JOURNAL OF INTERFERON RESEARCH 8:51-60 (1988)Mary Ann Liebert, Inc., Publishers

Detection of HumanLeukocyte Interferon-ceA and -c¿¿

Genes in Genomic DNAs by the Useof Deoxyoctadecyloligonucleotide Probes

KUNIMÖTO HOTTA,14 JOHN MONAHAN,2 3 KENNETH J. COLLIER,25and SIDNEY PESTKA16

ABSTRACT

Two deoxyoctadecyloligonucleotides complementary to the sequence spanning a singlebase substitution between human leukocyte interferon (HuIFN) aA and a2 genes were ef¬ficiently used as probes to distinguish between HuIFN- and -a2 genes. At 37°C or

42°C under aqueous conditions (0.9 M NaCI), hybridization between both probes andthe «A and a2 genes without any mismatch was strong, whereas the hybridization withone base mismatch (aA probe-a2 gene and a2 probe-aA gene) was very weak or negligi¬ble. Because the single base substitution of G in the a2 gene for A in the aA gene pro¬vides an extra Hinîl site in the a2 gene at the center of the sequence hybridizing to the a2

probe, digestion with Hinfl restriction endonuclease caused complete loss of the hybrid¬ization between the a2 probe and the a2 gene. PvwII digestion provides 298-bp fragmentshybridizing to the probes only from the aA and a2 genes among the known HuIFN- genes. Thus, with the use of these oligonucleotide probes in combination with Pvull andPvull-Hinfl restriction endonuclease digestion, the existence of the sequences corre¬

sponding to both IFN-aA and IFN-a2 genes in human genomic DNAs was demonstrated.The results also surprisingly indicate that these genes, formerly considered alíeles becauseof their essential identity (1 base pair difference in the coding sequence), are not likely tobe alíeles, but represent closely related distinct genes.

INTRODUCTION

Since their first cloning in 1980, many human leukocyte interferon (HulFN-a) genes havebeen cloned and characterized in terms of restriction maps, nucleotide sequence, and chromoso¬

mal location.<1_17> It is now known that HuIFN- genes are a family of closely related genes con¬

sisting of at least 13 nonallelic genes which are located on chromosome 9.<14-"->

'Roche Institute of Molecular Biology, Roche Center Center, Nutley, NJ 07110; department of MolecularGenetics, Hoffman-La Roche Inc., Nutley, NJ 07110.

Present addresses: 3Triton Biosciences Inc., 1501 Harbor Bay Parkway, Alameda, CA 94501; departmentof Antibiotics, National Institute of Health, 10-35 Kamiosaki 2-Chome, Shinagawa-Ku, Tokyo 141, Japan;'Department of Molecular Immunology, Merck & Co., Inc., P.O. Box 2000, Rahway, NJ 07065; 'Departmentof Molecular Genetics & Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ08854-5635.

51

HOTTA ET AL.

In addition to these distinct nonallelic genes, some cloned HuIFN- cDNAs and genes, such asHuIFN- and -a2 genes, with few nucleotide differences have been considered to be allelic. Thehuman IFN-aA cDNA was cloned from virus-induced mRNA of a continuous human myeloblastcell line, KG-1 cells,13·416-181 whereas the HuIFN-a2 cDNA was cloned from 12S RNA of virus-in¬duced human leukocytes" 51 and the IFN-a2 gene from a human genomic library in phage ."11 Thecoding sequence of these two genes is identical except for a single difference: A in the IFN-aAgene at position 137 becomes a G in the a2 gene. This results in one amino acid difference at aminoacid 23 (lysine in IFN-aA, and arginine in IFN-a2). Both proteins seem to exhibit similar activities.

To determine precisely if these two genes represent polymorphic variants of a single gene or dis¬tinct alíeles, it was useful to establish a simple and reliable method to distinguish them from eachother and from the other HuIFN- genes. Then, examination of human genomic DNAs from dif¬ferent individuals for the existence of the IFN-aA and IFN-a2 genes will answer whether or notthey are allelic.

