) in a 20μl reaction containing 66 mM Tris-HCl, pH 7.5, and 10 mM MgCl₂. The phage which hybridized intensely to the synthetic oligonucleotide probe were picked from the plates and stored in TY medium at 4° C. Samples of the intact phage were amplified by growth overnight on CGE5 cells, harvested by centrifugation, and subjected to electrophoresis in a 0.6% agarose gel containing 0.37 M Tris-glycine, pH 9.5, and stained with ethidium bromide after treatment in 0.2 N NaOH for one hour and neutralization in 0.5 M Tris-HCl, pH 7.4. The migration is inversely proportional to the log of the size of the phage DNA and allowed selection of phage carrying inserted IFN DNA of size of 1000 to 1200 base pairs. Double- stranded RFl DNA was prepared from the phage by the method of Moses et al. [Moses, P.B., Boeke, J.D. , Horuchi, K. and Zinder, N.D., Virology 104, 267-273 (1980)]. This DNA was cut with HinddIII restriction endonuclease and the resulting fragments analyzed on an agarose gel to confirm that the insert was in the Hindlll site and of the anticipated size.

One of the phage DNA' s which has an insert of about 1200 base pairs (bP) was chosen for further study. The DNA insert was sequenced by the method of Maxam and Gilbert [Maxam, A. M. and Gilbert , W. , Methods in Enzymol 68 , 499-560 (1980) ] .

6. Expression of LelFN in Saccharomyces cerevisiae

A plasmid , pCGS84 , designed to facilitate obtaining expression of LelFN in yeast was cons tructed. In order to produce the Le lFN in yeast , an ATG initiation codon was incorporated at the 5 ' -s ide of the f irst codon (TGT for cysteine) of mature , processed IFN. Based on the fact that Sau3AI cuts at the 3 ' -s ide of the f irst codon, an oligonucleotide (ACACATCGATGTGT) which is recognized by Clal and also contains the ATG-TGT sequence was synthesized by Collaborative Research, Inc . A Sau3Al fragment which codes the amino acid residues 2 to 61 was purified by digesting 30 μg of the Hindlll 1.2 kilobase fragment with 10 units Sau3Al restr iction endonuclease in a 50 μl reaction volume containing 10 mM Tris-HCl, pH 7.5 ; 10 mM MgCl₂ ; and 60 mM NaCl for 4 hours at 37 ° C. The DNA fragment was purified by polyacrylamide gel electrophoresis. The DNA was phenol extracted and precipitated with ice-cold absolute ethanol.

The cohesive ends were filled in by treating the DNA with 4 units E. coli DNA Polymerase I Klenow fragment and 0.1 mM each nucleoside triphosphate in 66 mM Tris-HCl, pH 7.5; 66 mM

NaCl; 66 mM MgCl₂ and 66 mM dithiothreitol, for 30 minutes at room temperature.

The above synthetic oligonucleotide was ligated onto the

Sau3Al fragment in 66 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; 10 mM 2-mercaptoethanol; ImM ATP with 500 pmole

³²P-oligonucleotide (5 μg) ; 4 pmoles DNA (20 μg) and 4 blunt-end units of T4 DNA ligase at 17° C overnight. This ligation created an ATG initiation codon and restored the first codon TGT. Clal polylinker was removed by treating the fragment with 20 units restriction endonuclease Clal for 3 hours at 37° C in a 20 μl reaction containing 10 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; and 1 mg/ml bovine serum albumin. The resulting fragment was cloned into the Clal site of plasmid pBR322. The plasmid (10 μg) was cut with the restriction endonuclease Clal (New England Biolabs, 20 units) for 2 hours at 37° C in a 20 μl reaction containing 10mM Tris-HCl, pH

7.5; 10 mM MgCl₂ and 1 mg/ml bovine serum albumin. The preparation of restriction cut plasmid was phenol extracted, ethanol precipitated and treated with calf intestinal phosphatase by the method of H. Goodman and R. J. MacDonald

