Gene encoding biologically active human interleukin 1

Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled, synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the interleukin 1 protein. Pools of host cells that provide a positive signal to the probe are identified, plated out and then employed in direct bacterial colony hybridization with the same probe, thereby to isolate the particular positive colony. Plasmid DNA is prepared from this colony and characterized by restriction enzyme mapping and sequencing by chain-termination method. The coding region for the IL-1 gene is inserted into a shuttle vector for amplification of the vector followed by expression of functional IL-1.

TECHNICAL FIELD 
The present invention relates to interleukin 1 (hereinafter "IL-1"), and 
more particularly to the cloning of a gene for human IL-1 by use of a 
synthetic oligonucleotide probe derived from the amino acid sequence of 
purified IL-1, to screen a complementary deoxyribonucleic acid ("cDNA") 
library synthesized from IL-1 messenger ribonucleic acid ("mRNA"); and, 
the characterization of the screened IL-1 gene. 
BACKGROUND OF THE INVENTION 
IL-1, formerly known in the literature as "lymphocyte activating factor" or 
"LAF," is a hormone secreted by macrophages while undergoing on immune 
response. This protein factor regulates a wide range of immunological and 
non-immunological responses. For instance, it is considered that IL-1 
mediates activities referred to as endogenous or leukocytic pyrogen, 
B-cell activating factor (BAF), epidermal cell thymocyte activating factor 
(ETAF), leukocyte endogenous mediator (LEM), bone resorption factor active 
in rheumatoid arthritis, and a variety of other activities. 
Although researchers have identified many of the biological properties of 
IL-1, the chemical nature of this hormone is not well understood. To date, 
this has been hampered, at least in part, by the unavailability of 
sufficient quantities of IL-1 in purified form to carry out necessary 
investigations. 
Attempts have been made in the past to purify and partially characterize 
IL-1 derived from both human and murine sources. For instance, Mizel, 122 
J. Immunol. 2167-2172 (1979), reported the production of murine IL-1 from 
the macrophage cell line, P388D.sub.1, cultured in a supplemented growth 
medium together with phorbol myristic acetate as an activating agent. The 
IL-1 from the culture fluid was subjected to ammonium sulfate 
precipitation, diethyl amino ethyl ("DEAE") cellulose column 
chromatography, ultrafiltration and Sephacryl S200 column chromatography. 
The resulting active fractions were analyzed by sodium dodecyl sulfate 
("SDS")-polyacrylamide gel electrophoresis ("PAGE") and were found to have 
a molecular weight in the range of 12,000 to 16,000 daltons. Through 
isoelectrofocusing ("IEF") in polyacrylamide gels, the pI of the IL-1 was 
found to be in the range of from 5.0 to 5.4. 
In a subsequent communication Mizel et al., 126 J. Immunol. 834-837 (1981), 
discussed purifying IL-1 from the same P388D.sub.1 cell line as used in 
Mizel, supra, to "apparent homogeneity" by ammonium sulfate precipitation, 
phenyl Sepharose chromatography, Ultrogel AcA54 gel filtration 
chromatography and preparative flat-bed IEF. From the IEF, the pI of the 
IL-1 was measured to be about 4.9 to 5.1. Through gel electrophoresis the 
molecular weight of the IL-1 molecule was determined to be approximately 
14,000 daltons. 
Researchers have also investigated IL-1 produced from human peripheral 
blood leukocytes and monocytes. Blyden et al., 118 J. Immunol. 1631-1638 
(1977), disclosed a protocol for concentrating IL-1 prepared from human 
peripheral blood leukocytes by Sephadex G-100 column chromatography. This 
procedure was reported to result in a four-to-five fold concentration of 
the crude IL-1. DEAE-Bio-Gel A anion exchange chromatography was employed 
to remove the albumin from the serum used during the preparation of the 
crude IL-1. Next, the collected active fractions were adsorbed onto a 
hydroxylapatite column. Fractions containing peak IL-1 activity were then 
applied to a CM-Bio-Gel A cationic exchange resin. By these procedures, 
about 20% of the initial IL-1 was recovered. The resulting IL-1 was found 
to have a molecular weight of about 13,000 daltons and a pI of 
approximately 6.8 to 7.2. 
Crude IL-1 prepared from human leukocytes by Togawa et al., 122 J. Immunol. 
2112-2118 (1979) was initially processed by membrane filtration and then 
applied to a Bio-Gel P-100 chromatography column which disclosed two major 
peaks of activity, one in the range of from 12,000 to 22,000 daltons and 
another in the range of bout 50,000 to 70,000 daltons. Active fractions in 
the lower molecular weight region of the Bio-Gel P-100 column were pooled, 
applied to a Blue Sepharose column, and then applied to a DEAE-cellulose 
ion-exchange chromatography column. Thereafter, the IL-1 containing 
fractions were pooled and applied to a hydroxylapatite chromatography 
column. Togawa et al. discovered that when the lower molecular weight IL-1 
activity resulting from each of these procedures was reconstituted with 2% 
human serum, concentrated and rechromatographed on Bio-Gel P-100, a 
significant portion of the higher molecular wight activity appeared. 
In a more recent study, Lachman, 42 Federation Proceedings 2639-2645 
(1983), reported preparing IL-1 by culturing peripheral blood monocytes or 
leukemic cells obtained from acute monocytic leukemia or acute 
myelomonocytic leukemia patients in a serum supplemented culture medium 
together with lipopolysaccharide ("LPS") to stimulate IL-1 production. 
Hollow fiber diafiltration and ultrafiltration were used to separate a 
lower molecular weight activity from most of the serum proteins. This 
lower weight activity was subjected to IEF in an Ampholine and sucrose 
gradient. From this procedure, the IL-1 activity was found to have a pI of 
about 6.8 to 7.2. The isoelectrofocused IL-1 activity was then subjected 
to SDS-PAGE which indicated that the human IL-1 being analyzed had a 
molecular weight of about 11,000 daltons. Lachman reported that the 
overall recovery of IL-1 activity from the above procedures was poor, in 
the range of about 4%. 
Applicants have purified IL-1 to homogeneity using a combination of 
ion-exchange chromatographic procedures together with dye-ligand binding 
chromatography. Use of these procedures resulted in elaboration of a 
17,500 dalton protein containing IL-1 activity. By trypsin degradation of 
the purified IL-1 protein, IL-1 peptides were liberated, one of which was 
subjected to amino acid sequence determination. Although applicants have 
been successful in purifying IL-1, the techniques developed for such 
purification remain costly and time consuming. 
The availability of adequate quantities of homogeneous human IL-1 could be 
valuable in investigations and possible treatment of autoimmune disorders 
such as arthritis and lupus erythematosis. Also, human IL-1 in greater 
purity and larger quantities than heretofore available, could prove useful 
in achieving successful wound and burn healing. 
One potential method of providing relatively large quantities of 
homogeneous human IL-1 is through recombinant DNA techniques. Recombinant 
DNA techniques have been developed for economically producing a desired 
protein once the gene coding for the protein has been isolated and 
identified. A discussion of such recombinant DNA techniques for protein 
production is set forth in the editorial and supporting papers in Vol. 196 
of Science (April, 1977). However, to take advantage of the recombinant 
DNA techniques discussed in this reference, the gene coding for human IL-1 
must first be isolated. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a gene coding for human IL-1 was 
isolated from a cDNA library with a synthetic oligonucleotide probe 
corresponding to a portion of the amino acid sequence of human IL-1. Total 
human RNA was extracted from cells thought to produce relatively high 
levels of IL-1. Polyadenylated mRNA was isolated from the total RNA 
extract. A cDNA library was constructed by reverse transcription of size 
separated polyadenylated mRNA with reverse transcriptase. The DNA was 
rendered double-stranded with DNA polymerase I and inserted into an 
appropriate cloning vector. Resultant recombinant cloning vectors were 
used to transform an appropriate host. 
Transformed hosts were identified and grouped into pools. Plasmid DNA 
prepared from these pools was hybridized with the oligonucleotide probe 
that had been radiolabeled. The pool(s) of clones that gave a positive 
signal to the probe was identified and then the putative pool subdivided 
and the hybridization screen repeated. By this procedure, a single 
transformant was eventually identified. Plasmid DNA was prepared from this 
transformant and characterized by restriction endonuclease digestion. The 
IL-1 gene was sequenced to establish its nucleotide and amino acid 
composition. Also the IL-1 gene was cloned in an E. coli/yeast cell system 
to express mature IL-1, and then biological assays were conducted to 
confirm that the expressed protein product is IL-1.