It has been shown that deoxyoligonucleotide-DNA duplexes with a single base mismatch have a

significantly lower thermal stability than those without any mismatch."9201 Accordingly, chemi¬cally synthesized deoxyoligonucleotides complementary to unique DNA segments have been used as

probes for the detection of site-specific mutations1201 and, in combination with specific restrictionendonuclease digestion, a single base substitution in genomic DNAs such as -antitrypsin,'2'1 ß-globin/221 and c-K-nw'231 genes. Based on these reports, we prepared two deoxyoctadecyloligonu¬cleotides complementary to the sequences spanning the region of the single base substitution be¬tween IFN-aA and IFN-a2 genes as hybridization probes.

In addition, we took advantage of recognition sites for PvwII and Hinfl restriction endonucle¬ases on the two genes. Digestion of the IFN-aA and IFN-a2 genes with PvwII restriction endonucle¬ase provides a unique small size 298-bp fragment containing the single base substitution as shown inFig. 1. Furthermore, the IFN-a2 gene contains an extra Hinfl site resulting from the single basesubstitution so that digestion with Hinfl restriction endonuclease eliminates the hybridization be¬tween the IFN-a2 gene and the specific oligonucleotide probe. In this report, we describe themethod for detecting the single base differences between the IFN-aA and IFN-a2 genes with the useof two deoxyoctadecyloligonucleotide probes in combination with PvwII and Hinfl restrictionendonuclease digestion of DNA. In addition, the results of this analysis of genomic DNAs fromdifferent individuals are presented.

102 197 340 400Pvull

A Gene: 5'

Hinfl Pvull

GATGAGGAAAATCTCTCT

A Probe: 3'CTACTCCTTTTAGAGAGA 5'

102 197 340 400Pvull Hinfl Hinfl Pvull

GATGAGGAGAATCTCTCT 2 Gene: 5'-

2 Probe: 3'CTACTCCTCTTAGAGAGA 5'

FIG. 1. Deoxyoctadecyloligonucleotide probes complementary to human leukocyte interferon Aand a2 genes.

52

DETECTION OF HUMAN IFN- GENES

EXPERIMENTAL PROCEDURES

Deoxyoctadecyloligonucleotides: Two deoxyoctadecyloligonucleotides (designated as IFN- and IFN-a2 probes, as shown in Fig. 1) complementary to the sequence spanning a single base sub¬stitution between human leukocyte interferon HuIFN- and HulFN-a2 genes were prepared bythe phosphoramidite method.'24251

Plasmids: Two plasmids, pLeIFA25 coding for HuIFN- '3-41 and pBR325-a2 coding forHuIFN-a2 in which the EcoKl fragment containing HuIFN-a2 coding sequence was subcloned intopBR325,<u) were prepared from Escherichia coli 294 containing these plasmids as previously de¬scribed.1261 The plasmid pBR325-a2 was supplied by Dr. D. Goeddel (Genentech, Inc.).

Human Genomic DNAs: DNA from a human myeloblast cell line, KG-1, was prepared fromfrozen cells stored in 10% glycerol/90% medium (H18/10% FCS) in a liquid nitrogen freezer.After the frozen KG-1 cells were thawed in an ice bath and washed twice with phosphate buffer,DNA was extracted according to the method described by Pellicer et al.121'

All the other genomic DNAs were prepared from blood supplied by the American Red Crossfrom North American individuals. Each individual sample was prepared separately. After lysis ofthe reticulocytes with ammonium acetate, the total white blood cell component was collected bylow-speed centrifugation. Each pellet was resuspended in 100 mM Tris«HCI (pH 8.0)/10 mMEDTA/0.1% SDS and incubated at 42°C for 1 h with proteinase (50 /¿g/ml). The solution wasthen extracted with phenol and the aqueous phase was precipitated with ethanol. The DNA pelletwas resuspended in 100 mM Tris · HC1 (pH 8.0)/10 mM EDTA, reextracted with phenol, and pre¬cipitated with ethanol.

Preparation of 32P-Labeled Deoxyoctadecyloligonucleotides: Octadecyloligonucleotides (25-30pmole ends) and [ -32 ] (Amersham, > 5,000 Ci/mmole, 50-60 pmoles) were incubated withT4 polynucleotide kinase (5 units) in kinase buffer'281 in a total volume of 30-50 µ\ at 37°C for 45min. The 32P-labeled octadecyloligonucleotides were precipitated with ethanol with the aid of am¬monium acetate and yeast tRNA, and redissolved and reprecipitated twice as described.1291 The re¬sultant precipitates were freed from ethanol under vacuum and dissolved in a small amount ofsterile deionized water.