[Goodman, H. M. and MacDonald, R. J. , Methods in Enzymology

68, 75-91 (1979)] to remove the terminal phosphates. Approximately 0. 5 pmole of the Cl a I fragment and 0.3 pmol e of the Cl al cut plasmid were l igated together at 15 ° C for 3 hour s in a 20 μl reaction containing 66 mM Tr is-HCl , pH 7.5 ;

6 mM MgCl₂; 10 mM dithiothreitol; ImM ATP; and T4 DNA ligase (New England Biolabs, 300 units) creating plasmid pCGE32. Transformation-competent E. coli strain CGE43 (LG90; F- (lac-pro)xlll) was prepared as described previously for CGE6, and 5μl of the ligated DNA was mixed with 200μl of the cells for 30 minutes at 0° C, heat treated at 37° C for 2 minutes, incubated at 18° C for 10 minutes, and diluted five-fold with fresh tryptone broth. After incubation for 30 minutes at 37° C with shaking, cells were plated on tryptone plates containing ampicillin (20 μg/ml). Ampicillin-resistant colonies were picked, and the plasmid DNA was prepared and analyzed by restriction enzyme digestion. By these criteria several cells carried the desired plasmid, pCGE32.

The rest of the IFN gene was put back together by using the EcoRl site located in the region coding for amino acid residue 37. Plasmid pCGE32 DNA (10 μg ) was cut with the restriction endonuclease HindIII (Collaborative Research, Inc., 12 units) for 2 hours at 37° C in a 20 μl reaction containing 10 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; 60 mM NaCl; and 1 mg/ml bovine serum albumin). This DNA was next digested with the endonuclease EcoRl (Collaborative Research, Inc., 15 units) for 3 hours at 37° C in a 20 μl reaction containing 100 mM Tris-HCl, pH 7.6; 10mM MgCl₂; 30 mM NaCl; and 1 mg/ml bovine serum albumin. The restriction cut DNA was phenol extracted, ethanol precipitated, redissolved in water and applied to a preparative horizontal 1.5% agarose gel. After electrophoresis for 2 to 3 hours in 40 mM Tris-acetate, pH 7.2, the gel was stained with ethidium bromide and examined under long wavelength ultraviolet light. The digested Hindlll to EcoRl band which codes the ATG-TGT to amino acid residue 37 was excised and the DNA extracted by freezing and thawing the gel pieces [Thuring, et al. , Anal. Biochem 66, 213 (1975)]. The DNA fragment was ethanol-precipitated and redissolved in water. The plasmid (20 μg) containing the IFN clone was cut with the restriction endonuclease Hindlll (New England Biolabs, 180 units) for 2 hours at 37° C as above and then the DNA

(12 μg) was cut with the restriction endonuclease EcoRl (New England Biolabs, 24 units) for 6 minutes at 37° C. The restriction cut DNA was phenol extracted, ethanol precipitated, and redissolved in water. This EcoRl to Hindlll fragment coding for amino acid residue 37 to the 3'-nontranslating region of IFN was analyzed by gel electrophoresis and excised from the gel (see above). Approximately 0.25 pmole of each fragment were ligated together into plasmid pBR322 opened at its Hindlll site (see above) for 4 hours at 14° C in a 20μl reaction containing 66 mM Tris-HCl, pH 7.6; 6.6 mM MgCl₂; 10 mM dithiothreitol; 1 mM ATP and T4 DNA ligase (New England Biolabs, 300 units). Transformation-competen t E. col i s train CGE43 cells were prepared exactly a s descr i bed above , and 5 μl o f the l igated

DNA was mixed wi th 100 μl of the cells for 30 minutes a t 0 °

C, heat treated at 37° C for 2.5 minutes, and diluted ten-fold with fresh tryptone broth. After incubation for 30 minutes at 37° C with shaking, cells were plated on tryptone plates containing ampicillin (20 μ g/ml) .

Ampicillin-resistant colonies were picked, and the plasmid DNA was prepared and analyzed by restriction enzyme digestion. By these criteria several strains carried the desired plasmid, pCGE38, which contained the ATG-α-IFN gene.

