Proc. Natl. Acad. Sci. USAVol. 81, pp. 6451-6455, October 1984Genetics

Human genomic library screened with 17-base oligonucleotideprobes yields a novel interferon gene

(DNA hybridization/synthetic oligonucleotides/DNA sequencing/lac promoter)

RICHARD M. TORCZYNSKI, MOTOHIRo FUKE*, AND ARTHUR P. BOLLONtDepartment of Molecular Genetics, Wadley Institutes of Molecular Medicine, Dallas, TX 75235

Communicated by S. M. McCann, July 2, 1984

ABSTRACT A method is presented that has permitted ahuman genomic library to be screened for low-copy genes us-ing 17-base synthetic oligonucleotides as probes. Parallelscreening with two different 17-base probes permitted the un-ambiguous identification of clones containing interferon-a(IFN-a) genes. The isolated human IFN-a genes were se-quenced, and one appears to be IFN-aL; the other is one notpreviously described, which we have designated IFN-aWA.The IFN-aWA sequence differs from those of IFN-a genes A-L at -10% of the positions and is most similar to IFN-aC,-aF, and -alH. IFN-aWA has been found to encode amino ac-ids that differ from those conserved at each of five positions inall previously reported IFN-a species. The IFN-aWA genecodes for an active interferon, which has been expressed inEscherichia coli using an M13-lacZ fusion as an expressionvector. About 5 x 106 units of IFN-aWA were obtained perliter of bacterial culture. The described screening procedureusing short probes should permit the isolation of genes forwhich sequence information is available from animal or plantgenomic libraries.

Although short synthetic oligonucleotides have been used toscreen cDNA libraries and cDNA probes have been used toscreen human genomic libraries (1, 2), synthetic oligonucleo-tides of less than 20 bases have not been used for isolation oflow-copy genes from human genomic libraries. The obviousadvantage of genomic libraries over cDNA libraries is thepresence of specific genes independent of gene expression.The synthesis of short oligonucleotides either by the phos-

photriester (3) or the phosphoramidite (4) method is a conve-nient technique for generating probes for screening pur-poses, especially since the chemistry involved has becomereliably automated. The improved sensitivity of hybridiza-tion techniques has resulted in the detection of single-basedifferences using probes less than 20 nucleotides long (5),permitting the use of such probes for diagnosis of geneticdiseases (6). They have also been used for screening cDNAlibraries that have been enriched in specific gene sequences(1).

Utilizing 17-base oligonucleotides, we have optimized theplaque-hybridization screening procedure and have beenable to isolate several interferon-a (IFN-a) genes from thehuman genomic library contained in Charon 4A (2, 7). Previ-ously, human IFN-a genes have been isolated from cDNAlibraries made using mRNA from virus-induced leukocytes(8, 9). Certain IFN-a genes isolated from such a humancDNA library and expressed in Escherichia coli are undergo-ing extensive analysis in preclinical (10) and clinical trials(11) of their utility in producing IFN-as as antiviral and anti-tumor agents. IFN-a genes have also been isolated from ahuman genomic library using IFN-a cDNAs as probes (12).A detailed comparison of the different IFN-a genes has per-

mitted us to identify conserved regions common to several ofthe genes and has served as the basis for constructing probesfor IFN-a genes (2, 7).One of the IFN-a genes that we have isolated from the

human genomic library has not been reported previously,and another appears to code for IFN-aL (13). A new IFN-agene, IFN-aWA, has been sequenced and compared to thelFN-a genes A-L previously described (9, 13). It is most ho-mologous overall with IFN-aC and with the 5' and 3' regionsofIFN-aF and -alH, respectively. The IFN-aWA gene codesfor a functional interferon, which has been expressed in E.coli using the M13-lacZ fusion as an expression vector.