Hybridization of Plasmid and Human Genomic DNAs with 32P-Labeled Deoxyoctadecyloligonu¬cleotides: For dot-blot hybridization, plasmid DNA solutions of pLeIFA25 and pBR325-a2 with orwithout digestion with restriction endonucleases were dotted on nitrocellulose filters (presoaked in2x SSPE consisting of 300 /NaCl/10 mM NaH2P04/l nuV/EDTA, pH 7.4) and air-dried. TheDNAs on the filters were denatured with 0.5 M NaOH/1.5 M NaCl for 10 min and neutralized with0.5 M Tris-HCl (pH 8.0)/1.5 M NaCI for 10 min. The filters were then air-dried and baked al80°C for 2 h.

For Southern blot hybridization, the above two plasmids (200 ng) or human genomic DNAs (20-30 /ig) digested to completion with restriction endonucleases were electrophoresed in agarose gelswith TAE (40 mM Tris ·acetate/2 mM EDTA, pH 8.0) and 0.5 ¿¿g/ml ethidium bromide. DNAfragments in agarose gels were denatured and neutralized in the solutions described above for 60min at room temperature. Then, DNA fragments were transferred to nitrocellulose filters by theSouthern blotting method.'301 The Southern blotted filters were air-dried and then baked at 80°Cfor 2 h.

The baked filters were incubated at designated temperatures in the hybridization solution with32P-labeled deoxyoctadecyloligonucleotide aA or a2 probes after prehybridization in the hybridiza¬tion solution at 37 or 42°C for 2-6 h. The hybridization solution consisted of 180 mM Tris «HO(pH 8.0), 0.9 M NaCl, 12 mM EDTA, 0.1°7o SDS, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2%bovine serum albumin (Pentex Fraction V), and 100 /¿g/ml E. coli DNA. The oligonucleotides were32P-labeled to a specific activity of > 1 10s cpm/^g and each was added to the hybridization solu-

53

HOTTA ET AL.

tion at a concentration of 5-10 ng/ml. After hybridization, the filters were washed with threechanges of 100 or 200 ml of 4x SSC (1 SSC: 150 mM NaCl/15 mM sodium citrate, pH 7.0) atdesignated temperatures (1 h for each wash) and air-dried. For autoradiography, X-ray films(Kodak X-Omat film XAR-2) were exposed to the filters at -80°C for the times designated in thelegends to the figures.

RESULTSEffect of temperature and Hin// restriction endonuclease digestion on thehybridization between HuIFN-aA and HuIFN-a2 geneswith the deoxyoligonucleotide probes

Figure 2 shows the hybridization at different temperatures of the oligonucleotide aA and a2probes with plasmids pLeIFA25 and pBR325-a2 with or without digestion by PvwII or Hinfl re¬striction endonucleases. At all temperatures (25, 37, and 42°C) tested, strong hybridization was ob¬served between the aA probe and pLeIFA25 or between the a2 probe and pBR325-a2 with the com¬

plete base pair match. These hybridizations were so strong that the probes were not removed bywashing the filters with 4 SSC at the calculated dissociation temperature (Td, 50°C for the aAprobe and 52°C for the a2 probe) as reported'311 for 15 min. On the other hand, the hybridization

PROBEUSED

TREAT¬MENT

OFPLASMID

HYBRIDIZATION TO PLASMÎD DNAs AT

25°C( a2

37°C«A aZ

42°C a2

aA

None

Pvull

Hinfl

: « oc2

None Pvull · ·

Hinfl

#

FIG. 2. Effect of temperature and restriction endonuclease digestion on hybridization of humanleukocyte interferon aA and a2 genes to the aA and a2 oligonucleotide probes. Plasmid DNAs (50ng) from pLeIFA25 and pBR325-a2 coding for HulFN-aA and -a2 genes, respectively, with orwithout digestion with PvwII and Hinfl were spotted onto nitrocellulose filters. After hybridizationwas performed for 40 h, the filters were washed at 25°C and subjected to autoradiography for 12 h.