DESCRIPTION OF THE INVENTION 
Sources of Human IL-1 Producing Cells 
Preferably, a cDNA library, from which genes coding for human IL-1 will be 
sought, is constructed from cells previously found to produce relatively 
high levels of other lymphokines, under the assumption that they might 
also produce human IL-1. These sources may include malignant cell lines, 
such as acute myelogenous leukemia cells. 
Activated human peripheral blood adherent mononuclear cells also 
potentially may be a source of IL-1 molecules. For use in the present 
invention, the peripheral blood mononuclear cells can be separated from 
whole blood by standard techniques, such as by Ficoll-Hypaque 
centrifugation. Adherent cells are selected by plastic adherence and 
stimulated with Escherichia coli ("e. coli") LPS in vitro in a 
serum-containing medium. 
Applicants found that stimulation of such adherent cells with E. coli LPS 
leads to elaboration of significant quantities of IL-1. As set forth 
infra, applicants have successfully isolated the IL-1 gene from a cDNA 
library prepared from LPS activated adherent leukocytes. 
Preparation of RNA from Human IL-1 Producing Cells 
Total RNA from human potentially IL-1-producing cells is extracted by 
standard methods, such as disclosed by Chirgwin et al., 18 Biochemistry 
5294 (1979), and Maniatis et al., Molecular Cloning, a Laboratory Manual, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). 
As is well known, when extracting RNA from cells, it is important to 
minimize ribonuclease ("RNase") activity during the initial stages of 
extraction. One manner in which this is accomplished is to denature the 
cellular protein, including the RNase, at a rate that exceeds the rate of 
RNA hydrolysis by RNase. In the procedures of Chirgwin et al., supra, and 
Maniatis et al., supra at 196, this is carried out by use of guanidinium 
thiocyanate, together with a reducing agent, such as 2-mercaptoethanol (to 
break up the protein disulfide bonds). The RNA is isolated from the 
protein by standard techniques, such as phenol/chloroform extraction, 
ethanol precipitation or sedimentation through cesium chloride. 
Next, polyadenylated mRNA is separated from the extracted protein. Although 
several techniques have been developed to carry out this separation 
process, one preferred method is to chromatograph the polyadenylated mRNA 
on oligo (dT)-cellulose as described by Edmonds et al., 68 Proc. Natl. 
Acad. Sci. 1336 (1971); Aviv and Leder, 69 Proc. Natl. Acad. Sci. 1408 
(1972); and, Maniatis et al., supra at 197. The oligo (dT)-cellulose 
column is prepared with a loading buffer and then the mRNA applied to the 
column. Thereafter, the column is initially washed with a buffer solution 
to remove the non-polyadenylated mRNA and then the polyadenylated mRNA is 
eluted from the column with a buffered, low ionic strength eluant. The 
integrity of the polyadenylated mRNA is verified by gel electrophoresis. 
The polyadenylated mRNA is then sized by electrophoresis through 
methylmercury agarose and gel fractions corresponding to different size 
classes of mRNA are then translated in vitro, by use of standard rabbit 
reticulocyte lysates technique, such as described by: Palmiter, 248 J. 
Biol. Chem. 2095 (1973); Pelham and Jackson, 667 Eur. J. Biochem. 246 
(1976); and, Lee et al., 253 J. Biol. Chem. 3494 (1978). Kits for the 
rabbit reticulocyte assay are commercially available from many sources, 
such as from Bethesda Research Laboratories, Gaithersburg, Md. 
Alternatively, the mRNA translation can be carried out by microinjection 
of the mRNA into frog Xenopus laevis ("X. laevis") oocytes using standard 
techniques, such as described by Stoma et al., 79 Meth. Enzym. 68 (1981). 
Fluids liberated by either reticulocyte lysate translations, or by mRNA 
microinjected oocytes are then tested for the presence of IL-1 activity. 
mRNA gel fractions which, when translated in vitro gave rise to IL-1 
activity, are selected as a source of mRNA for cDNA construction. 
In the X. laevis oocyte translation procedure, approximately 50 nanoliters 
("nl") of mRNA (dissolved in sterile H.sub.2 O at a concentration of 0.5-1 
mg/ml) is injected into each oocyte. The oocytes are harvested from X. 
laevis (Nasco, Fort Atkinson, Wis.) and incubated in 150 ml of oocyte 
incubation medium (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO.sub.3, 0.82 mM 
MgSO.sub.4.7 H.sub.2 O, 0.33 mM Ca (NO.sub.3).sub.2 . 4H.sub.2 O, 0.41 mM 
CaCl.sub.2 . 6H.sub.2 O, 7.5 mM Tris base, 18 units/ml (11 ug/ml) 
penicillin G potassium, and 18 ug/ml streptomycin). The final pH of the 
medium is adjusted to 7.6 with HCl and then sterilized by filtration. 
After injection, the oocytes are placed in 0.1 ml of fresh oocyte 
incubation medium and incubated for 18 hours at 23.degree. C. in a 1.5 ml 
sterile conical polypropylene tube. After incubation, the oocytes are 
homogenized manually in the same conical tube and then the resulting 
extract is centrifuged. 
Thymocyte Proliferation Assay 
As noted above, mRNA translations by the rabbit reticulocyte lysates or the 
X. laevis oocytes are assayed by testing for the presence of IL-1 activity 
in the fluids liberated by the lysates or the oocyate extract. A first 
assay involves ascertaining the capacity of a sample of the lysate or 
oocyte extract to induce proliferation of thymocytes derived from CD-1 
mice. In this assay, approximately 1.times.10.sup.6 thymocyte cells, 
obtained from 10 to 12 week old CD-1 mice (Charles River Breeding 
Laboratories, Wilmington, Md.), re seeded in round bottom microplate wells 
(Corning Plastics, Corning, N.Y.) in the presence of three-fold serial 
dilutions of the IL-1 containing fluid samples. The thymocytes are 
cultured in 150 microliters ("ul") of MEM containing 50 U/ml penicillin, 
50 micrograms ("ug")/ml streptomycin, 2 millimolar ("mM") glutamine, 0.2 
mM gentamycin, 10 mM HEPES buffer, ("Supplemented MEM"), pH 7.4, together 
with 3% v/v human serum and 10.sup.-5 M 2-mercaptoethanol. The samples are 
cultured for 72 hours at 37.degree. C. in an atmosphere of 5% CO.sub.2 in 
air. Thereafter the cultures are pulsed for approximately 4 hours with 0.5 
microcuries ("uCi") of tritiated thymidine (".sup.3 H-Tdr"), (New England 
Nuclear, Boston, Mass., 2 Ci/mM specific activity), after which the 
cultures are harvested onto glass fiber filter strips, for instance with 
the aid of a multiple-automated sample harvester. .sup.3 H-Tdr 
incorporation is then measured by liquid scintillation counting. Details 
of this procedure are set forth by Gillis et al., 120 J. Immunol. 2027 
(1978) and in U.S. Pat. No. 4,411,992. 
By this thymocyte proliferation assay procedure, only the CD-1 thymocytes 
cultured in the presence of IL-1 incorporate .sup.3 H-Tdr in a dose 
dependent manner. CD-1 cells cultured in the absence of IL-1 incorporate 
only background levels of .sup.3 H-Tdr. IL-1 activity is calculated from 
the linear portion of the .sup.3 H-Tdr incorporation data in a manner 
similar to the procedure used by Gillis et al. supra, for determining 
interleukin-2 activity. Units of IL-1 activity are determined as the 
reciprocal dilution of a sample which generates 50% of maximal thymocyte 
.sup.3 H-Tdr incorporation as compared to a laboratory standard. For 
example, if a sample generates 50% of maximal thymocyte .sup.3 H-Tdr 
incorporation at a dilution of 1:15, then one unit ("U") of IL-1 is found 
in 1/15 of the 150 ul assay volume or 10 ul is said to contain one U of 
activity. The total sample would, therefore, contain 100 U [1,000 
(ul/ml).div.10 ul (per U)] of IL-1 activity/ml. See Gillis et al., supra. 
