Proc. Nat. Acad. Sci. USAVol. 71, No. 6, pp. 2251-2255, June 1974

The Somatic Cell Genetics of Human Interferon: Assignment of HumanInterferon Loci to Chromosoines 2 and 5

(somatic cell hybridization/regulation)

Y. H. TAN, R. P. CREAGAN, AND F. H. RUDDLE

Department of Biology, Yale University, Kline Biology Tower, New Haven, Connecticut 06520

Communicated by Edward A. Adelberg, March 18, 1974

ABSTRACT A total of 40 mouse-human cell hybridswere induced with poly(I) * poly(C) and with Newcastledisease virus. Ten hybrid clones produced low levels ofhuman interferon in response to bqth the virus and poly-(I)-poly(C). Concurrent enzymic and chromosomal an-alysis of the hybrid clones indicated a concordant segrega-tion of these levels ofhuman interferon with chromosomes2 and 5. The presence of both chromosomes 2 and 5 wasnecessary for interferon production; neither was sufficientalone. Based on this finding, we postulate that there areat least two asyntenic gene loci for the expression of inter-feron in human cells.

In previous studies using human-mouse somatic cell hybrids,we have provided evidence for the syntenic association be-tween the gene that codes for the dimeric form of indophenoloxidase (IPO-A) and a genetic factor that codes for the anti-viral state (A VP) induced by interferon, and for their assign-ment to chromosome 21 in human cells (1). Anti-viral proteinis the presumed protein factor that mediates the inhibitionof virus replication in interferon-treated cells (2). The assign-ment of AVP and IPO-A to chromosome 21 is consistent withan earlier unpublished result by Nabholz (3) that the anti-viral action of interferon segregated concordantly with IPO-Ain mouse-human hybrid clones. Indophenol oxidase was re-

cently shown by Beckman (4) and by Brewer (5) to be a

cytoplasmic form of superoxide dismutase (EC 1.15.1.1) whosepresumed physiological function is to oxidize free oxygen

radicals (02-) in aerobic organisms (6).In this paper, we report experiments that show the existence

of two or more genetic factors assignable to chromosomes 2and 5, both of which are required for the expression of hu-man interferon. In addition, evidence will be presented tostrengthen the hypothesis that the gene(s) for expression ofthe anti-viral state and the gene(s) for the expression of in-terferon synthesis are located in separate chromosomes, as

earlier reported (1, 7).

MATERIALS AND METHODS

Cell Hybridization and Culture Methods. Hybrid cell popula-tions were produced by mixing mouse and human cells to-gether in a 1:4 or 1:10 human-to-mouse cell ratio at a totaldensity of 4 X 106 cells per 25-cm2 Falcon (Oxnard, Calif.)culture flask or at a cell density of 4 X 101 cells per ml andstimulated to fuse by fl-propiolactone-inactivated Sendai virus

Abbreviations: NDV, Newcastle disease virus; AVP, IPO-A, andHuIF, genes for anti-viral protein, indophenol oxidase, and humaninterferon, respectively.

2251

(1 ml) at a concentration of 1000 hemagglutinating units perml (8). Hybrids were selected inAA medium (7 .g/ml of alano-sine and 50 j&M adenine) (9) or in HAT medium (0.1 mM hy-poxanthine, 10 pM aminopterin, 40 juM thymidine, and 10,uM glycine) (10) in Dulbecco-Vogt modified Eagle's minimalmedium (DVME) from Grand Island Biologicals and supple-mented with 10% fetal-calf serum, 100 units/ml of penicillin,and 100 ug/ml of streptomycin. The hybrids can be isolatedfrom the human parental cells by their different morphologyand their more rapid growth rate over the human parentalfibroblasts. In those hybrids that were formed from a humanleucocyte parent and a mouse fibroblast, fusion was performedwith the cells in suspension (8).