The 1.1 kilobase HindIII fragment containing the gene for LelFN was isolated from plasmid pCGE38 (30 JULq) by cutting the plasmid with restricton endonuclease HindIII for 1.5 hours at 37° C as above. The restriction cut DNA was phenol extracted, ethanol precipitated, redissolved in water and applied to a preparative 1% agarose gel. After electrophoresis in 40 mM Tris-acetate, pH 7.2, the gel was stained with ethidium bromide and examined under long wavelength ultraviolet light. The 1.1 kilobase band was excised and the DNA extraced by freezing and thawing the gel pieces [Thuring, et al. , Anal. Biochem. 66, 213 (1975)]. The DNA fragment was ethanol precipitated and redissolved in water. Approximately 0.5 pmole of the HindIII fragment was ligated into plasmid pCGS40 opened at its HindIII site adjacent to the P_SUC2 region (see above) for 2.5 hours at 14° C in a 20 μl reaction containing 66 mM Tris-Hcl, pH 7.6; 6.6 mM MgCl₂; 10 mM dithiothreitol; 1 mM ATP and T4 DNA ligase (Collaborative Research, Inc., 10 units) . The plasmid, pCGS40, comprises most of YIp5 containing a DNA replication origin and β-lactamase gene for selection in E. coli, with a 1.6 kilobase fragment of the yeast 3μ plasmid containing an initiation site for replication in yeast, with a 1.1 kilobase fragment from the yeast chromosomal DNA carrying a URA3 gene for selection in yeast and with a 0.9 kilobase fragment from yeast chromosomal DNA containing the SUC2 promoter of the yeast invertase gene. The plasmid pCGS40 was constructed by first cutting 60 μq of plasmid pRB118 [Carlson, M. and Botstein, D., Cell 28, 145-154

(1982)] with restriction endonuclease Hindlll for 30 minutes at 37° C and then with restriction endonuclease EcoRl (see above). The restriction cut DNA was phenol extracted, ethanol precipitated, redissolved in water and purified by gel electrophoresis. The digested EcoRl to Hindlll 0.9 kilobase band which contains the promoter for the SUC2 invertase gene was excised and the DNA extracted by glass beads. [Vogelstein, B. and Gillespie, D., PNAS 76, 615-619

(1979) .] The 0.9 kilobase DNA fragment containing the SUC2 promoter was placed on the plasmid YIp5 (a shuttle vector which can be selected for and maintained in yeast due to the presence of the URA3 gene or E. coli due to the presence of the Amp gene) [Struhl, D. , Stinchcomb, D. T. , Scherer, S. and Davis, R. W., Proc. Nat. Acad. Sci. USA 76, 1035-1039 (1979)]. The resulting plasmid, pCGS46, obtained after ligation and transformation (as described above) was purified and its structure verified by analysis with restriction endonucleases. The plasmid, pCGS40, was the result of cutting the plasmid pCGS46 with restriction endonuclease PvuII for 1 hour at 37° C. A 1.56 kilobase fragment of 3μ, DNA from plasmid YEpl3, obtained from R. Davis, Stanford University, was removed by cutting YEpl3 with Hpal and Hindlll. Ihe resulting fragment was gel purified, phenol extracted, ethanol precipitated, and treated with T4 DNA polymerase (see above) in order to create blunt ends at the Hindlll restriction cut. After phenol extraction and ethanol precipitation, the PvuII cut DNA and blunt-ended 3μ DNA fragment were purified by gel electrophoresis and ligated together overnight. The resulting plasmid, pCGS40, can be grown and its presence can be selected for in either E. coli or Saccharomyces cerevisiae.