MATERIALS AND METHODSStrains and General Methods. M13mp8 bacteriophage and

E. coli K-12 JM103 (Alac-pro thi strA endA sbcB15 hsdR4supE F'traD36 proAB lacI ZA mlS) were obtained from Be-thesda Research Laboratories. M13mpll bacteriophageDNA was obtained from P-L Biochemicals. Restriction en-donucleases were obtained from Bethesda Research Labora-tories except for Xmn I, which was obtained from New En-gland Biolabs. Interferon was assayed at the InterferonProduction Laboratory at Wadley Institutes using the cyto-pathic effect-reduction assay (14) on WISH cells (obtainedfrom Flow Laboratories) challenged with vesicular stomati-tis virus.

Oligonucleotide Synthesis and Labeling. Probe A (see Re-sults) was obtained from BioLogicals (Toronto). Probe B(see Results) was synthesized by the solid-phase phospho-triester method using a Bachem DNA Synthesizer (BachemFine Chemicals, Torrance, CA) and purified as described byItakura and Riggs (3). The two probes were labeled at the 5'ends with [y-32P]ATP (New England Nuclear; 5000 Ci/mmol, 1 Ci = 37 GBq) using T4-infected E. coli polynucleo-tide 5'-hydroxyl-kinase (P-L Biochemicals). The end-labeledDNA probes were separated from unincorporated [y-32P]ATP by gel filtration through a Sephadex G-50 columnand filtration through a 0.2-gm Acrodisc filter (Gelman) (2,7). Specific activities of the probes were 2-5 x 108 cpm/,ug.Screening the A Phage Library for IFN Clones. The human

genomic library in Charon 4A (15) was obtained from T.Maniatis. Phage were used to infect E. coli DP-50 [supFsupE dapD8 lacY (gal-uvrB) thyA nalAr hsd] at a density of 1x 104 plaque-forming units (pfu) per 80-cm2 dish. A total of180,000 pfu were screened separately with each probe usinga modification (7) of the plaque-hybridization method ofBenton and Davis (16). Duplicate nitrocellulose-filter(Schleicher & Schuell BA85-SD) copies were made fromeach plate. The DNA was alkali-denatured on filter paper

Abbreviations: IFN, interferon; pfu, plaque-forming units; kb, kilo-base pair(s).*Present address: Biotechnology Division, Phillips Research Cen-ter, Phillips Petroleum Company, Bartlesville, OK 74004.tTo whom correspondence and reprint requests should be ad-dressed.

6451

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Proc. NatL Acad. Sci. USA 81 (1984)

saturated with 0.5 M NaOH/1.5 M NaCl, neutralized on pa-per saturated with 1 M Tris HCl, pH 7.5, and again neutral-ized on paper saturated with 0.5 M Tris HCl, pH 7.5/1.5 MNaCl. The filters were baked in a vacuum oven at 80'C for 2hr. Prehybridization was performed at 37TC in 0.9 MNaCl/6.0 mM Na2EDTA/19.8 mM Tris-HCl, pH 8.0/0.1%Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albu-min/0.1% NaDodSO4/10% dextran sulfate. After overnightprehybridization, the solution was removed and new hybrid-ization solution (identical to prehybridization solution) con-taining labeled probe at a concentration of 2 ng/ml was add-ed. Hybridizations were performed at 370C for 20 hr withconstant agitation by rotation. Filters were washed fourtimes (20 min per wash) at room temperature in 0.9 MNaCl/90 mM sodium citrate, pH 7, and twice at 450C (1 hrper wash) in the same buffer containing 0.1% NaDodSO4.Autoradiography was done for 64 hr at -80'C using KodakXAR film plus two intensifying screens (DuPont Cronex Hi-Plus). Positive clones were identified and isolated by severalcycles of plaque purification and phage DNA stocks wereprepared as described (17).