IL-1 Conversion Assay 
A second alternative assay for IL-1 activity may be employed which takes 
advantage of the fact that IL-1 was found by applicants to convert an 
interleukin 2 ("IL-2") nonproducer murine tumor cell line, LBRM-33-145, to 
an IL-2 producer. In this assay LBRM-33-aA5 cells, ATCC No. CRL-8079, are 
inactivated by addition of 50 ug/ml of mitomycin C and incubated for 1 
hour at 37.degree. C. 100 ul of the inactivated LBRM-33-1A5 cells 
(5.times.10.sup.5 cells/ml) are cultured in 96-well flat-bottomed plates 
in the presence of an equal volume of the mitogen, phytohemagglutinin 
("PHA") (1% final concentration) together with serial dilutions of IL-1 
containing fluid samples. At hourly time intervals the existence of IL-2 
activity, generated by IL-1 triggered, mitomycin C - inhibited LBRM-33-1A5 
cells (and thus IL-1 activity), is directly ascertained by adding 50 ul of 
IL-2 dependent CTLL-2 cells (8.times.10.sup.4 cells/ml). The microwell 
cultures are then incubated for 20 additional hours followed by a 4 hour 
pulse with 0.5 uCi of .sup.3 H-Tdr (New England Nuclear, Boston, Mass., 2 
Ci/mM specific activity). Thereafter, the thymidine-pulsed cultures are 
harvested onto glass fiber filter strips with the aid of a multiple 
automated sample harvester (MASH II; Microbiological Associates, Bethesda, 
Md.). .sup.3 H-Tdr incorporation is measured by liquid scintillation 
counting. Details of this procedure are set forth in Gillis et al. supra, 
and in U.S. Pat. No. 4,411,992. In this assay, only the CTLL-2 cells 
cultured in the presence of IL-2 incorporate .sup.3 H-Tdr in a dose 
dependent manner. CTLL-2 cells cultured in the absence of IL-2 (and thus 
IL-1) incorporate only background levels of .sup.3 H-Tdr. This 
"conversion" assay has the advantage of being quicker (completion within 
24 hours) and approximately 1000 to 10,000 times more sensitive than the 
above-discussed thymocyte proliferation assay. Nevertheless, both the 
"conversion" and "proliferation" assays may be employed in conjunction 
with the present invention. 
Preparation of cDNA from mRNA 
A library of double-stranded cDNA corresponding to the mRNA, as prepared 
and assayed above, is constructed by known techniques employing the enzyme 
reverse transcriptase. One such procedure which may be employed in 
conjunction with the present invention is detailed by Maniatis et al., 
supra at 230. Briefly, the polyadenylated mRNA is reverse transcribed by 
using oligo-dT, that has been hybridized to the polyadenylated tail of the 
mRNA, as a primer for a first cDNA strand. This results in a "hairpin" 
loop at the 3' end of the initial cDNA strand that serves as an integral 
primer for the second DNA strand. Next, the second cDNA strand is 
synthesized using the enzyme DNA polymerase I and the hairpin loop is 
cleaved by S1 nuclease to produce double-stranded cDNA molecules. The 
double-stranded cDNA is fractionated by any convenient means to remove the 
shorter strands, thereby avoiding the needles cloning of small cDNA 
fractions. 
It is to be understood that in accordance with the present invention, 
alternative standard procedures may be employed to prepare double-stranded 
cDNA from mRNA. One such alternative technique is disclosed by Land et 
al., 9 Nucl. Acids Res. 2251 (1981). In the Land et al. protocol, the 
hairpin loop is not used as a primer for the second cDNA strand. Rather, 
the 3' end of the first cDNA strand is tailed with dCMP residues using 
terminal deoxynucleotidyl transferase ("TdT"). This produces a 3' tail of 
poly-C residues. Then the synthesis of the second strand is primed by 
oligo-dG hybridized to the 3' tail. This technique is said to help avoid 
losing portions of the 5' tail of the second cDNA strand which might occur 
if the hairpin is cleaved with S1 nuclease, as in the Maniatis et al. 
protocol. 
Cloning of cDNA 
Next, the double-stranded cDNA is inserted within a cloning vector which is 
used to transform compatible prokaryotic or eukaryotic host cells for 
replication of the vector. Thereafter, the transformants are identified 
and plasmid DNA prepared therefrom. 
To carry out the present invention, various cloning vectors may be 
utilized. Although the preference is for a plasmid, the vector may be a 
bacteriophage or a cosmid. If cloning occurs in mammalian cells, viruses 
also can be used as vectors. 
If a plasmid is employed, it may be obtained from a natural source or 
artificially synthesized. The particular plasmid chosen should be 
compatible with the contemplated transformation host, whether a bacteria 
such as E. coli, yeast or other unicellular microorganism. The plasmid 
should have the proper origin of replication for the particular host cell 
to be employed. Also, the plasmid should have a phenotypic property that 
will enable the transformed host cells to be readily identified and 
separated from cells that do not undergo transformation. Such phenotypic 
characteristics can include genes providing resistance to growth 
inhibiting substances, such as an antibiotic. Plasmids are commercially 
available that encode genes resistant to various antibiotics, including 
tetracycline, streptomycin, sulfa drugs, penicillin and ampicillin. 
If E. coli is employed as the host cell, many possible cloning plasmids are 
commercially available which may be used in conjunction with the present 
invention. A preferred plasmid for performing the present invention is 
pBR322. This plasmid has ben fully sequenced, as set forth in Sutcliffe, 
43 Cold Spring Harbor Symp. Quant. Biol. 77 (1979). A significant 
advantage of this plasmid is that it has 11 known unique restriction 
sites, including the Pst I site in the ampicillin resistant gene. This 
feature is particularly useful for cloning by the homopolymer tailing 
method. 
If a bacteriophage is used instead of a plasmid, such phages should have 
substantially the same characteristics noted above for selection of 
plasmids. This includes the existence of a phenotypic marker and ligatable 
termini for attachment of foreign genes. 
Preferably, in the present invention, the double-stranded cDNA, having 
blunt ends, is inserted into a plasmid vector by homopolymeric tailing. As 
is well known in the art, in this technique, complementary homopolymer 
tracks are added to the strands of the cDNA and to the plasmid DNA. The 
vector and double-stranded cDNA are then joined together by hydrogen 
bonding between complementary homopolymer tails to form open, circular 
hybrid molecules capable of transforming host cells, such as E. coli. 
In one procedure for homopolymeric tailing, approximately 50 to 150 dA 
nucleotide residues are added to the 3' ends of linearized plasmid DNA. A 
similar number of dT nucleotide residues are added to the 3' ends of the 
double-stranded cDNA and then the cDNA and plasmid joined together. 
In an alternative and preferred method, dG tails are added to the 3' ends 
of the cloning vector that has been cleaved with an appropriate 
restriction enzyme. For instance, if the pBR322 plasmid is employed, the 
restriction enzyme Pst I may be used to digest the plasmid at the 
ampicillin resistant gene. Complementary dC tails are added to the 3' ends 
of the double-stranded cDNA prior to insertion of the cDNA segment in the 
plasmid with an appropriate annealing buffer. 
It is to be understood that the double-stranded cDNA may be inserted within 
plasmid cloning vectors by other various standard methods. One such 
alternative technique involves attaching synthesized nucleotide linkers to 
the ends of the cDNA strands by using DNA ligase. The linkers are cleaved 
with a restriction enzyme to generate cohesive termini for insertion 
within a plasmid cleaved with the same restriction enzyme. Scheller et 
al., 196 Science 177-180 (1977); Maniatus et al., supra at 219. 
The recombinant DNA plasmids, as prepared above, are used to transform host 
cells. Although the host may be any appropriate prokaryotic or eukaryotic 
cell, it is preferably a well-defined bacteria, such as E. coli or a yeast 
strain. Such hosts ar readily transformed and capable of rapid growth in 
culture. Other forms of bacteria, such as salmonella or pneumococcus, may 
be substituted for E. coli. In place of bacteria, other unicellular 
microorganisms may be employed, for instance, fungi and algae. Whatever 
host is chosen, it should not contain a restriction enzyme that would 
cleave the recombinant plasmid. 
If E. coli is employed as a host, preferable strains are MM294 and RR1. 
Protocols for transformation of the MM294 host by a plasmid vector are 
well known, as set forth in Maniatis et al., supra at 255; and, Hanahan, 
166 J. Mol. Biol. 557 (1983). Protocols for transformation of the RR1 host 
by a plasmid vector are also well known as set forth in Bolivar et al., 2 
Gene 95 (1977) and Peacock et al., 655 Biochem. Biophys. Acta. 243 (1981). 