The yeast strain CGY123 (MATa, leu 2-3, leu 2-112, ura 3-50) was transformed with the plasmid DNA by the method of A. Hinnen, J. B. Hicks, and G. Fink [Hinnen, A., Hicks, J. B. and Fink, G. F. , Proc. Nat. Acad. Sci. USA 75, 1929-1933 (1978)]. Yeast transformants CGY144, capable of growth without added uracil due to the presence of URA3 gene on the plasmid, were picked and the plasmid DNA pCGS84 was prepared following amplification of these transformants. The plasmid

DNA was analyzed by restriction enzyme digestion with restriction endonucleases HindIII and EcoRl and the proper

IFN DNA insert and orientation were determined. (Strain

CGY144 bearing plasmid ρCGS84 is on deposit with the American

Type Culture Collection (ATCC) and its Accession number is

20644.) The yeast cells were grown at 30° C with agitation in a medium containing 6.7 g/l yeast nitrogen base, 30 mg/l

L-leucine and 2% glucose. The synthesis of interferon was induced by collecting cells grown to Klett = 50 (10⁷ cells/ml) by centrifugation and resuspeπding in minimal medium containing 0.05% glucose. After growing 2.5 hours at

30° C with agitation, the cells were collected by centrifugation, resuspended in 0.25 ml 0.05 M Tris-HCl, pH 7.6, 20% glycerol and 1 mM PMSF, and frozen at -20° C. The cells were disrupted by glass beads by the method of M Rose, et al. [Rose, M., Casadaban, M. J. and Botstein, D., Proc. Nat. Acad. Sci. USA 78, 2460-2464 (1981)] and the amount of interferon activity in the cellular extract was determined by conventional methods to be 10 units/l.

The sequencing information for the human leukocyte interferon gene produced is shown in Table 5.

While the specific embodiments of the invention have been shown and described, many variations are possible. For example, the present..invention is mainly concerned with the production of human leukocyte interferon of a particular type. Obviously, other interferons can be obtained and expressed using the SUC2 promoter of this invention in the operative relationship defined. Moreover, the ability to use a passive signal to initiate expression of a desired polypeptide such as interferon is important for production of polypeptide products in large volume. Thus, expression can be obtained without introduction into a growing culture of an external inducer to stimulate expression. The stimulus can be depletion of glucose as in the present example, whereupon the culture, as a self-contained system, will initiate expression in high quantities of a desired polypeptide. Such polypeptides may be enzymes or other biologically active proteins. The promoters may also differ. The present example is but one example of passive production.

It should be understood that the advantages of passive production have been known in nature and in the production of antibiotics. However, to date, this methodology has not been used, insofar as is known, to produce large quantities of polypeptide products during cell growth without adversely interfering with the growth or maintenance of cells prior to the production phase of the desired polypeptide. The present invention has provided a methodology for carrying this out in yeast. The passive signal within the cell maximizes efficiency of production of the desired polypeptide, which signal is given, as for example in the present specific embodiment, by the exhaustion or depletion to a desired level of glucose.

The SUC2 promoter has been linked to genes other than interferon to promote their expression in yeast. Thus far, comparative studies show the levels of expression of interferon to be far greater than those of the few other genes tested. It is expected that SUC2 can promote the expression of a variety of genes at levels high enough for commercial applications.

What is claimed is:

Claims

1. A recombinant DNA segment containing a SUC2 promoter l inked to a g ene o ther than invertase for directing the expression of said gene within a yeast cell.

2. In a method of expression of a gene other than invertase in yeast, said method comprising introducing a SUC2 promotor in a DNA segment, said segment being linked to said gene in a chromosome or vector in such a fashion that said chromosome or vector is replicated and carried by the cell as part of its genetic information and said gene is expressed.

3. A DNA segment containing a SUC2 promoter linked to an interferon gene for directing the expression of the gene within a yeast cell.

4. In a method of expression of interferon in yeast, said method comprising introducing a SUC2 promotor in a DNA segment, said segment being linked to an interferon gene in a chromosome or vector in such a fashion that said chromosome or vector is replicated and carried by the cell as part of its genetic information and said interferon gene is expressed.

5. A method as in claim 4, wherein said yeast is of the strain Saccharomyces cerevisiae.

6. A method of obtaining expression of interferon in yeast by the use of a SUC2 promoter in a DNA segment linked to an interferon gene, which DNA segment is incorporated in yeast cells, growing said yeast cells in a medium containing glucose, wherein said yeast cells metabolize said glucose, and permitting said cells to express interferon when glucose in said medium is at or near depletion, which expression occurs automatically without addition of further expression inducers when said glucose is at or near depletion.