Restriction and Hybridization Analysis of Cloned DNA.Phage DNA from isolated recombinants was digested withBamHI, Xmn I, EcoRI, or Dde I under the conditions recom-mended by the supplier. The digested DNA was fractionatedon agarose gels, transferred to nitrocellulose paper as de-scribed by Southern (18), and hybridized with probes A andB under the conditions described for the screening proce-dure.DNA Sequence Determination. A 2.1-kilobase (kb) EcoRI

fragment from clone X-85 and a 1.8-kb EcoRI fragment fromclone X-77 (see Table 1) were ligated into M13mp8 DNA,using DNA ligase (Bethesda Research Laboratories) fromT4-infected E. coli. DNA sequence was determined by themethod of Sanger et al. (19) using probes A and B and select-ed restriction fragments as primers. Second-strand sequenc-ing of IFN-aWA was completed using two synthetic oligo-mers (C and D; obtained from Systec, Minneapolis, MN).Probe C is 5'-C-C-A-T-C-T-C-C-A-G-T-A-G-C-C-3' andProde D is 5'-G-A-G-A-C-C-C-T-C-C-T-A-G-A-C-3'. TheNew England Nuclear DNA Sequencing System (Sangermethod) and the large (Klenow) fragment of DNA polymer-ase I (Bethesda Research Laboratories) were used for DNAsequencing by the dideoxy method (19). The sequence ofcertain regions was confirmed by the method of Maxam andGilbert (20).

Construction of IFN-Expressing Clone. Replicative-formDNA of M13 clones with inserts containing IFN gene se-quences were isolated from infected JM103 cells using thecleared-lysate technique (21) followed by two cycles of CsClequilibrium centrifugation. After the digestion of mp8-85(which contains the 2.1-kb EcoRI fragment from X-85) withAlu I/EcoRI and agarose gel electrophoresis, a 1-kb frag-ment was isolated. This fragment was cloned into M13mpllcleaved with HincII/EcoRI, and the ligated product wasused to transform JM103 (22). Isolated phage clones contain-ing the 1-kb insert were designated M13mpll-IFN-a WA.

Induction of IFN-aWA in E. coli. E. coli JM103 cultureswere grown to OD6N = 0.8 in 2 x YT medium (22) and wereinfected with M13mpll-IFN-aWA bacteriophage at a multi-plicity of infection (moi) of 100. After incubation for 5 min at30°C, the culture was diluted 1:100 in 2 x YT medium andallowed to grow for 2 hr at 37°C. Isopropyl B-D-thiogalacto-side was added to a final concentration of 0.5 mM to inducethe bacteria; 2 hr later the cells were harvested by centrifu-gation and resuspended in 0.1 volume of ice-cold 50 mMTris HCI, pH 8.0/30 mM NaCl. Lysozyme (Sigma) was add-ed to a final concentration of 1 mg/ml and the suspensionwas kept for 30 min at 00C. After three cycles of freeze-thawing, cell debris was removed by centrifugation at

100,000 x g for 1 hr at 40C. The supernatant was assayed forIFN activity as described above.

RESULTSConstruction of Synthetic Probes. Part of the strategy em-

ployed in our isolation of human IFN-a genes from a humangenomic library involved the construction of short syntheticprobes homologous to well-conserved regions of IFN-agenes A-H (9). From the published sequences for part of theIFN-a genes A-H (9), two (G + C)-rich regions of homologycontained within the gene sequences defined by bases 401-500 were selected as the bases for probe synthesis.Probe A (5'-C-A-G-C-C-A-G-G-A-T-G-G-A-G-T-C-C-3')

is homologous to IFN-aB, -aC, -aE, -aH, and -aL and con-tains one mismatch with IFN-aA, -aD, -aF, and -aK and twomismatches with IFN-aG (9, 13). Probe B (5'-C-C-T-C-C-C-A-G-G-C-A-C-A-A-G-G-G-3') is homologous to IFN-aA,-aC, -aD, -aH, -aK, and -aL and has one mismatch withIFN-aB, -aE, -aF, and -aG.-Screening the Human Genomic Library. A human genomic

library contained in Charon 4A (15) was screened for IFN-agenes using the synthetic probes A and B. From -180,000pfu, the phage DNA of five foci appeared to hybridize toboth probe A and probe B. One example is shown in Fig. 1.When phage from the five foci were replated at lower den-

sities (500 pfu/dish), three clones were isolated that hybrid-ized to both A and B and were designated X-77, X-85, and X-105. Filtration of the probe solutions and inclusion of dex-tran sulfate in the hybridization solution, as described inMaterials and Methods, optimized the signal-to-noise ratio.The addition of E. coli DNA or yeast tRNA to the hybridiza-tion mixture did not reduce background noise and, therefore,was not incorporated in the procedure.