Other strains of E. coli which also could serve as suitable hosts include 
DH1 (ATCC No. 33849) and C600. These strains and the MM294 and RR1 strains 
are widely commercially available. 
In transformation protocols, including those disclosed by Maniatis et al., 
supra, and Hanahan, supra, only a small portion of the host cells are 
actually transformed, due to limited plasmid uptake by the cells. The 
cells that have been transformed can be identified by placing the cell 
culture on agar plates containing suitable growth medium and a phenotypic 
identifier, such as an antibiotic. Only those cells that have the proper 
resistance gene (e.g., to the antiobiotic) will survive. If the 
recombinant pBR322 plasmid is used to transform E. coli strain MM294, 
transformed cells can be identified by using tetracycline as the 
phenotypic identifier. 
Preparation of a Synthetic Oligonucleotide Screening Probe 
A radiolabeled synthetic oligonucleotide corresponding to a portion of the 
known amino acid sequence of human IL-1 is used as a probe to screen the 
cDNA library. The hybridization of the synthetic oligonucleotide probe 
with plasmid cDNA prepared from the library clones is subsequently 
identified by autoradiography. 
The N-terminal portion of the amino acid composition of the IL-1 molecule 
was initially sequenced by applicants and found to be composed of the 
residues: NH.sub.2 
-Ala-Pro-Val-Arg-Ser-Leu-Asn-Cys-Thr-Leu-Arg-Asp-Ser-Gly-Gln-Lys-Ser-Leu-V 
al-Met-Ser-Gly-Pro-Tyr-Glu-Leu-Lys-Ala-Leu-His-Leu-Gln-Gly 
-Gln-Asp-Met-Glu-Gln-Gln-Val, is employed as the basis for the synthetic 
oligonucleotide probe. This particular portion of the amino acid sequence 
of IL-1 has the advantage of being short enough to be relatively easily 
chemically synthesized, while also being long enough to provide sufficient 
information to be useful in preparing a direct probe for the IL-1 gene. 
Also, this sequence corresponds to a particular codon composition that is 
relatively free of ambiguity. 
Applicants developed a synthetic oligonucleotide from the above amino acid 
sequence for use as a probe to screen plasmid DNA thought to contain the 
IL-1 gene. The probe is composed of the following sequence which 
corresponds to the antisense sequence coded for by the above amino acid 
sequence downstream from the Met residue: 5'-AC TTG TTG TTC CAT GTC TTG 
GCC TTG CAG GTG CAG GGC TTT CAG TTC GTA GGG GCC GGA CAT-3'. This probe has 
the advantage of being short enough to be easily synthesized, while being 
long enough to contain sufficient information to be useful as a probe for 
the IL-1 gene. 
Although the described oligonucleotide sequence is a preferred composition 
of the synthetic probe of the present invention, it is to be understood 
that probes of other compositions corresponding to other segments of amino 
acid sequence of the IL-1 molecule can be employed without departing from 
the spirit or scope of the present invention. 
The synthetic oligonucleotide probes may be chemically synthesized by 
well-known techniques, such as by phosphodiester or triester methods. The 
details of the triester synthesis technique are set forth in Sood et al., 
4 Nucl. Acid Res. 2557 (1977); and, Hirose et al., 28 Tet. Lett. 2449 
(1978). After synthesis, the oligonucleotide probe is labeled with T4 
polynucleotide kinase and .sup.32 P-ATP. A standard protocol from the 
labeling procedure is set forth in Maniatis et al., supra at 122. 
Advantageously, the oligonucleotide probes can be synthesized with OH 5' 
termini thereby avoiding the phosphatase procedure typically required. 
Screening of cDNA Library 
In the screening procedure of the present invention, the transformants are 
pooled into groups each composed of approximately 2,000 transformants. The 
replicated plasmids are extracted from the transformants using any one of 
several well-known techniques, such as by alkaline lysis. Plasmid DNA is 
prepared by cleaving the plasmids at the Pvu II and Hind III restriction 
sites, both being unique sites on the hybrid plasmid. The resulting DNA 
segments are fractionated by electrophoresis on agarose gel and then 
directly analyzed by Southern blotting as described in Southern, 98 J. 
Mol. Biol. 503 (1975). The DNA that binds to the nitrocellulose filter in 
the Southern blotting procedure is hybridized with the labeled 
oligonucleotide probe. The specific DNA fragments that hybridize to the 
probe are identified by autoradiography. 
The particular pool(s) of clones that give a signal following 
autoradiography are plated out and used in direct bacterial colony 
hybridization on a nitrocellulose filter with the same above-identified 
oligonucleotide probes. After completion of the hybridization, the 
nitrocellulose filter is monitored by autoradiography to identify a 
positive colony. In the present invention, applicants discovered one such 
positive colony. Plasmid DNA designated as IL-1 Z-14 is prepared from the 
particular positive colony identified. 
Characterization of Screened cDNA 
The plasmid DNA prepared above is characterized by restriction enzyme 
mapping. Various strategies for restriction enzyme mapping are discussed 
by Maniatis et al., supra at 374. One standard technique involves the 
partial digestion of end-labeled fragments of linear DNA. This technique 
was developed by Smith and Birnstiel, 3 Nucl. Acids Res. 2387 (1976). A 
partial restriction enzyme map of the IL-1 X-14 plasmid in the region of 
the IL-1 gene is shown in FIG. 1. The distance between restriction sites 
is given in base pairs 9"bp"). The Pst I restriction sites shown in the 
brackets are those generated by the cloning procedures. 
The mapped plasmid cDNA illustrated in FIG. 1 was sequenced using the 
chain-termination method. This method of nucleotide sequencing was 
originated by Sanger et al., 70 Proc. Natl. Acad. Sci. (USA) 5463 (1977). 
See U.S. Pat. No. 4,322,499. Methods for chain-termination sequence 
determination are set forth in the Amersham Handbook entitled, M13 Cloning 
and Sequencing, Blenheim Cresent, London (1983) (hereinafter "Amersham 
Handbook"); Messing, 2 Recombinant DNA Technical Bulletin, NIH Publication 
No. 79-99, 2, 43-48 (1979); Norrander et al., 26 Gene 101 (1983); Cerretti 
et al., 11 Nucl. Acids Res. 2599 (1983); and, Biggin et al., 80 Proc. 
Natl. Acad. Sci. (USA) 3963 (1983). M13 filamentous phage are employed as 
vectors to clone the DNA sequences of interest. These phage vectors 
provide single-stranded DNA templates which are readily sequenced by the 
chain-termination method, which involves priming a single-stranded 
template molecule with a short primer strand having a free 3' hydroxyl 
group and then using DNA polymerase to copy the template strand in a chain 
extension reaction using all four deoxyribonucleotide triphosphates, i.e., 
dATP, dCTP, dGTP, and dTTP (collectively referred to as "dNTPs"), with one 
of them being radiolabeled. In the synthesis reaction, a nucleotide 
specific chain terminator lacking a 3'-hydroxyl terminus, for instance, a 
2',3' dideoxynucleotide triphosphate ("ddNTP"), is used to produce a 
series of different length chain extensions. The terminator has a normal 
5' terminus so that it can be incorporated into a growing DNA chain, but 
lacks a 3' hydroxyl terminus. Once the terminator has been integrated into 
the DNA chain, no further deoxynucleotide triphosphates can be added so 
that growth of the chain stops. Four separate synthesizing reactions are 
carried out, each having a ddNTP of one of the four nucleotide dNPTs, 
i.e., dATP, dCTP, dGTP and dTTP. One of the normal dNTPs is radiolabeled 
so that the synthesized strands after having been sorted by size on a 
polyacrylamide gel, can be autoradiographed. The chain extensions from the 
four reactions are placed side by side in separate gel lanes so that the 
pattern of the fragments from the autoradiography corresponds to the DNA 
sequence of the cloned DNA. 
The DNA and corresponding amino acid sequences of the plasmid cDNA in FIG. 
1, as determined by the above techniques, is illustrated in FIG. 2. The 
nucleotides are numbered from the beginning of the sequence shown in FIG. 