Six different series of mouse-human hybrid clones wereused: (i) J hybrids, resulting from fusion of human peripheralleukocytes with a hypoxanthine phosphoribosyltransferasedeficient mouse-cell line (RAG) (11); (ii) JBA (1) and (iii)JFA hybrids, derived, respectively, from the fusion of humanleukocytes (JBA) and skin fibroblasts (JFA) obtained from acarrier of a 14/22 fusion product, with the mouse L-cell lineA-9, which is spontaneously deficient in adenine phosphori-bosyltransferase as well as being deficient in hypoxanthinephosphoribosyltransferase (1, 9, 12); (iv) WA hybrids, derivedfrom the fusion of A-9 with the human lung fibroblast cellstrain WI-38 (1, 9); (v) RK hybrids, derived from the fusionof human fibroblasts (KOP-1 and KOP-2), which carryreciprocal X/14 translocations with RAG (13); and (vi) WIGhybrids, derived from the fusion of RAG with WIL-2 (14), ahuman spleen-derived lymphocytic line. The WIG hybridswere derived in the laboratory of J. Eisenstadt, Dept. ofHuman Genetics, Yale College of Medicine. A total of 34clones and 12 subclones were tested for the production ofmouse and human interferon. The RK series (six clones) wassubsequently lost and is not included in our present data ex-cept that five of the six RK hybrid' clones could be inducedNewcastle disease virus for a low level of human interferonproduction (16-48 units of interferon per 2 X 106 cells),and that chromosomal analysis showed that chromosome 5was present in all the five hybrid clones that tested positivefor human interferon with chromosome 2 present in at-leasttwo of these positive clones.

Gel Assays of Human Enzyme Markers. Hybrid clones wereexamined for the human forms' of the following 22 enzymes bystarch-gel and acrylamide-gel electrophoresis (15, 16): adeno-sine deaminase (EC 3.5.4.4), adenine phosphoribosyltrans-ferase (EC 2.4.2.7), aspartate aminotransferase (EC 2.6.1.1),

Proc. Nat. Acad. Sci. USA 71 (1974)

FIG. 1. Karyotype of a cell from clone JFA 14a-13 that produced human interferon. The human chromosomes (numbered chromosomes)include both chromosomes 2 and 5.

glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose-phosphate isomerase (EC 5.3.1.9), hypoxanthine phosphori-bosyltransferase (EC 2.4.2.8), isocitrate dehydrogenase (EC1.1.1.42), indophenol oxidase-B (tetrameric form) (mito-chondrial superoxide dismutase), indophenol oxidase-A(dimeric form) (cytosol superoxide dismutase), lactate de-hydrogenase-A (EC 1.1.1.27), lactate dehydrogenase-B (EC1.1.1.27), malate oxidoreductase (decarboxylating) (EC1.1.1.40), malate dehydrogenase (EC 1.1.1.37), mannose-phosphate isomerase (EC 5.3.1.8), purine-nucleoside phos-phorylase (EC 2.4.2.1), peptidase-A, peptidase-B, peptidase-C, peptidase-D, phosphogylcerate kinase (EC 2.7.2.3), phos-phoglucomatase-1 (EC 2.7.5.1), and thymidine kinase (EC2.7.1.21).

Chromosome Analysis. The human chromosomes were iden-tified in metaphase cells prepared to reveal quinacrine mustardbanding (17) (Fig. 1). To facilitate the identification ofchromosomes, metaphases that were photographed with ul-traviolet illumination for quinacrine mustard banding werealso photographed with dark-field illumination in the visiblespectrum.

Interferon Inducers. Newcastle disease virus (NDV, Hertsstrain) was grown in chick embryos (Spafas, Inc., Norwich,Conn.). A virus preparation in 0.85% (w/v) NaCl was ob-tained by centrifugation at 80,000 X g for 2 hr and contained1.0 X 108 plaque-forming units per ml, as titrated on chickembryonic fibroblast cultures (18). For the induction of in-terferon, mouse-human hybrid clones (2 X 106) were incu-bated at 370 for 1 hr with 0.2 ml of NDV.