Characterization of IFN-a Clones. The X-77, X-85, and X-105 clones were characterized by restriction and Southernhybridization analyses (18). Restriction fragments that hy-bridized with both A and B were identified (Table 1). Thehybridization ofA and B to single fragments from each cloneconfirmed that the two probes were recognizing closelylinked regions ofDNA and suggested that the isolated clonescontained IFN or IFN-like genes.EcoRI fragments from X-77 and X-85 (1.8 and 2.1 kb, re-

spectively) were subcloned into M13mp8 (23). A 3.5-kb XbaI fragment from A-105, which hybridized to probes A and B(data not shown), was cloned into M13mpll (23). Thesethree clones were designated mp8-77, mp8-85, and mpll-105, respectively.Further characterization of clones mp8-77, mp8-85, and

A B

.........1; . ..

FIG. 1. Plaque hybridizations for X-105. Duplicate filter copieswere made from an 80-cm2 dish containing 10,000 plaques of thehuman genomic library. Filters were treated with probe A or probeB, washed, and autoradiographed. Positive clones were identifiedby common signals on both filters, as indicated by the arrows.

6452 Genetics: Torczynski et aL

Proc. NatL Acad Sci USA 81 (1984) 6453

Table 1. Characterization of cloned IFN genes

Restriction enzyme*

Clone BamHI Dde I EcoRIX-77 20 1.1 1.8X-85 20 1.0 2.1X-105 8 ND 7.0

*Numbers are sizes (in kb) of restriction fragments that hybridizedwith both probes A and B by Southern hybridization. ND, notdone.

mpll-105 was based on the observation that the Xmn I sitewas conserved in IFN-a genes A-L (excepting IFN-aG andIFN-aE) at a location between the hybridization sites forprobes A and B. Southern analysis ofXmn I-restricted DNAof these three clones was done using A or B as hybridizationprobes (Fig. 2). Clone mp8-77 clearly contains an Xmn I siteat the predicted location, since probes A and B hybridized todifferent restriction fragments. Clones mp8-85 and mpll-105lack the predicted Xmn I site; for each of these clones,probes A and B hybridized to the same restriction fragment.These clones could potentially contain IFN-aE or -aG al-though these genes contain mismatches with the probes.Alternatively, they could represent new IFN genes.DNA Sequence Determination of IFN-a Genes. DNA se-

quence analysis was performed on clones mp8-77, mp8-85,and mpll-105. Probes A and B (also C and D for mp8-85) andrestriction fragments from mp8-85 were used as primers inthe dideoxy sequencing method (19). Select sequences wereconfirmed by the Maxam and Gilbert method (20). The strat-egy employed for the determination of the IFN-aWA se-quence is shown in Fig. 3.The IFN-a gene sequence contained within the X-85

EcoRI fragment is presented in Fig. 4. A sequence compari-son of this IFN-a gene with those ofIFN-a genes A-L previ-ously described indicates that this IFN-a gene in X-85 is dif-ferent. This new IFN-a gene has been designated IFN-aWAand shows -88% DNA sequence homology with the IFN-aA, -aB, -aD, and -aE genes and 92-94% homology with theIFN-aC, -aF, -aG, -aH, and -aL genes. The 150 bases at the

C.)