7. Yeast strain as deposited in the American Type Culture Collection under Accession Number 20644, Strain Designation CGY144.

8. The synthetic DNA sequence

A A G C T T A T A T G T G T

9. The recombinant DNA sequence

A A G C T T A T A T G T G T wherein said sequence follows a DNA segment containing a SUC2 promoter for invertase in yeast.

10. The recombinant DNA sequence as in claim 9, wherein the last three bases TGT in said sequence are the intial codon for an interferon gene , said interferon gene following said sequence.

11. A DNA segment comprising a SUC2 promoter derived from the yeast genome that carries the promoter for the major regulated messenger RNA transcript of the invertase gene linked to an interferon gene .

12. A DNA segment as in claim 11, wherein said SUC2 promoter has a 0.9 kilobase DNA sequence.

13. A plasmid comprised of plasmid YIp5 having modifications comprising : a fragment of the yeast 2μ plasmid containing an initiation site for replication in yeast at the PvuI I site of said plasmid, and a fragment from yeast chromosomal DNA containing the SUC2 promoter of the yeast invertase gene between the EcoRl and Hindlll sites of said plasmid.

14. A plasmid as in claim 13, wherein said gene for selection in yeast is a DNA segment comprising the Ura3 gene.

15. A plasmid as in claim 13 having an interferon gene linked to said SUC2 promoter.

1'6. A plasmid comprised of plasmid YIp5 having modifications comprising a fragment from yeast chromosomal DNA containing the SUC2 promoter of the yeast invertase gene between the EcoRl and HindIII sites of said plasmid.

17. A plasmid as in claim 16 having an interferon gene linked to said SUC2 promoter.

18. The synthetic DNA sequence

A A G C T T A T C G A T G T G T

19. The recombinant DNA sequence

A A G C T T A T C G A T G T G T wherein said sequence follows a DNA segment containing a SUC2 promoter for invertase in yeast.

20. The recombinant DNA sequence as in claim 19, wherein the last three bases TGT in said sequence are the intial codon for an interferon gene, said interferon gene following said sequence.

21. Yeast cells capable of producing polypeptide products which can be obtained therefrom at a preselected stage of cell growth, said cells each containing a promoter, which promoter is in operative relation to a gene, the relationship of which is not found in nature, but the promoter or gene has been introduced into the cells or into ancestors of the cells for the purpose of permitting a passive signal to turn on synthesis of a desired polypeptide product by expression of said gene upon activation of the promoter after a period of promoter inactivity during cell growth.

22. Yeast cells as in claim 21, further comprising said gene being a mammalian gene.

23. A method of producing large quantities of a desired polypeptide product from cells growing in a medium, said method comprising growing cells capable of producing a desired polypeptide product which can be obtained therefrom in a preselected stage of cell life.

said cells each containing a promoter, which promoter is in operative relation to a gene which is not found in nature in such relationship, but the promoter or gene has been introduced into the cells or into ancestors of the cells for the purpose of permitting a passive signal, to turn on synthesis of the desired polypeptide product by expression of the gene upon activation of the promoter after a period of promoter inactivity during cell growth.

24. The recombinant DNA material found in the yeast strain identified as American Type Culture Collection Accession Number 20644 , Strain Designation CGY144.

25. Recombinant DNA material as in claim 24, wherein said material comprises a SUC2 promoter linked to an interferon gene.

26. A vector carrying a SUC2 promoter linked to an interferon gene capable of insertion in yeast and bacteria.

27. The recombinant DNA material comprising the following human leukocyte interferon nucleotide sequence:

28. The polypeptide produc t produced by expression of th e nucleotide sequence of claim 27.

29. A recombinant DNA segment comprising a 1.6 kilobase Hpal to Hindlll fragment of the yeast 2μ plasmid, wherein said fragment initiates replication of genetic material in yeast.