2. The amino acids are numbered beginning from the mature NH.sub.2 
-terminus of the IL-1 protein, i.e., the Ala residue, marked with a star, 
and extending to the Ser residue (No. 153) located adjacent the 
termination codon TAA. The coding region of the IL-1 gene, extending from 
the Ala codon to the TAG termination codon, is shown as a box portion in 
FIG. 1. The restriction enzyme cleaving sites identified in FIG. 1 ar also 
indicated in FIG. 2. 
In preparation for the sequencing procedures, the plasmid cDNA section 
shown in FIG. 1 is digested with various restriction endonucleases and 
then the resulting DNA fragments cloned into M13 phage vectors to form 
single stranded DNA templates. A universal primer is used to sequence 
upstream and downstream from intermediate locations of the sense and 
antisense strands. Rather than relying on the sequencing results obtained 
from sequencing the entire length of the fragments with a single chain 
termination procedure, additional synthetically produced primers are used 
to initiate the chain termination procedure from other intermediate 
locations along the lengths of the strands. By this process, both strands 
of the plasmid cDNA shown in FIG. 1 are sequenced in overlapping fashion, 
thereby serving to redundantly confirm the sequences. 
It is to be understood that rather than employing the chain-termination 
technique outlined above, other known methods may be utilized to sequence 
the IL-1 gene without departing from the spirit or scope of the present 
invention. For instance, the chemical degradation method of Maxam and 
Gilbert as set forth in 74 Proc. Nat'l Acad. Sci. (USA) 560 (1977) can be 
used. 
Amino acid sequences studies of IL-1 prepared as above and purified were 
conducted according to the method of Stern et al., Proc. Natl. Acad. Sci. 
(USA) 871 (1984). The endopeptidase, cyanogen bromide was used to cleave 
the IL-1 at the methionine residues and then the resulting fragments 
analyzed by standard Edman degradation method. By this procedure, 
applicants have confirmed that the C-terminal of the IL-1 protein is 
composed of the amino acid sequence: Gln-Phe-Val-Ser-Ser. This establishes 
that the "natural" IL-1 is not processed by removal of amino acids from 
this end of the molecule after translation from mRNA. This is important 
because it is clear that much of the RNA sequence is removed from the 
N-terminus of the IL-1 gene during the maturation of IL-1 from its 
precursor. 
Expression of Functional IL-1 From cDNA Clone 
To determine whether the cDNA coding region of the IL-1 X-14 clone could 
encode functional IL-1, the clone is expressed in a prokaryotic/eukaryotic 
host system. A hybrid cDNA fragment containing the coding region of the 
IL-1 X-14 clone is inserted into a shuttle expression vector having two 
sets of replication sequences, a first sequence for amplification of the 
vector in prokaryotic host cells, and a second sequence for high level 
expression of the foreign structural protein, i.e., IL-1, in eukaryotic 
host cells. The transformed eukaryotic host cells are harvested and 
assayed for expression of mature IL-1 by use of the above detailed 
thymocyte proliferation assay and IL-2 conversion assay. 
Various types of shuttle vectors have been developed. A common type 
includes an origin of replication and promoter sequences that signal DNA 
replication in prokaryotic cells, typically E. coli and a comparable 
origin of replication and promoter sequences that signal DNA replication 
in eukaryotic cells, most commonly yeast cells. The shuttle vector also 
includes a phenotypic marker, such as a drug resistant gene, for selection 
of the transformed prokaryotic cells. The shuttle vector has a comparable 
phenotypic marker change for selection of transformed eukaryotic cells. 
Ideally, for high level expression of IL-1, all protein coding sequences 
are removed from the eukaryotic promoter sequence to avoid expression of 
an undesired protein. Also, to this end, a natural or synthetic initiator 
codon sequence, i.e., ATG, is attached to the 5' end of the inserted 
coding region of the IL-1 gene. 
A preferable shuttle vector for carrying out the present invention is 
designated as pY ADH. As illustrated schematically in FIG. 3, the pY ADH 
plasmid includes an origin of replication (from plasmid pBR322) for high 
copy DNA expression in E. coli, and an ampicillin ("Amp.sup.R ") resistant 
gene for selection of transformed E. coli cells. The shuttle vector also 
includes the 2u circle origin of replication and a yeast Trp I gene for 
selection of transformed yeast hosts in yeast (trp minus) trp auxotrophs. 
The shuttle vector further includes the yeast promoter sequence from the 
alcohol dehydrogenase gene ("ADH") for propagation of the plasmid in both 
yeast and E. coli hosts. This promoter sequence is especially advantageous 
for use in the present invention due to the high level expression of this 
gene in yeast, and because the complete DNA sequence of this gene is 
known. All protein coding sequences, including the initiator ATG codon, 
have been removed from the ADH promoter fragment. The pY ADH shuttle 
vector includes a number of unique substrate sites for cleavage with 
restriction enzymes, i.e., Eco RI and Stu I. 
As illustrated in FIG. 3, the pY ADH IL-1 plasmid is prepared as an 
expression vector for expression of IL-1 gene by insertion of the coding 
region of the IL-1 gene in plasmid pY ADH. Samples of this shuttle vector 
are on deposit with the American Type Culture Collection ("ATCC"), 12361 
Parklawn Drive, Rockville, Md. 20852, under Accession No. 39967. The 
coding region of the IL-1 gene is removed from the cDNA plasmid, prepared 
above. Due to the absence of a unique restriction enzyme cleavage site at 
precisely the 5' end of the coding region of the IL-1 gene, a major 
portion of the coding region is cleaved from the plasmid cDNA with the 
restriction enzymes Hpa II and Pst I. The Hpa II site is located slightly 
downstream from the 5' end of the gene coding region. Thereafter, a 
synthetic oligonucleotide containing the cleaved 5' end of the gene is 
chemically synthesized with a Hpa II cohesive 3' terminal for convenient 
ligation to the "natural" major IL-1 cDNA fragment. Since, as noted 
above, all protein coding sequences were removed from the ADH promoter 
sequence, the synthetic oligonucleotide is synthesized with an ATG 
initiation codon at its 5' end. 
The IL-1 cDNA fragment together with the synthetic oligonucleotide are 
inserted in shuttle vector pY ADH which previously has been digested with 
appropriate restriction enzymes corresponding to the configurations of the 
5' terminal of the synthetic oligonucleotide and the 3' terminal of the 
major IL-1 cDNA fragment. The resultant recombinant shuttle vector pY ADH 
IL-1 is used to transform a prokaryotic host, e.g., E. coli, for high copy 
amplification of the shuttle vector. After this initial transformation 
process, the recombinant shuttle vector is isolated from the E. coli host 
and then employed to transform a eukaryotic host, e.g., yeast cells for 
high level expression of IL-1. The transformed yeast hosts are harvested 
and the resulting supernatant is assayed for biological activity utilizing 
the above described thymocyte proliferation and/or IL-1 conversion assays. 
The processes and products of the present invention are further illustrated 
by the following examples. 
EXAMPLE 1 
Preparation of Polyadenylated mRNA 
Leukocyte concentrates of a volume of 350-400 ml, obtained from human whole 
blood (mixture from Portland, Ore. Red Cross), were mixed with and diluted 
in Ca.sup.++, Mg.sup.++ free phosphate buffered saline ("PBS") layered 
onto Histopaque (Sigma Chemical Company, St. Louis, Mo.) and then 
centrifuged at 600.times.g for 30 minutes at room temperature. The 
interface layer, consisting of the leukocytes, was recovered, washed with 
PBS and centrifuged at 400.times.g for 10 minutes at room temperature. The 
cells were washed two more times in Ca.sup.++, Mg.sup.++ free PBS and 
centrifuged at 200.times.g for 10 minutes after each washing. 
Cells were then added to plastic culture flasks in Roswell Park Memorial 
Institute ("RPMI")-1640 medium together with 10% fetal bovine serum (v/v). 
Following a two-hour incubation at 37.degree. C., nonadherent cells were 
decanted and the flasks were then replenished with additional serum 
supplemented RPMI-1640 medium containing 20 ug/ml E. coli LPS at 20 ug/ml. 
Sixteen hours later, adherent LPS stimulated cells were harvested for RNA. 
Total RNA was extracted from the adherent mononuclear cells by the method 
as described by Chirgwin et al., supra. In this procedure guanidinium 
thiocyanate was used to denature the cellular protein including the RNase 
at a rate that exceeds the rate of RNA hydrolysis by RNase. The mRNA was 
removed from the cellular protein by ultracentrifugation through a dense 
cushion of cesium chloride. 