M13mp8

kb

21.2 -

4.9 -4.23.5 -

2.0 -

1.9 -1.6 -1.4 -

0.95 -

0.83 -

0.56-

A

1 2 3

B

1 2 3

FIG. 2. Southern hybridization analysis of IFN-a clones. PhageDNA from M13 clones was digested with Xmn I and electropho-resed on a 1.0% agarose gel. DNA was transferred to nitrocellulosepaper and hybridized with probe A or B. Duplicate filters werewashed three times in 0.9 M NaCl/90 mM sodium citrate (pH 7.0) atroom temperature, then once at 45°C in the same buffer containipg0.1% NaDodSO4 and autoradiographed for 3 hr at -80°C using twointensifying screens. (A) Filter hybridized with probe A; (B) filterhybridized with probe B. Shown are digests of mp8-77 (lanes 1),mp8-85 (lanes 2), and mpll-105 (lanes 3). Sizes (in kb) of 32P-end-labeled HindIII/EcoRI fragments of phage X DNA (left lane in A)are shown at the left.

5' end of IFN-aWA are most homologous (99%) with IFN-aF; this homology decreases toward the 3' end. In contrast,the 125 bases at the 3' end of IFN-aWA are most homolo-gous (98%) with IFN-aH, and the sequences diverge towardthe 5' termini. A comparison of the amino acid sequence en-coded by IFN-aWA (Fig. 4) to those encoded by IFN-agenes A-L (9) indicates that five positions (42, 56, 57, 110,and 133) that are conserved in the other IFN-a are differentin IFN-aWA. Three of these substitutions maintain hydro-phobic residues (positions 56, 57, and 110) whereas the othertwo replace acidic residues with hydrophobic residues.

a x

M13mp.M13mp8

_- _-9 Oc4

,v2 - .r

3 = = =EI I I

E 7//////////////// /////7// IA

D ---_ . ~~~~D_

100 bp

FIiFIG. 3. Restriction map and sequencing strategy for the IFN-aWA gene contained in mp8-85. The upper line shows the major restriction

sites of a 2.1-kb EcoRI fragment isolated from X-85 and cloned into M13mp8. An expanded restriction map of the IFN-aWA gene (indicated bythe hatched box) is shown. Primers used for dideoxy sequencing (19) are indicated by thick lines at the origins of the arrows. A, B, C, and D aresynthetic oligomers. HinfI fragments (1 and 2) were isolated from mp8-85. A, B, and 1 were used with mp8-85 single-stranded DNA, and C, D,and 2 were used with mp8-85R single-stranded DNA, which contains the 2.1-kb EcoRI fragment in the orientation opposite that in mp8-85. Thefilled circles are at the origins of arrows representing 5'-labeled DNA sequenced by the method of Maxam and Gilbert (20). Arrows showdirection of sequence determination. bp, Base pairs.

Genetics: Torczynski et aL

0-4

LV

1.

CO-4 CB11! 1:

M: x

I I I

Proc. NatL Acad. Sci. USA 81 (1984)

S1 S10 S20 1MET ALA LEU SER PHE SER LEU LEU MET ALA VAL LEU VAL LEU SER TYR LYS SER ILE CYS SER LEU GLY CYS ASP LEU PRO GLN THR

CAACATCCCA ATG GCC CTG TCC TTT TCT TTA CTG ATG GCC GTG CTG GTG CTC AGC TAC AAA TCC ATC TGT TCT CTG GGC TGT GAT CTG CCT CAG ACT-10 1 50

10 20 30HIS SER LEU GLY ASN ARG ARG ALA LEU ILE LEU LEU ALA GLN MET GLY ARG ILE SER HIS PHE SER CYS LEU LYS ASP ARG TYR ASP PHE GLY PHE PROCAC AGC CTG GGT AAT AGG AGG GCC TTG ATA CTC CTG GCA CAA ATG GGA AGA ATC TCT CAT TTC TCC TGC CTG AAG GAC AGA TAT GAT TTC GGA TTC CCC