54

DETECTION OF HUMAN IFN- GENES

between the probe aA and pBR325-a2 or between the probe a2 and pLeIFA25, each pair with a

single base mismatch, was very weak when hybridization was performed at 37°C or 42°C. Signifi¬cantly strong hybridization of the above mismatched pairs was observed at 25°C. However, themismatched probes were removed by washing with 4x SSC at the elevated temperatures (37°C or42°C). Thus, a single base difference between HuIFN- and -a2 genes was clearly detected by per¬forming the hybridization with these deoxyoligonucleotide probes at 37°C or 42°C. On the otherhand, upon digestion with restriction endonucleases, the hybridization was not influenced by PvwIIbut by Hinfl as predicted from the restriction maps (Fig. 1) of the IFN-aA and IFN-a2 genes. Nosignificant hybridization was detected between ///wfl-digested pBR325-a2 and the a2 probe. This isdue to the fact that the IFN-a2, but not the IFN-aA, sequence contains an extra Hinfl recognitionsite (GAATC) at the center of the sequence complementary to the a2 probe because of the singlebase substitution. These results were confirmed by Southern blot hybridization as shown in Fig. 3.As expected from the restriction maps of pLeIFA25,(41 in which a PvwII site located at the 5' side(Fig. 1) was eliminated during construction of this expression plasmid, and pBR325-a2,"" theprobe aA hybridized strongly and exclusively to the 2.6-kb PvwII fragment and 1.2-kb Hinfl frag¬ment of pLeIFA25, whereas the probe a2 hybridized only to 298-bp PvwII fragment of pBR325-a2.

<tt.

a

3 c 3 a> ·— > ·—

cu X cu X

CMO10MCO

§a

3 tía, X

CM<

a>

a.

— 2.6 kb

— 1.2 kb

0.3 kb

aA PROBE a 2 PROBE

FIG. 3. Fragments of PvwII or Hinfl digestion of plasmids coding for aA and a2 genes hybridiz¬ing to the oligonucleotide probes.

55

HOTTA ET AL.

Detection of the sequence corresponding to HuIFN-aA and HuIFN-a2genes in human genomic DNA

Many HuIFN- genes reported so far have sequences"closely similar to those of the IFN-aA andIFN-a2 genes in terms of the sequences corresponding to the probes as shown in Table 1. It is clearthat the IFN-aA gene yields a 298-bp fragment containing the unique sequence complementary tothe probe aA by PvwII digestion. On the other hand, the IFN-aH gene has the same sequence com¬

plementary to the probe a2 except for the two bases located at the 5' and 3' ends. However, uponPvwII digestion, the IFN-aH yields a fragment larger than 900 bp containing the sequence whichcan easily be distinguished from the 298-bp fragment available from the IFN-a2 gene. All the otherIFN-a genes contain three or more mismatches with the aA or a2 probes. Thus, it is expected thatdigestions of human genomic DNA with PvwII and Pvull-Hinfl provide a 298-bp PvwII fragmentand a 237-bp Pvull-Hinfl fragment, respectively, hybridizable to the aA probe only if the HuIFN-aA gene is present. Similarly, only a 298-bp PvwII fragment hybridizable to the a2 probe is ex¬

pected if the HuIFN-a2 gene is present, when genomic DNA is digested with PvwII and PvwII-Hinfl.

Genomic DNA preparations obtained from normal human leukocytes of 13 different NorthAmerican individuals were subjected to Southern blot hybridization with the oligonucleotideprobes. Typical results with four are shown in Fig. 4. Positive bands were detected with the aAprobe at the position equivalent to 300 bp corresponding to PvwII-digested DNA and at a positionslightly smaller than 300 bp on the lanes represented by DNA digested with PvwII and Hinfl. Onthe other hand, positive bands were detected with the a2 probe at the position of about 300 bp on

lanes corresponding to PvwII-digested DNA, but not on the lanes corresponding to DNA digestedwith both PvwII and Hinfl. The same results were obtained with the other nine leukocyte genomicDNAs. Therefore, it appeared that all the leukocyte genomic DNAs contain both HuIFN- andHuIFN-a2 genes. In addition, the hybridization of KG-1 cell genomic DNA with the aA and a2

probes was also performed with similar results indicating that the sequences corresponding to bothHuIFN- and HuIFN-a2 genes are present in KG-1 cell genomic DNA from which the IFN-aA gene was cloned into E. coli as a cDNA'3 4) (data not shown). Besides the positive bands so fardescribed, several other positive bands with higher molecular sizes were also observed in the hybrid¬izations with both aA and a2 probes (Fig. 4).