Double-stranded poly(I) -poly(C) (P.L. Biochemicals, Wis-consin) at 100 ,ug/ml admixed with DEAE-dextran (20,ug/ml)in half-strength modified Eagle's medium without calf serumwas freshly prepared for each experiment. Two milliliters ofthe mixed poly(I) * poly(C) and DEAE-dextran was incubatedfor 3 hr with each hybrid clone culture to induce interferonproduction. To superinduce interferon production, cultureswere incubated with poly(I) -poly(C) and DEAE-dextran inthe presence of cycloheximide (25 gg/ml) for 3 hr and thenwashed twice with Hank's balanced salt solution at 5 ml perwash. Actinomycin D was added to the cultures at hr 7. Afterthe cultures were induced with poly(L) - poly(C) and the finalconcentration of actinomycin D was 2.5 jsg/ml at hr 8, thecultures were washed four times with Hank's solution at 5ml per wash and refed with 2 ml of modified Eagle's medium.The rationale and details of superinduction have been de-scribed (18-21). After interferon induction, the virually in-duced cultures were washed three times, whereas the poly(I).poly(C)-induced cultures were washed four times at 5 ml perwash. Two Falcon flasks (25-cm2) containing a monolayerof 2 to 3 X 106 hybrid cells were used for each induction, andfluids from the two cultures were pooled for interferon titra-tion. Subsequent to the harvest of fluid for interferon fromthe cultures, the cultures were disrupted by alternate freezingand thawing of the monolayers three times and then harvest-ing of the lysed cells in 1 ml of modified Eagle's medium witha sterile rubber scraper. The concentrations of cycloheximideand actinomycin D used inhibited the incorporation of [14C]-aminoacids and the incorporation of [3H]uridine about 90and 92%, respectively, in the parental mouse cells. All sam-ples of NDV-induced interferon were dialyzed against 0.15

2252 Genetics: Tan et al.

Somatic Cell Genetics of Human Interferon 2253

TABLE 1. Correlation of human interferom production itmththe presence of individual human chromosome

Con- Dis-cordant cordantclones clones

Human Interferon/chromosome (+/+ (+/-chromo- and andsome +/+ +/- -/+ I - /-1-) -/+)

1 2 8 12 18 20 202 10 0 9 21 31 93 2 8 6 24 26 144 0 10 8 22 22 185 10 0 2 28 38 26 2 8 7 23 25 157 1 9 9 21 22 188 5 5 7 23 28 129 0 10 0 30 30 1010 2 8 8 22 24 1611 7 3 7 23 30 1012 2 8 10 20 22 1813 2 8 5 25 27 1314 6 4 7 23 29 1115*16 7 3 17 13 20 2017 7 3 11 19 26 1418 10 0 13 17 27 1319 2 8 6 24 26 1420 4 6 6 24 30 1021 2 8 10 20 22 1822 5 5 11 19 24 16X 0 10 14 16 16 24Y 0 10 0 20 20 20

(+/+) HuIf positive/chromosome positive; (+/-) HuIfpositive/chromosome negative; (-/+) HuIf negative/chromo-some positive; (-/-) Huff negative/chromosome negative.

* Chromosome l5 could not be reliably identified in the mouse-human clones.

M HCl-KCl buffer at pH 2.0 for 5 days at 4°. The sampleswere readjusted to physiological pH by dialysis for 4-8 hr inmodified Eagle's medium without calf serum. All sampleswere centrifuged at 2000 X g for 15 min to remove cell debrisbefore they were titrated for interferon. The samples treatedat pH 2.0, when titrated on chick embryonic fibroblasts, didnot show any virus plaques. The treated interferon sampleswere then assayed for mouse and for human interferon onmouse A-9 or RAG cells and on human foreskin fibroblasts bya semi-microassay procedure (21). For each titration, an in-ternal standard of mouse and human interferon was used, andall values were adjusted with respect to these standards.