100 15040 50 60 70

GLN GLU VAL PHE ASP GLY ASN GLN PHE GLN LYS ALA GLN ALA ILE SER ALA PHE HIS GLU MET ILE GLN GLN THR PHE ASN LEU PHE SER THR LYS ASPCAG GAG GTG TTT GAT GGC AAC CAG TTC CAG AAG GCT CAA GCC ATC TCT GCC TTC CAT GAG ATG ATC CAG CAG ACC TTC AAT CTC TTC AGC ACA AAG GAT

200 25080 90 100

SER SER ALA ALA TRP ASP GLU THR LEU LEU ASP LYS PHE TYR ILE GLU LEU PHE GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL THR GLN GLU VAL GLYTCA TCT GCT GCT TGG GAT GAG ACC CTC CTA GAC AAA TTC TAC ATT GAA CTT TTC CAG CAA CTG AAT GAC CTA GAA GCC TGT GIG ACA CAG GAG GTT GGG

300 350110 120 130

VAL GLU GLU ILE ALA LEU MET ASN GLU ASP SER ILE LEU ALA VAL ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU MET GLY LYS LYS TYR SER PROGTG GAA GAG ATT GCC CTG ATG AAT GAG GAC TCC ATC CTG GCT GTG AGG AAA TAC TTT CAA AGA ATC ACT CTT TAT CTG ATG GGG AAG AAA TAC AGC CCT

400 450140 150 160 166

CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER PHE SER PHE SER THR ASN LEU GLN LYS GLY LEU ARG ARG LYS ASP STOP AAACTTGT GCC TGG GAG GTT GTC AGA GCA GAA ATC ATG AGA TCC TTC TCT TTT TCA ACA AAC TTG CAA AAA GGA TTA AGA AGG AAG GAT TGA

500 550

FIG. 4. Sequence of the IFN-aWA gene and its derived amino acid sequence. The putative signal peptide is indicated by positions S1-S23.Amino acid position is indicated by numbers above and nucleotide position is indicated by numbers below the sequences. Underlined nucleo-tide sequences: 412-428 is complementary to probe A, 480-4% is complementary to probe B.

DNA sequence determination of nucleotides 1-406 of theX-77 EcoRl fragment indicates complete homology with thesequence of IFN-aL, which has been shown to contain astop codon in the signal sequence (13). Partial DNA se-quence analysis of X-105 indicates that this clone containsanother distinct IFN gene since the sequences do not corre-spond exactly to those of any of the previously identifiedIFN-a genes or to IFN-aWA.

Expression of IFN-aWA in E. Coli. Since the analysis ofthe IFN-aWA gene (Fig. 4) failed to reveal any terminationcodons in either the signal or the mature IFN sequences, thegene was engineered for expression in E. coli to determine ifthe gene product was an active interferon. The IFN-aWAgene was inserted into an M13mpll expression vector sys-tem (Fig. 5) (24). M13mpll was used because it contained asite for in-frame insertion of the IFN-aWA gene. The con-struction involved fusion at the HincII site of M13mpll andthe Alu I site of the IFN-aWA gene. The 3'-terminal side ofthe IFN-aWA gene was fused to the M13mpll vector byligation at an EcoRI site. The encoded peptide product con-tains part of the IacZ gene product from methionine to argi-nine, the COOH-terminal part of the NH2-terminal IFN sig-

M13MP11

nal peptide (from serine to glycine), and the mature IFN-aWA sequence (Fig. 5).

E. coli JM103 were infected with the M13mpll bacteri-ophage containing the lacZ-IFN-aWA fusion, induced withisopropyl ,f-D-thiogalactoside, and lysed by freeze-thawing.The cell debris was removed by centrifugation and the super-natant was assayed for IFN activity. Utilizing the M13expression system, about 5 x 106 units of IFN-aWA wereobtained per liter of bacterial culture.

DISCUSSIONDescribed is a procedure using short synthetic probes for theisolation of select, low-copy genes from animal or plant ge-nomic libraries. It is possible that this method will permit theisolation from genomic libraries of most genes for whichDNA or amino acid sequences are known. This may includegenes containing introns, assuming that the sequences corre-sponding to the probes are not interrupted by introns. Clear-ly, multiple probes could be used. Short probes should offerthe opportunity for isolating a specific subset of genes thatcomprise a multigene family.