Thereafter, polyadenylated mRNA was separated from the extracted protein on 
an oligo (dT)-cellulose chromatography column using the method disclosed 
by Maniatis et al., supra at 197. Briefly, the column was prepared with 
application buffer composed of 20 mM Tris-Cl (pH 7.6), 0.5M NaCl, 1 mM 
ethylene diamine tetraacetate ("ETDA") and 0.1% sodium dodecyl sulfate 
("SDS"). The protein pellet was dissolved in water and application buffer 
and then loaded onto the column. The nonadsorbed material was eluted by 
initial washings with application buffer followed by additional washings 
with application buffer containing 0.1M NaCl. The retained polyadenylated 
mRNA was eluted with buffers of reduced ionic strength composed of 10 mM 
Tris-Cl (pH 7.5), 1 mM EDTA and 0.05% SDS. The eluted polyadenylated mRNA 
was precipitated at -20.degree. C. with 1/10 volume sodium acetate (3M, pH 
5.2) and 2.2 volumes of ethanol. After elution of the polyadenylated mRNA 
from the oligo (dT)-cellulose column, the integrity of the polyadenylated 
mRNA was confirmed by electrophoresis through agarose gels as detailed in 
Maniatis et al., supra at 199. 
The polyadenylated mRNA was sized by electrophoresis through methylmercury 
agarose. Gel fractions corresponding to different size classes of mRNA 
were then translated in vitro, either by use of rabbit reticulocyte 
lysates or by injection in frog X. laevis oocytes as described above. 
Fluids liberated by either reticulocyte translations or by mRNA injected 
oocytes were then tested for the presence of IL-1 activity using the 
assays set forth above. mRNA gel fractions which, when translated in vitro 
gave rise to IL-1 activity, were selected as a source of mRNA for cDNA 
construction. 
EXAMPLE 2 
Construction of cDNA Library 
A library of double-stranded cDNA corresponding to the mRNA was prepared 
from the purified mRNA in Example 1 by employing the standard procedure 
detailed by Maniatis et al., supra at 229. Oligo-dT was hybridized to the 
polyadenylated tail of the mRNA to serve as the primer for the reverse 
transcription of the first cDNA strand. The enzyme avian myeloblastosis 
virus ("AMV") reverse transcriptase synthesized the first DNA strand by 
using the mRNA as a template. This procedure resulted in a hairpin loop 
being formed at the 3' end of the initial cDNA strand that served as an 
integral primer for the second cDNA strand. After the mRNA strand had been 
degraded with NaOH, the second cDNA strand was synthesized with DNA 
polymerase I. The hairpin was then removed with nuclease S1 to produce 
double-stranded cDNA molecules. 
The double-stranded cDNA was fractionated into size classes by Sephacryl 
S-400 (Pharmacia Fine Chemicals) column chromatography and monitored by 
analysis using alkaline agarose electrophoresis employing end-labeled 
fragments of pBR322 DNA as molecular-weight markers. DNA strands having a 
length of less than 500 bp were culled out to avoid needles cloning of 
these undersized cDNA fractions. 
The double-stranded cDNA fractions, as prepared above, were inserted into 
the Pst I site of the pBR322 plasmid (Pharmacia Fine Chemicals) by the 
method disclosed by Maniatis et al., supra, beginning at 239. In this 
procedure the double-stranded cDNA was tailed with poly (dC) at its 3' 
ends. The plasmid pBR322 was digested with Pst I endonuclease and then 
tailed with poly (dG) at its 3' ends. The tailed plasmid DNA and the 
tailed cDNA were annealed with annealing buffer (0.1M NaCl, 10 mM Tris-Cl 
(pH 7.8) and 10 mM ETDA) to form novel recombinant plasmids. All 
restriction enzymes described herein are commercially available from New 
England Biolabs, Beverly, Mass. 
The recombinant plasmids were transformed into E. coli strain MM294 by 
using the procedure of Hanahan, supra in which the E. coli cells were 
prepared by growth in elevated levels of Mg.sup.2+. The transformation 
hosts were plated and then transformants were identified by use of 
tetracycline as a phenotypic identifier. By use of this technique, 
applicants obtained approximately 2.times.10.sup.6 independent 
transformants. 
EXAMPLE 3 
Preparation of Synthetic Oligonucleotide Screening Probes 
A synthetic oligonucleotide was employed as a probe in screening the cDNA 
library prepared as set forth above in Example 2. The probe was composed 
of the following composition: 5' AC TTG TTG TTC CAT GTC TTG GCC TTG CAG 
GTG CAG GGC TTT CAG TTC GTA GGG GCC GGA CAT 3'. The oligonucleotide probe 
was chemically synthesized by triester method as detailed by Sood et al., 
supra and Hirose et al., supra. 
After chemical synthesis has been completed, the 5' ends of the 
oligonucleotide probes were labeled with .sup.32 P. To facilitate 
labeling, the 5' ends of the oligonucleotide were synthesized with OH 
termini, thereby eliminating the phosphatase treatment which typically 
must be employed when labeling DNA fragments. The labeling protocol 
included adding 1 ul of the synthetic oligonucleotides to 16 ul of .sup.32 
P - ATP (3000 ci/mM), 1 microliter ("ul") (10 U) of T4 polynucleotide 
kinase and 2 ul of 10.times.kinase buffer I. The 10.times.kinase buffer I 
was composed of 0.5M Tris-Cl (pH 7.6), 0.1M MgCl.sub.2, 50 mM 
dithiothreitol, 1 mM spermidine and 1 mM ETDA. The reaction was carried 
out at 37.degree. C. for 30 minutes, and thereafter the synthesized 
oligonucleotides were extracted with phenol/chloroform. The labeled probes 
were separated from unlabeled oligonucleotides by chromatography on or 
centrifugation through Sephadex G-50 columns (Pharmacia Fine Chemicals). 
EXAMPLE 4 
Screening of cDNA Library 
To facilitate initial screening of the cDNA library prepared in Example 2 
above, the transformed bacteria cultures were pooled into groups each 
having approximately 2,000 transformants of different clones. Plasmid DNA 
was removed from samples of the host bacteria by standard alkaline lysis 
method detailed by Ish-Horowicz and Burke, 9 Nucl. Acids Res. 2989 (1981). 
The isolated plasmids were separated into two fragments. This was 
accomplished by initially digesting the plasmids to completion with Pvu II 
and Hind III. To this end, the plasmids were redissolved in 20 ul of 
1.times.Hind III buffer (7 mM Tris, (pH 7.4), 7 mM magnesium chloride, 60 
mM NaCl) and then 1 ul of Pvu II and 1 ul of Hind III restriction 
endonucleases are added. This mixture was incubated at 37.degree. C. for 
two hours. 
Next, the plasmid digests were fractionated by electrophoresis through 0.8% 
agarose gel with markers of appropriate size. The agarose gel was blotted 
onto nitrocellulose filter using the standard method described by 
Southern, supra. After the transfer process, the filter was air dried and 
baked for two hours at approximately 80.degree. C. under a vacuum to bind 
the DNA fragments to the nitrocellulose. 
The bound DNA was next hybridized with the labeled oligonucleotide probes. 
Briefly, the baked nitrocellulose was presoaked in 6.times.saline sodium 
citrate ("SSC") (20 X SSC is composed of 175.3 g of NaCl and 88.2 g of 
sodium citrate in 800 ml of H.sub.2 O, with pH adjusted to 7.0 with 10N 
NaOH) and then incubated at 50.degree. C. for 2-4 hours in 
prehybridization buffer composed of 6.times.SSC, 0.5% NP40 detergent, 0.1% 
sarcosyl, 5.times.Denhardt's solution (0.02% Ficoll, 0.02% polyvinyl 
pyrrolidone, 0.02% BSA) and 100 ug/ml denatured salmon sperm DNA (Sigma 
Type III, sodium salt). The filter was then incubated overnight at 
50.degree. C. with the .sup.32 P-labeled oligonucleotide probe (10.sup.6 
cpm/ug) (from Example 3) in hybridizing solution as above. After overnight 
hybridization, the filter was washed extensively with 6.times.SSC at room 
temperature and then for 5 minutes at 50.degree. C. with 6.times.SSC. 
After air drying, the filter was subjected to autoradiography at 
-70.degree. C. 