RESULTSA total of 28 independent derived mouse-human hybridclones and 12 subclones were induced with poly(I) - poly(C)and NDV in order to correlate the human interferon pheno-type with the presence of specific human chromosomes and/orhuman enzymes. Correlation tests between human interferon,human chromosomes, and human enzymes showed an absenceof concordance with human interferon and any of the humanlinkage groups tested individually (Table 1). However humanchromosomes 2, 5, and 18 were present in all clones that pro-duced human interferon for which the chromosome constitu-tion was known. Chromosome 18 could be ruled out based on

TAIBLE 2. Correlation of human interferon production withchromosomes 2 and 5-

Poly(I) * poly(C)-NDV-induced induced

interferon interferon(no. of clones) (no. of clones)

Human chromosomes + - + -

A. Primary clones2+ 5+ 5 0 5 02+ 5- 0 5 0 52- 5+ 0 2 0 22- 5- 0 16 0 16

B. Subclones2+ 5+ 5 0 5 02+ 5- 0 4 0 42- 5+ 0 0 0 02- 5- 0 3 0 3

enzyme data from additional clones in which the chromosomeconstitution was not examined. The gene for human peptidaseA has been assigned to chromosome 18, and in three of theseadditional clones that produced human interferon, peptidaseA was absent. Chromosome 2 and particularly chromosome5 showed a markedly higher degree of concordance withhuman interferon expression in comparison to the otherchromosomes. We reorganized the data in order to test thecombined effect of chromosomes 2 and 5 on human interferonexpression. The primary clone data showed that only clonesthat possessed both chromosomes 2 and 5 together expressedhuman interferon. The clones were carefully examined forthe frequencies of these chromosomes in the hybrids, wherefrequency represents percentage of cells that possess one ormore of the chromosomes in question. In human interferon-negative clones that lacked chromosome 2 but possessedchromosome 5, chromosome 5 was present at a frequency of50% or greater. The same quantitative relationship obtainedin human-negative clones that lacked chromosome 5 but re-tained chromosome 2. In order to confirm the relationship be-tween chromosomes 5 and 2 and human interferon production,two primary clones, JFA-16a and JFA-14a, which retainedchromosomes 2 and 5, were subeloned. These subelones segre-gated chromosome 5, but not chromosome 2. The clones thatlost chromosome 5 also lost the capacity to produce humaninterferon when challenged by poly(I) poly(C) or NDV. Thecombined data from primary clones and subelones werearranged to test the combined effect of chromosomes 2 and 5on human interferon (Table 2).We cannot presently reliably detect human chromosome 15

either cytologically or inferentially by isoenzyme assay. Webelieve it to be unlikely that chromosome 15 plays a role inthe regulation of human interferon, because of the positivecorrelations already demonstrated for chromosomes 2 and 5.Still, it must be kept in mind that the possible involvement ofchromosome 15 has not yet been formally excluded. In sum,we believe the data strongly support the hypothesis thatchromosome 2 and chromosome 5 carry genetic factors bothor which are required simultaneously for the induction ofhuman interferon with poly(I) * poly(C) and NDV.The genes for the anti-viral protein (A VP) and indophenol

oxidase-A (IPO-A) (super oxide dismutase, cytosol) have been

Proc. Nat. Acad. Sci. USA 71 (1974)

Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 3. Into(units produced by 2 X 108 cei

Induction ofinterferon

poly(I) epoly(C) +DEAE-

Cell type dextra

Parental cellsWI38 <2Human

fibroblasts* <2Human

fibroblastst <2Human

leukocyte <2Mouse A9 <2Mouse RAG <2

Primary clonesJFA 12a N.D.JFA 14a <2JFA 15f <2JFA 16a <2JFA 19 <2WaIa <2WaIla <2WaIIIa <2WaIVa <2WaVa <2WaVIa <2WaVIIa <2WaVIIIa <2WaIXa <2JBA-la <2J3S N.D.W-1 <2W-4 <2W-5 <2W-8 <2W-10 <2W-12 <2W-13 <2W-15 <2W-17 <2W-20 <2W-21 <2W-23 <2

Subclones of J1OHtJ1OH7 N.D.J1OH9 N.D.JlOH12 N.D.