H iNC IIATG ACC ATG ATT ACG CCA AGC TTG GGC TGC AGG TCG ACMET THR MET ILE THR PRO SER LEU GLY CYs ARG SER

LAC Z

M13mP11- IFN-ctWA ATG ACC ATG ATT ACG CCA AGC TTG GGCMET THR MET ILE THR PRO SER LEU GLY

LAC Z

IFN-oWA SIGNALTGC AGG TCC TAC AAA TCC ATC TGT TCT CTG GGC TGT GATCYs ARG SER TYR LYs SER ILE CYS SER LEU GLY CYs As_

MATURE IFN-cvWA

ALuI MATURE IFN-(XWAIFN-axWA AGC TAC AM TCC ATC TGT TCT CTG GGC TGT GAT

SER TYR LYs SER ILE CYS SER LEU GLY, CYs AsPIFN-oXWA SIGNAL

FIG. 5. Construction of the Ml3mpll-IFN-aWA expression vector. The IFN-aWA gene was fused to part of the lacZ gene contained in theM13mpll vector by ligation at the HincHI site of M13mpll and the Alu I site of the IFN-aWA gene. The arrows indicate the identity of therespective sequences.

6454 Genetics: Torczynski et aL

Proc. NatL Acad. Sci. USA 81 (1984) 6455

IFN-aWA was isolated from a genomic library by usingshort probes but was not isolated by others from an inducedleukocyte cDNA library (9) or a human genomic library (12)by using cDNA probes. Clearly, the use of short probes inscreening genomic libraries permits the isolation of genes

that may not be expressed in a particular cDNA library.Since WFfN-,WA had not previously been isolated from a

cDNA library made using leukocytes induced with vesicularstomatitis virus or Sendai virus, either the IFN-aWA gene

was not expressed under the inducing conditions used or itwas expressed at such a low level that it was not detected in

the cDNA library.In view of the isolation ofIFN-aWA from the human geno-

mic library, several points are worth considering. The isola-tion of new IFN-a genes may reflect even greater diversity.Although one of the three genes isolated in this work is noveland another appears novel, it should be pointed out that only180,000 clones were tested. Theoretically, about 500,000clones would need to be tested to isolate all of the possibleIFN-a genes homologous to the specific probes since the av-

erage size of the human DNA insert in the Charon 4A libraryis 15 kb. Why IFN-aWA was not isolated from the human

gbnomic library in Charon 4A in prelious studies (9, 12) is

not clear. Either the cDNA probe failed to recognize IFN-

aWA under the hybridization conditions employed (12) or

the hybridization signal was very weak and not detected.Since the IFN-aWA gene codes for an active interferon in

bacteria, .one can speculate that either it is expressed in cul-tured cells or humans under conditions that have not beenidentified or that the gene is not expressed due to repressionof transcription or translation. Based on previous studieswith various gene systems, it seems more likely that theIFN-aWA gene is expressed and conceivably may correlatewith specific disease states. The characterization of IFN-aWA and analysis of its expression in different tissues andunder various induction conditions may help resolve thisquestion.

The authors gratefully acknowledge discussions with Drs. N, 0.

Hill and Susan Berent. The authors thank Dr. Paul Bragg for synthe-sis of one of the 17-base probes, L. Cheryl Hendrix for assistance inthe IFN-aWA expression studies, Dr. Kurt Berg and Pia Jensen atWadley Institutes for interferon assays, and Carol Crumley for prep-

aration of the manuscript. Some of this work was carried out in par-

tial fulfillment of the requirements for the Ph.D. degree of R.M.T. at

the University of Texas at Dallas. This work was performed in theOree Meadows Perryman Laboratory for Cancer Research at Wad-ley Institutes and has been funded by the Meadows Foundation andin part by a National Institutes of Health grant to A.P.B.

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Genetics: TO'rczynski et aL