Table 1. Partial Sequences of Human Leukocyte Interferon GenesComplementary to the Deoxyoligonucleotide Probes

Pvull fragmentGene Sequence complementary to the probes (bp)aA 5-GATGAGGAAAATCTCTCT-3' 298a2 G 298aB A C A G C >374aC A G A G C >970aD A C G C 301aF A G A G C >351aH A G C >900al A G A G C >1,100aJ A G A G C > 1,100aK A G A G C >920aL A G A G C > 1,050

56

DETECTION OF HUMAN IFN- GENES

300 bp

aA PROBE a 2 PROBE

FIG. 4. Hybridization of human leukocyte genomic DNAs digested with PvwII or Pvull-Hinfl tothe oligonucleotide probes. Restriction endonuclease digests were electrophoresed on a 2.0% aga¬rose gel. The Southern blotted filters were hybridized at 42°C for 65 h and washed with threechanges of 200 ml of 4 SSC at 45°C. X-ray film was exposed for 4 weeks.

DISCUSSION

As expected from the report by Wallace el al.,"'" two deoxyoctadecyloligonucleotides comple¬mentary to the sequence spanning a single base difference of G for A between HuIFN- andHuIFN-a2 genes hybridized to these genes strongly if, and only if, there is no single base pair mis¬match and weakly if there is a single base pair mismatch. Therefore, the HuIFN- gene was

clearly distinguishable from the HulFN-a2 gene when hybridization with plasmids containing thesegenes was carried out at 37°C or 42°C under the condition described. Hinfl digestion, which cutsthe lFN-a2 gene at the center of the sequence hybridizing to the a2 probe, results in the eliminationof the hybridization and provided supplementary evidence for [he existence of the IFN-a2 gene se¬

quence. It was found that 2-5 pg of the IFN-aA and IFN-a2 genes in plasmid DNAs were easily de¬tectable and distinguishable. Furthermore, the hybridization without any base pair mismatch was

so strong that the probes were not removed from the nitrocellulose filters by washing with 4 SSC,even at elevated temperatures (i.e., 55°C) higher than the dissociation temperatures (Pd) of the aA(50°C) and a2 (52°C) probes calculated as previously reported."" Therefore, more stringent condi¬tions for hybridization (temperatures between 42°C and 55°C) and washing (temperatures between42°C and 55°C or prolonged time) might be belter, especially to minimize the level of background.In fact, washing temperatures as high as the were used in recent papers to detect single base sub¬stitutions in genomic DNAs with deoxyoligonucleotide probes.'21 2"

Because very high backgrounds at the region of high molecular size were observed in genomicblot hybridization with oligonucleotide probes,"1 221 restriction fragments of relatively small sizewere generated for our analyses. In addition, among the known HuIFN- genes, the IFN-aH genemight hybridize to the a2 probe because the aH gene contains the sequence complementary to theoctadecyl a2 probe except for the terminal nucleotides at both 5' and 3' ends (Table 1). Therefore,to avoid such predictable problems, we also utilized differences in restriction maps of the known

57

HOTTA ET AL.

human leukocyte interferon genes to assist the analysis. Among the known HuIFN- genes otherthan IFN-aA and IFN-a2, PvwII digestion provides a fragment of about 300 bp containing the se¬

quence complementary to the probes only from IFN-aD as shown in Table 1. However, the IFN-aD sequence has three base differences which should result in no hybridizatiion to the aA and a2

probes under the hybridization condition used. On the other hand, the IFN-aH gene has the same

target sequence as the a2 probe except for two base substitutions at the 5' and 3' ends which mightresult in reasonable hybridization to the a2 probe. However, PvwII digestion of IFN-aH provides a

fragment longer than 900 bp which contains the target sequence to the a2 probe. Thus, it turnedout that PvwII digestion and Hinfl digestion were very useful for demonstrating the presence of theIFN-aA and IFN-a2 gene sequences in human genomic DNA. Therefore, positive bands to the aAand a2 probes detected by genomic DNA blot hybridization probably represent the IFN-aA andIFN-a2 genes.