Subelones of JFA-14a2a6b1323

<2N.D.N.D.N.D.

erferon production assigned to chromosome 21. Two human interferon-positivells) in mouse-human cell hybrids clones, JFA-14a and JFA-16a, which retained chromosomes 2

mouse Induction of human and 5 but lacked chromosome 21, were protected by mouse,by interferon by but not human, interferon against a vesicular stomatitis virus

challenge. This result further confirms the asyntenic relation-Poly(I) * ship between the AVP and the human interferon loci.

ply(CA) + The amount of mouse interferon produced as a consequence

NDV dextran NDV of NDV induction was comparable between mouse parentsand hybrid clones, with two exceptions (Table 3). Clones JFA-16a and JBA-1 produced 10-16 times more mouse interferon

2 4 12 than the mouse parents and hybrids. We have been unable tocorrelate this high level of production with other attributes

2 98 502 of these clones, such as chromosome constitution and isozyme

2 90 256 expression, except that both of these clones had a doublemurine chromosome constitution (i.e., 2s). Other clones with

2 <2 1200 2s inputs were not, however, high producers. The results sug-60 <2 <2 gest a possible interaction between the mouse and human98 <2 <2 genome that could involve dosage effects for either the inter-

feron structural or regulatory genes of either parent. Ten of72 <2 <2 the human interferon-positive clones produced human inter-32 4, 8, 4§ 4, 8, 16§ feron at levels 1-10% of levels produced in the human parental32 <2 <2 cells (Table 3). We cannot currently explain the reduced pro-

512 16, 32, 8§ 16, <4, 4§ duction of human interferon, although at least three possibili-64 <2 <2 ties come to mind. (i) The intracellular milieu of the hybrid32 <2 <2 cells may in some way repress human interferon synthesis.32 <2 <2 (ii) The mouse and human interferon may form a heteropoly-48 <2 <2 meric complex with low human specific activity. The demon-

32 <2 <2 stration by Carter (22) that human interferon forms a homo-

48 <2 <2 polymer is consistent with this suggestion. (iii) The human

64 <2 <2 chromosomes are present in only a fraction of the cells in any32 <2 <2 clone, and, moreover, any human gene is invariably present at64 <2 <2 a lower dosage than any corresponding mouse gene. Because

960 <2 <2 the production of human interferon was low in the hybrid cells,256 <2 <2 the clones were also superinduced for interferon production.128 4, 8§ 32, 4, 16§ The hybrid clones positive for human interferon produced120 4, 8§ 16, 16, 64§ higher amounts of human interferon upon superinduction,

256 <2 <2 whereas negative clones still showed no detectable amounts of128 <2 <2 human interferon. This result further substantiates the view

128 <2 <2 that human interferon-negative clones result from a loss of

128 <2 <2 specific human genetic components, and not as a consequence164 <2 <2 of an insensitive assay system.128 <2 <2 Human and mouse parental cells of different epigenetic128 <2 <2 types respond differently to interferon induction by NDV and128 <2 <2 poly(I) * poly(C). The mouse parental cell lines used responded128 8, 16, <2§ 2, 4, <2§ to NDV but not to poly(I) - poly(C). Human primary, diploid

fibroblasts responded both to NDV and poly(I) - poly(C),256 <2 <2 whereas the human transformed pseudodiploid lymphocyte256 N.D. <2 cell line Wil-2a responded to NDV but not to poly(I) *poly(C).256 N.D. <2 In the hybrids, mouse interferon was produced only in re-

sponse to NDV. Thus, the murine genome responds in a way256 <2 <2 consistent with the response of the mouse parental cells. Hy-

N.D. <2 <2 brid cells positive for human interferon responded both toN.D. 16, 4, 16§ 4, 4§ NDV and poly(I) -poly(C) irrespective of the parental cellN.D. <2 <2 origin. Thus, even when the human chromosomes were derived

Subelones of JFA-16A5 <2 625 4, 16§8 <2 62 64, 200§19 <2 62 64, 32§21 <2 80 <210 <2 125 32

4, 16§44

<24

(Notes to Table 3 are at the bottom of the next column.)