From the autoradiography, applicants found several hybridizing bands. The 
pool of clones from which the plasmid DNA that produced the hybridizing 
bands was plated out and then used in direct bacterial colony 
hybridization on nitrocellulose paper with the labeled oligonucleotide 
probe under the same hybridizing conditions as above. By this process, a 
single positive colony was identified. 
EXAMPLE 5 
Restriction Enzyme Mapping of Screened cDNA 
Plasmid, designated as IL-1 X-14, was prepared from the identified positive 
colony by the procedures set forth in Example 4. Samples of the IL-1 X-14 
plasmid transformed into E. coli strain RR1 are on deposit with the ATCC 
under Accession No. 39925. Thereafter, the IL-1 X-14 plasmid was analyzed 
by restriction enzyme mapping using the technique developed by Smith and 
Birnstiel, supra, involving partial digestion of end-labeled fragments of 
the linearized DNA. The DNA fragments were labeled at their 5' termini 
with .sup.32 P-phosphoryl groups using polynucleotide kinase and .sup.32 P 
- ATP. The labeled DNA strands were then cleaved asymmetrically with a 
suitable restriction enzyme to provide two fragments, each labeled at only 
one of its ends. These labeled fragments were isolated by gel 
electrophoresis. Each of the two fragments was partially digested by 
appropriate restriction enzymes. Although a large spectrum of digestion 
fragments may be produced, the labeled fragments form a simple overlapping 
series each having a common labeled terminus. These fragments were 
fractionated by gel electrophoresis and then examined by autoradiography. 
The locations of the fragments on the gel correspond directly to the order 
of the restriction sites along the plasmid DNA. 
By this procedure, applicants have partially mapped the restriction sites, 
as shown in FIG. 1, of the IL-1 X-14 plasmid in the region of the IL-1 
gene. The numbers shown between the restriction sites of the gene 
correspond to the approximate distances between the sites, in base pairs. 
EXAMPLE 6 
Sequencing of Screened cDNA 
The DNA segment shown in FIG. 1 was sequenced by the dideoxy 
chain-termination method essentially as described in the Amersham Handbook 
supra, with the variations set forth below. The DNA segment was digested 
with Hind III and Pst I restriction endonucleases and then the resulting 
DNA fragments were cloned into strains mp18 and mp19 of the M13 
single-stranded filamentous phage vector (Amersham, Arlington Heights, 
Ill.). The mp18 and mp19 phage vectors, as set forth in Norrander et al. 
supra, contain the following unique cloning sites: Hind III; Sph I; Pst I; 
Sal I; Acc I; Hinc II; Xba I; BamHI; Xma I; Sma I; Kpn I; Sst I; and, 
EcoRI. The composition of the mp18 and mp19 vectors are identical, with 
the exception that the order of the above-identified restriction sites are 
reversed in the mp19 vector so that both strands of a DNA segment may be 
conveniently sequenced with the two vectors. The mp18 mp19 vectors, with 
fragments of the cDNA segment of FIG. 1 inserted therein, were used to 
transform E. coli MJ103 and JM105 of the strain K12 (Bethesda Research 
Laboratories, Bethesda, Md.) to produce replicate single-stranded DNA 
templates containing single-stranded inserts of the sense and antisense 
strands. 
The synthetic universal primer: 5'-CCCAGTCACGACGTT-3' (P-L Biochemicals, 
Milwaukee, Wis.), was annealed to the single-strand DNA templates and used 
to prime DNA synthesis upstream and downstream from a location between 
nucleotides 476 and 477 (FIG. 2) as described above at page 16. 
Thereafter, the extension fragments were size-separated by gel 
electrophoresis and autoradiographed from which the nucleotide sequences 
of the fragments were deduced. Three additional primers were employed to 
prime synthesis from intermediate locations along the sense strands of the 
DNA segment in FIG. 2. A primer having the composition: 
5'-CTGGAGAGTGTAGATCC-3', corresponding to nucleotides 671 through 688 
(FIG. 2), was used to prime synthesis of the sense strand in the 
downstream direction from nucleotide No. 688. The composition of this 
primer strand was established from the sequencing information previously 
obtained by use of the universal primer. A second synthetic primer of the 
composition: 5'-GATATAACTGACTTCAC-3' (corresponding to nucleotides 851 
through 868 in FIG. 2) was used in sequencing the sense strand in the 
downstream direction from nucleotide No. 868. A third primer having the 
sequence: 5'-GATTCGTAGCTGGATGC-3' (corresponding to nucleotides No. 235 
through No. 218) was employed to sequence the antisense strand in the 
upstream direction from nucleotide No. 218. 
By the above "walk down" method, both strands of the plasmid cDNA in FIG. 1 
were sequenced in an overlapping, redundant manner thereby confirming 
their nucleotide sequence. It is to be understood that other synthetic 
primers could have been employed to initiate chain extensions from other 
locations along the strands without departing from the scope of the 
present invention. The above primer strands were chemically synthesized by 
triester method as detailed by Sood et al., supra and Hirose et al., 
supra. It is to be understood, however, that other well-known techniques, 
such as by phosphodiester method, may be employed to synthesize the primer 
strands. 
Deoxyadenosine 5' (alpha-[.sup.35 S] thio) triphosphate (hereinafter "dATP 
[alpha-.sup.35 S]") was used as the radioactive label in the dideoxy 
sequencing reactions. Also, rather than using the gel set forth at page 36 
of the Amersham Handbook, a 6% polyacrylamide gel was employed (6% 
polyacrylamide gel, 0.4 mm thick, containing 7 M, urea 100 mM Tris borate 
(pH 8.1), and 2 mM EDTA). 
As noted above, the nucleotide sequence of the plasmid DNA in FIG. 1 is 
illustrated in FIG. 2. This segment of DNA was found to include the region 
of the IL-1 gene coding for mature IL-1. The nucleotides are numbered from 
the beginning of the DNA segment in FIG. 2. The corresponding amino acids, 
as determined by the nucleotide sequence and by protein sequence analysis, 
are set forth above the appropriate codons. The amino acid composition of 
the IL-1 gene extends from the mature NH.sub.2 -terminus of the IL-1 
molecule, i.e., the Ala residue, in IFG. 2 (from which the numbering of 
the amino acid residues begins), to the Ser residue (No. 153) immediately 
preceding the termination codon TAA. Various restriction enzyme cleaving 
sites are also indicated in FIG. 2. The coding region of the IL-1 gene in 
FIG. 2 is illustrated as a boxed section in FIG. 1. 
Amino acid sequence studies of IL-1 were conducted according to the method 
of Stern et al., supra, wherein cyanogen bromide was employed to cleave 
the IL-1 at the methionine residues. The resulting fragments were 
separated by size by standard ion-exchange methods. The isolated peptide 
fragments were then sequenced by automated amino terminal Edman 
degradation using an Applied Biosystems Model 470 protein sequencer. By 
this process, applicants have confirmed the results obtained by nucleotide 
sequencing that the C-terminal of the IL-1 protein is composed of the 
amino acid sequence: Gln-Phe-Val-Ser-Ser. This establishes that the 
"natural" IL-1 is not processed by removal of amino acids from this end of 
the molecule after translation from mRNA. This is significant since from 
the nucleotide sequence of the IL-1 gene in FIG. 2, it is clear that a 
significant amount of RNA sequence is removed from the N-terminus of the 
IL-1 gene during the maturation of IL-1 from its precursors. 
EXAMPLE 7 
Expression of Mature IL-1 
The coding region of the IL-1 gene was removed from the cDNA clone of FIG. 
1 and then inserted into the pY ADH shuttle vector to form the recombinant 
expression plasmid pY ADH IL-1. The restructuring scheme for preparing the 
pY ADH IL-1 shuttle expression vector is shown in FIG. 3. This plasmid was 
amplified in E. coli host cells and then employed to transform yeast host 
cells for high level expression of mature IL-1. The functionality of the 
expressed IL-1 was confirmed by using the thymocyte proliferation and IL-2 
conversion assays, detailed above. 
A major portion of the coding region of the IL-1 gene from the Hpa II site 
(base #457 in FIG. 2) to the 3' flanking region of the gene was removed 
from the cDNA plasmid segment illustrated in FIGS. 1 and 2 by use of the 
Hpa II and Pst I restriction enzymes in the standard protocol set forth in 
Maniatis et al., supra at 104. The IL-1 gene segment was cleaved from the 
cDNA clone at the Hpa II site, which is located 29 nucleotides downstream 
from the 5' end of the gene, because no convenient restriction site was 
found to correspond precisely with the 5' terminal of gene. The 3'-Pst I 
site of the excised IL-1 gene segment was filled in with T4 DNA polymerase 
to create a blunt end compatible with the Stu I site of the shuttle 
vector, discussed below. 