These sequences were detected in all human genomic DNAs tested, suggesting a wide codistri-bution of the aA and a2 genes in human genomic DNA. Detection of both gene sequences in all 14human genomic DNAs suggests that they are not allelic because homozygous segregante which con¬tain only one of them should be detected more frequently if these genes were allelic. It is of interestthat both IFN-aA and IFN-a2 genes were detected in KG-1 cell genomic DNA. From KG-1 cells,only the IFN-aA gene was cloned via cDNA.'3 41 These results suggest that only the IFN-aA gene isexpressed in KG-1 cells. There exist several other positive bands to the IFN-aA probe. Since the se¬

quence of the aA gene hybridizing to the aA probe is unique among the known HulFN- genes,they might represent unidentified aA-type interferon genes, or possibly unrelated genes.

The IFN-a2 gene isolated from a human genomic DNA library in phage contains several differ¬ences in sequence from the IFN-aA cDNA clones isolated.'3·41" In addition to the single base dif¬ference in the coding sequence, there are two base differences in the 3' noncoding region and fourbase differences in the 5' noncoding region between positions 48 and 61." 4 '" These differenceswere discounted and considered insignificant so that IFN-aA and IFN-a2 were considered to beallelic variants."11 However, our results are consistent with the differences in sequence that wouldindicate that IFN-aA and IFN-a2 are, indeed, distinct nonallelic genes. If, indeed, many of theminor variants of the IFN-a genes are also distinct, the total number of IFN-a genes will increasesubstantially. Since IFN-aA and IFN-a2 are separate genes, the total number of apparently sepa¬rate HuIFN- genes is at least 14.'321 A cDNA closely related to lFN-a2 and IFN-aA was clonedfrom induced Namalva cells."3341 This [Arg34]IFN-a2 differs from IFN-a2 only in position 34where Arg-34 is substituted for His-34. It differs from IFN-aA in two positions: Arg-23 for Lys-23and Arg-34 for His-34. Whether [Arg34]IFN-a2 represents another gene, an alíele of IFN-a2 or IFN-aA, or a variant thereof, requires further study. Todokoro et al.l35) reported two human IFN-agenes, IFN-a, and IFN-a,,, with identical coding sequences, but different noncoding sequences.Thus, it is clear that closely related or even identical IFN-a products can be the product of distinctgenes.

Since the HuIFN- and HuIFN-a2 are extremely closely related, their divergence (if not conver¬

gence) must be of relatively recent evolutionary vintage. Accordingly, examination of their chromo¬somal location and detailed structure should provide useful insights into relatively recent evolution¬ary events.

Although IFN-aA was isolated as a cDNA cloned from mRNA induced in a tumor cell and IFN-a2 as a cDNA from normal induced leukocytes, it is likely their isolation from different sources are

completely fortuitous. On the other hand, because all the cDNA clones from the KG-1 cells repre¬sented IFN-aA, it is possible there is differential regulation of the IFN-aA and IFN-a2 genes.Future studies might provide valuable insights into the regulation of these genes in normal and ma¬

lignant cells.

ACKNOWLEDGMENTS

We thank Dr. Jerome Langer for providing frozen cells of human myeloblast cell line KG-1.

58

DETECTION OF HUMAN IFN- GENES

REFERENCES

1. NAGATA, S., TAIRA, H., HALL, ., JOHNSRUD, L., STREULI, M., ECSÖD1, J., BOLL, W.,CANTELL, K., and WEISSMAN, C. (1980). Synthesis in E. coli of a polypeptide with human leukocyteinterferon activity. Nature 284, 316-320.

2. MANTEI, N., SCHWARZSTEIN, M., STREULI, M., PANEM, S., NAGATA, S., and WEISSMANN,C. (1980). The nucleotide sequence of a cloned human leukocyte interferon cDNA. Gene 10, 1-10.

3. MAEDA, S., McCANDLISS, R., GROSS, M., SLOMA, ., FAMILLETTI, P.C., TABOR, J.M.,EV1NGER, M., LEVY, W.P., and PESTKA, S. (1980). Construction and identification of bacterial plas¬mids containing nucleotide sequence for human leukocyte interferon. Proc. Nati. Acad. Sci. USA 77,7010-7013; 78, 4648 (1981).