TABLE 3 (notes).Human interferon was assayed on human foreskin fibroblasts;

mouse interferon was assayed on mouse A9 or mouse RAG cells.N.D., not done.

* Contain a 14/22 centric fusion.t Contain a 14/X translocation.t J1OH was not assayed for interferon production.§ Repeated assays.

an

2254 Genetics: Tan et al.

Somatic Cell Genetics of Human Interferon 2255

from a lymphocyte parent, the hybrid cell responded bothto NDV and poly(I) poly(C). In such instances, the hybridcontinued to respond only to NDV in terms of mouse inter-feron production. We cannot yet explain these results, butthe patterns suggest the presence of a genetic factor that isactive in human lymphocytes that represses induction bypoly(I) poly(C). If this is so, then it must be asyntenic withchromosomes 2 and 5. The data would also presuppose speciesspecificity and noncrossreactivity, since in many hybridsmouse interferon can only be induced by NDV, whereashuman interferon can be induced both by NDV and poly-(I) * poly(C).

DISCUSSIONFrom this study we have traced human interferon productionto two human chromosomes, chromosomes 2 and 5, by con-cordant segregation. In all the hybrid clones and the derivedsubclones tested we found that human interferon productioninduced by NDV and by poly(I) poly(C) segregated con-cordantly, suggesting that the same genetic factors are in-volved in the inducibility of interferon production by the twoinducers. The assignment of human interferon to the twohuman chromosomes, we believe, represents the first assign-ment of an inducible phenotype to more than one chromo-some.Assuming that chromosome 15 is not involved in the ex-

pression of human interferon, there are at least three possibleexplanations for the assignment of human interferon to thetwo human chromosomes. First, one of the two chromosomesmay contain a genetic factor that codes for a specific receptorsite necessary for the processing of interferon inducers intosignals that activate the structural gene for interferon produc-tion; the other human chromosome may contain the struc-tural gene for interferon. Second, both chromosomes may con-tain genes that code for interferon subunits. Third, one chro-mosome may contain a structural gene for a precursor inter-feron such as preinterferon (23), with the other containinga gene for a factor involved in the processing of the precursorto an active form.The assignment of the genes for interferon production re-

ported here provides'a unique opportunity to compare homol-ogous linkage groups in humans and the lower primates,specifically the African green monkey, Cercopithecus aethiops.Cassingena et al. (7) reported that the structural gene for in-terferon in African green monkey could be assigned to a smallsubtelocentric chromosome, probably A8 or A9 according tothe classification of Stock and Hsu (24). Stock and Hsu (24),in comparing the banding patterns of the rhesus macaque

(Macaca mulatta), African green monkey, and human chromo-somes, indicated homology of rhesus chromosome A7, Africangreen monkey chromosomes A8 and A9, and human chromo-some 5. Such comparisons can provide useful insights intothe evolution and conservation of linkage groups. More ex-tensive data for a number of primates comparing homologouschromosomal banding patterns synteny groups, and chromo-some assignments of genes should prove to be valuable fordetermining the mechanisms of chromosomal aberration andkaryotypic evolution.

We thank B. Carritt, D. Wallace, C. Tsai, and J. Eisenstadtfor making the WIG cells available. We thank S. Chen and E.Nichols for excellent technical assistance and M. Reger for herpatient preparation of the manuscript. This work was supportedunder USPHS GM 19952.

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Proc. Nat. Acad. Sci. USA 71 (1974)