A synthetic oligonucleotide was chemically synthesized to add back the 5' 
terminal portion of the coding region of the IL-1 gene and also to create 
a translation initiation codon at the 5' end of the coding region. The 
composition of the oligonucleotide, as shown in Table 1 below, includes an 
Eco RI cohesive 5' terminal followed by an ATG initiation codon and then 
the 5' end of the coding region of the IL-1 gene (to the Hpa II site). 
Although the oligonucleotide shown in Table I was chemically synthesized 
by triester technique as detailed by Sood et al., supra and Hirose et al., 
supra, it is to be understood that the oligonucleotide can be prepared by 
other methods, such as by phosphodiester method. 
TABLE 1 
__________________________________________________________________________ 
ECO R1 Met Ala Pro Val Arg Ser Leu Asn Cys Thr Leu 
__________________________________________________________________________ 
5'-AATTCAAC 
ATG GCA CCT GTA CGA TCA CTG AAC TGC ACG CCT -3' 
GTTG TAC CGT GGA CAT GCT AGT GAC TTG ACG TGC GAGGC 
Hpa II 
__________________________________________________________________________ 
Also, rather than cleaving the coding region of the IL-1 gene at the Hpa II 
site, the plasmid cDNA in FIG. 2 could be cleaved at a restriction enzyme 
site in the 5' flanking region of the gene. Thereafter, the nucleotides of 
the flanking region can be sequentially removed by standard techniques. 
The pY ADH shuttle vector was prepared for ligation to the synthetic 
oligonucleotide and the excised major portion of the coding region of the 
IL-1 gene by digestion of the vector to completion with the restriction 
endonucleases Eco RI and Stu I by standard techniques, as set forth in 
Maniatis et al., supra at 104. The desired larger fragment from the 
digestion of the pY ADH plasmid was isolated by electrophoresis on 0.7% 
agarose gel at 100 volts at 22.degree. C. for two hours. 
As shown in FIG. 3, the synthetic DNA oligomer, the excised major portion 
of the coding region of the IL-1 gene and the desired linearized pY ADH 
fragment were ligated together in a reaction mixture composed of 100 ug of 
the pY ADH vector fragment (Eco RI - Stu I), 40 ug of the major IL-1 cDNA 
fragment (Hpa II, Pst I [blunt]), 5 ug of synthetic oligonucleotide (Eco R 
I - Hpa II), 1 ug of T4 DNA ligase and sufficient T4 ligase buffer (0.4M 
Tris [pH 7.4]0.1 MgCl.sub.2, 0.1M dithiothreitol, 10 mM spermidine, 10 mM 
ATP and 1 mg/ml BSA) to form a 20 ul reaction volume. The reaction was 
carried out by incubation at 15.degree. C. for 15 hours. 
The resulting recombinant plasmid, designated as pY ADH IL-1, was then 
transformed into E. coli strain RR1 using standard transformation 
techniques, such as set forth in Bolivar et al., supra and Peacock et al., 
supra. The host cells were cultured to amplify the pY ADH IL-1 plasmid, 
and then the plasmids were removed from the host bacteria by standard 
alkaline method as detailed by Maniatis et al., supra at 368 and by 
Ish-Horowicz and Burke, supra. The plasmid DNA was purified by 
centrifugation to equilibrium in cesium chloride-ethidium bromide density 
gradients, as set forth in Maniatis et al., supra at 93. It is to be 
understood that other techniques for extracting/concentrating the 
amplified plasmid DNA from the E. coli may be employed without departing 
from the scope or spirit of the present invention. 
The amplified pY ADH IL-1 plasmid, as prepared above, was then employed to 
transform the protease deficient yeast strain 20B-12 (alpha, pep 4.3, Trp 
1) of S. Cerevisiae by standard techniques. Prior to transformation, the 
20B-12 strain was grown in culture in YP-glucose medium (200 ml) to 
cultures of 2.times.10.sup.7 cells/ml. The cells were harvested by 
centrifugation at 1000.times.g for 5 minutes at 22.degree. C., and then 
the resulting pellet washed with sterile distilled water. 
The yeast cells were then concentrated by resuspending in 20 ml of SED (1M 
sorbitol, 25 mM ETDA [pH 8.0], and 50 mM dithiothreitol) and incubated for 
10 minutes at 30.degree. C. The cell-buffer mixture was then centrifuged 
for 5 minutes at 300.times.g. The pellet was washed once with 200 ml of 1M 
sorbitol and the cells resuspended in 20 ml of SCC (1M sorbitol, 0.1M 
sodium citrate [pH 5.8], 0.1M ETDA). Glusulase, to break down the cell 
walls, in an amount of 0.2 ml was added to the solution and then the 
solution incubated at 30.degree. C. for 30 minutes with occasional gentle 
shaking. 
The presence of spheroplast was assayed by diluting 10 ul of yeast cells 
into a drop of 5% sodium dodecyl sulfate (SDS) (wt./vol.) on a microscope 
slide to observe for "ghosts" at 400.times.phase contrast. 
The cell mixture was then centrifuged at 300.times.g for 3 minutes. The 
resulting pellet was twice washed with 20 ml of 1M sorbitol. The pellet 
was then washed once with STC (1M sorbitol, 10 mM, CaCl, 10M Tris HCl [pH 
7.5]). 
The yeast spheroplasts were then transformed with the previously prepared 
plasmid vector in a procedure adapted from Beggs, 275 Nature (London) 104 
(1978). The pelleted protoplasts are suspended in 1.0 ul of STC and then 
divided into 100 ml aliquots in 10 ul disposable tubes (Falcon #2059). 
Then, from 1 to 10 ul of the DNA plasmids were added to each aliquot (0.5 
to 5 ug). The mixture was rested at room temperature for 10 minutes and 
then 1 ml of PEG (20% PEG 4000, 10 mM CaCl.sub.2, 10 mM Tris - HCl [pH 
7.4]) was added to each aliquot to promote DNA uptake. After 10 minutes at 
room temperature, the mixture was centrifuged for 5 minutes at 
350.times.g. The resulting pellet was resuspended in 150 ul of SOS (10 ml 
of 2M sorbitol, 6.7 ul of YEP [0.13 ml of 1M of CaCl, 27 ul of 1% 
leucine, and 3.7 ml of H.sub.2 O]). This mixture was incubated for 20 
minutes at 30.degree. C. 
Thereafter the protoplast/DNA mixture was plated in the presence of yeast 
minimal medium containing 1.2M sorbitol and 3% agar at 45.degree. C. and 
without tryptophan. The minimal medium was composed of 0.67 Difco yeast, 
Nitrogen Base, 0.5% casamino acids, 2% glucose. By maintaining the 
protoplast/DNA mixture in this medium, only transformants survived, i.e., 
those that contained the Trp 1 gene. 
Prior to biological assay, the transformants were inoculated from the 
minimal medium into rich medium (1% yeast extract, 2% peptone, 2% glucose) 
and grown at 30.degree. C. for 15-20 hours until the late exponential 
phase. At the time of harvest, the protease inhibitor phenyl methyl 
sulfonyl fluoride (PMSF) was added to 1 mM. The culture was then 
centrifuged at 400.times.g to pellet the cells. Thereafter, the cells were 
washed once in 0.1 vol. cold H.sub.2 O. For breakage, the cells were 
resuspended in 0.01 vol. cold H.sub.2 O containing 1 mM PMSF and vortexed 
with glass beads (1/3 vol.) for 2 minutes. The cell debris and glass beads 
were pelleted by centrifugation. The resulting supernatant was found to 
exhibit IL-1 activity. This was ascertained by utilizing the supernate in 
both of the thymocyte proliferation and IL-1 conversion assays, discussed 
above. 
As will be apparent to those skilled in the art in which the invention is 
addressed, the present invention may be embodied in forms other than those 
specifically disclosed above without departing from the spirit or 
essential characteristics of the invention. The particular embodiments of 
the present invention, described above, are therefore to be considered in 
all respects as illustrative and not restrictive. The scope of the present 
invention is as set forth in the appended claims rather then being limited 
to the examples contained in the foregoing description.