4. GOEDDEL, D., YELVERTON, E., ULLRICH, ., HEYNECKER, H.L., MIOZZAWRI, G.,HOLMES, W., SEEBURG, P.H., DULL, T., MAY, L., STEBBING, N., CREA, R., MAEDA, S., Mc¬CANDLISS, R., SLOMA, ., TABOR, J.M., GROSS, M., FAMILLETTI, P.C., and PESTKA, S.(1980). Human leukocyte interferon produced in E. coli is biologically active. Nature 287, 411-416.

5. STREULI, M., NAGATA, S., and WEISSMANN, C. (1980). At least three human type a interférons:Structure of al. Science 209, 1343-1347.

6. NAGATA, S., MANTEI, N., and WEISSMANN, C. (1980). The structure of one of the eight or more

distinct chromosomal genes for human interferon-a. Nature 287, 401-408.7. GOEDDEL, D.V., LEUNG, D.W., DULL, T.J., GROSS, M., LAWN, R.M., McCANDLISS, R., SEE-

BURG, P.H., ULLRICH, ., YELVERTON, E., and GRAY, P.W. (1981). The structure of eight dis¬tinct cloned human leukocyte interferon cDNAs. Nature 290, 20-26.

8. MAEDA, S., McCANDLISS, R., CHIANG, T.R., COSTELLO, L., LEVY, W.P., CHANG, N.T., andPESTKA, S. (1981). Sequences of a human fibroblast interferon gene and two linked human leukocyte in¬terferon genes, in: Developmental Biology Using Purified Genes. D. Brown and C.F. Fox (eds.). NewYork: Academic Press, pp. 85-96.

9. BRACK, C, NAGATA, S., MANTEI, N., and WEISSMANN, C. (1981). Molecular analysis of the hu¬man interferon- gene family. Gene 15, 379-394.

10. LAWN, R.M., ADELMAN, J., DULL, T.J., GROSS, M., GOEDDEL, D., and ULLRICH, A. (1981).DNA sequence of two closely linked human leukocyte interferon genes. Science 212, 1159-1162.

11. LAWN, R.M., GROSS, M., HOUCK, CM., FRANKE, A.E., GRAY, P.V., and GOEDDEL, D.V.(1981). DNA sequence of a major human leukocyte interferon gene. Proc. Nati. Acad. Sci. USA 78,5435-5439.

12. ULLRICH, ., GRAY, ., GOEDDEL, D.V., and DULL, T.J. (1982). Nucleotide sequence of a portionof human chromosome 9 containing a leukocyte interferon gene cluster. J. Mol. Biol. 156, 467-486.

13. LUND, B., EDLUND, T., L1NDENMA1ER, W., NY, T., COLLINS, J., LUNDGREN, E., and VONGABAIN, A. (1984). Novel cluster of a-interferon gene sequences in a placenta! cosmid DNA library.Proc. Nati. Acad. Sci. USA 81, 2435-2439.

14. OWERBACK, D., RUTTER, W.J., SHOWS, T.B., GRAY, P., GOEDDEL, D.V., and LAWN, R.M.(1981). Leukocyte and fibroblast interferon genes are located on human chromosome 9. Proc. Nati. Acad.Sci. USA 78, 3123-3127.

15. TRENT, J.M., OLSON, S., and LAWN, R.M. (1982). Chromosomal localization of human leukocyte, fi¬broblast, and immune interferon genes by means of in situ hybridization. Proc. Nati. Acad. Sci. USA 79,7809-7813.

16. PESTKA, S. (1983). The human interferons —from protein purification and sequence to cloning and ex¬

pression in bacteria: Before, between and beyond. Arch. Biochem. Biophys. 221, 1-37.17. LANGER, J.A., and PESTKA, S. (1984). Purification, bacterial expression and biological activities of the

human interferons. J. Invest. Dermatol. 83, 128-136s.18. FAMILLETTI, P.C., McCANDLISS, R., and PESTKA, S. (1981). Production of high levels of human

leukocyte interferon from a continuous human myeloblast cell culture. Antimicrob. Agents Chemothcr.20, 5-9.

19. WALLACE, R.B., SHAFFER, J., MURPHY, R.F., HIROSE, T., and ITAKURA, K. (1979). Hybridiza¬tion of synthetic oligodeoxyribonucleotides to 0 174 DNA: The effect of single base pair mismatch. Nu¬cleic Acids Res. 6, 3543-3557.

20. WALLACE, R.B., SCHÖLD, M., JOHNSON, M.J., DEMBEK, P., and ITAKURA, K. (1981). Oligonu¬cleotide directed mutagenesis of the human /3-globin gene: A general method for producing specific pointmutations in cloned DNA. Nucleic Acids Res. 9, 3647-3656.

21. K1DD, Y.J., WALLACE, R.B., ITAKURA, K., and WOO, S.L.C. (1983). al-Antitrypsin deficiency de-

59

HOTTA ET AL.

tection by direct analysis of the mutation in the gene. Nature 304, 230-234.22. CONNER, B.J., REYES, A.A., MORIN, C, ITAKURA, K., TEPLITZ, R.L., and WALLACE, R.B.

(1983). Detection of sickle cell as-globin alíele by hybridization with synthetic oligonucleotides. Proc.Nati. Acad. Sci. USA 80, 278-282.

23. GUERRERO, I., VILLASANTE, ., CORCES, V., and PELLICER, A. (1984). Activation of a c-K-rasoncogene by somatic mutation in mouse lymphomas induced by gamma radiation. Science 225, 1159-1162.

24. BEAUCAGE, S.L., and CARUTHERS, M.H. (1981). Deoxynucleotide phosphoramidites-a new class ofkey intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859-1862.

25. CHOW, F., KEMPE, T., and PALM, G. (1981). Synthesis of oligodeoxyribonucleotides on silica gel sup¬port. Nucleic Acids Res. 9, 2807-2817.

26. DAUGHERTY, B., MARTIN-ZANCA, D., KELDER, B., COLLIER, K., SEAMANS, T.G., HOTTA, ., and PESTKA, S. (1984). Isolation and bacterial expression of a murine alpha leukocyte interferongene. J. Interferon Res. 4, 635-643.

27. PELLICER, ., WIGLER, M., AXEL, R., and SILVERSTEIN, S. (1978). The transfer and stable inte¬gration of the HSV thymidine kinase gene into mouse cells. Cell 14, 133-141.

28. MAXAM, A.M., and GILBERT, W. (1980). Sequencing end-labeled DNA with base-specific chemicalcleavages. Methods Enzymol. 65, 499-560.

29. HOTTA, K., COLLIER, K., and PESTKA, S. (1986). Detection of a single base substitution between hu¬man leukocyte interferon aA and a2 genes with octadecyl deoxyoligonucleotide probes. Methods Enzy¬mol. 119, 481-485.

30. SOUTHERN, E. (1975). Detection of specific sequences among DNA fragments separated by gel electro-phoresis. J. Mol. Biol. 98, 503-517.

31. SUGGS, S.U., H1ROSE, T., MIYAKE, T., KAWASHIMA, E.H., JOHNSON, M.J., ITAKURA, .,and WALLACE, R.B. (1981). Use of synthetic oligodeoxyribonucleotides for the isolation of specific-cloned DNA sequences, in: Developmental Biology Using Purified Genes. D. Brown and C.F. Fox (eds.).New York: Academic Press, pp. 683-693.

32. PESTKA, S. (1986). Interferon from 1981 to 1985. Methods Enzymol. 119, 3-14.33. DWORK1N-RASTL, E., DWORKIN, . ., and SWETLY, P. (1982). Molecular cloning of human alpha

and beta interferon genes from Namalwa cells. J. Interferon Res. 2, 575-585.34. BODO, G., and FOGY, I. (1985). Characterization of different molecular species in affinity purified re-

combinant human interferon alpha 2, in: The Interferon System, vol. 24. F. Dianzani and G. Rossi (eds.).Serono Symposia Publications, New York: Raven Press, pp. 23-27.

35. TODOKORO, K., KIOUSSIS, D., and WEISSMANN, C. (1984). Two non-allelic human interferon-alpha genes with identical coding regions. EMBO J. 3, 1809-1812.

Address reprint requests to:Dr. Sidney Pestka

University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School

Department of Molecular Genetics and MicrobiologyPiscataway, NJ 08854-5635

Received 31 July 1985/Accepted 18 June 1987

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