JOURNAL OF INTERFERON RESEARCH 5:391-395 (1985)Mary Ann Liebert, Inc., Publishers

Subchromosomal Localization of Mouse IFN-aGenes by In Situ Hybridization

LINDA A. CAHILLY, DONNA GEORGE, BRUCE L. DAUGHERTY,1and SIDNEY PESTKA1

ABSTRACT

We have utilized in situ hybridization techniques to determine the chromosomal locationof alpha-interferon (IFN-a) genes in the mouse. The results allow regional assignment ofthe IFN-a gene family to a position close to the center of chromosome 4, in bands C3-C7.

INTRODUCTION

Interferons comprise a family of proteins that are produced by animal cells in response to viralinfection as well as to a number of other substances.'1·2' These proteins can be divided into three

classes: leukocyte or alpha-interferons (IFN-a), fibroblast or beta-interferons (IFN-/3), and immuneor gamma-interferon (IFN- ). The mouse provides a convenient animal model system to explore thegenetic interactions affecting the expression of and cellular responses to the various interferons. Re¬cently, sequences comprising the murine IFN-a gene family have been assigned to chromosome 4based on hybridization of radiolabeled IFN-a cDNA probes to DNAs from Chinese hamster-mousesomatic cell hybrids in Southern blot analyses.'3·4' We have confirmed and extended these studies todetermine the subchromosomal position of IFN-a by in situ hybridization of a mouse IFN-a molecu¬lar probe to mouse metaphase chromosomes.

EXPERIMENTAL PROCEDURES

Metaphase chromosome spreads for in situ hybridization were prepared from the 416B murine cellline, which was established from a long-term culture of Friend murine leukemia virus-infected fe¬male BDFt bone marrow cells.'5' A trypsin-Giemsa banded karyotype of the 416B cells is presentedelsewhere.'6' The 416B cells were chosen for this study because the chromosome constitution differslittle from that of normal mouse cells in terms of chromosome number and G-banding pattern, andthey have a high mitotic index. The cells exhibit two Robertsonian translocations, one involvingchromosomes 2 and 10, and another involving chromosomes 12 and 18. Thus, the chromosomenumber has been reduced to 38 from the usual 40. In addition, a small amount of extra material ispresent at the distal end of the nontranslocated chromosome 12 and of an X chromosome. All otherchromosomes have banding patterns that are indistinguishable from normal chromosomes. Tetra-ploidy was observed in less than 1% of the 300 metaphases examined.

The plasmid probe used for the in situ hybridization analysis, pBD2, contains a 675-bp insertion in

Department of Human Genetics, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104.'Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.

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CAHILLYETAL.

pBR322. The insert includes the entire sequence of the mouse IFN-aA gene.'7' The fragment was

originally isolated from a mouse genomic library on the basis of homology to a human IFN-a clone(Hu-IFN-a/1).'2' The sequence of the Mu-IFN-aA gene exhibits greater than 90% nucleotide homol¬ogy with the sequences of other published murine IFN-a clones.<8.99a) Thus, in situ hybridizationanalysis with this probe is likely to detect other IFN-a. genes in addition to the gene from which it was

derived.In situ hybridization and 3H-labeling of probe DNA were performed according to a modifica¬

tion'10' of a standard protocol.'11' Nick-translation was carried out with 1 ¿ig of plasmid pBD2in50mM Tris-Cl (pH 7.2), 10 mM MgS04, 0.1 mM dithiothreitol containing 20 µ each of [3H]dATP,[3H]dCTP, [3H]dGTP, and [3H]dTTP (Amersham, sp. act. 12-30 Ci/mmol). The reaction was

started with 5 units of DNA polymerase I (Amersham) and 0.1 ng of DNase (Amersham) and incu¬bated at 15°C for 2 h. The solution was extracted once with phenol and loaded into a Sephadex G-25column. Elution was performed with 50 mM Tris-Cl (pH 8.0), 10 mMEDTA, 0.1% SDS, and 150mMNaCl. Fractions of 0.5 ml were collected. Two peak fractions were pooled. The specific activityattained was about 1.7 IO7 cpm//¿g.

Chromosomes were harvested in the presence of 5 /¿g/ml ethidium bromide"2' to minimize chro¬mosome contraction and increase the number of metaphase spreads with well-banded chromosomes.We had determined previously that such treatment does not alter the efficiency or pattern of in situhybridization (L.A. Cahilly and D. George, unpublished results).

RESULTS AND DISCUSSION

The results of the in situ hybridization analysis are given in Fig. 1. We found significant hybridiza¬tion to mouse chromosome 4. Of 117 labeled sites present on 92 metaphase spreads, 31 (26%) were

located on chromosome 4 (Fig. 1 A). The remainder of the grains were randomly distributed amongthe other chromosomes. Of the grains specific for chromosome 4, 84°7o (26/31) were located over

bands C3-C7 (Fig. IB). Representative metaphase spreads demonstrating hybridization of pBD2 tothis region are shown in Fig. 2. These data therefore confirm the assignment of IFN-a to mouse

chromosome 4, and provide strong evidence that the gene family can be assigned to bands C3-C7.With the use of a panel of somatic cell hybrids from Chinese hamster and murine cells, some of

which contained only part of murine chromosome 4, Van Der Korput et /."3' have localized theMu-IFN-a gene family and the Mu-IFN-ß gene between the centromere and the locus for 6-phospho-gluconate dehydrogenase (PGD), a marker located at the distal end of chromosome 4. <14) The resultsof a study by Dandoy et al. '"' suggest that the Mu-IFN-a gene family is closely linked to the histo-compatibility locus H-15 on chromosome 4. This conclusion was based on Southern blot analysis ofDNAs isolated from inbred strains of mice that exhibited restriction fragment length polymorphismsassociated with IFN-a genes. On genetic linkage maps, the mouse H-15 locus is approximately 55map units from the centromere, or slightly distal to the midpoint of the chromosome. Although dis¬tances on linkage maps are not directly comparable to those of cytologie maps based on bandedmetaphase chromosomes, the position of H-15 corresponds well to the cytologically defined locationof the IFN-a genes determined by our in situ hybridization analysis. Thus, these results <13·15» are

consistent with our assignment by in situ hybridization of the Mu-IFN-a locus to a position near thecenter of mouse chromosome 4, in bands C3-C7.

ACKNOWLEDGMENT

This work was supported by NIH Grants CA-34462 and GM-32592. L.C. is supported by NIHPredoctoral Training Grant 5T32-GM-07071.

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FIG. 1. In situ hybridization of 3H-labeled IFN-a probe pBD2 to 416B mouse chromosomes. A.Distribution of 117 labeled sites among chromosomes from 92 metaphase spreads. Each chromo¬some was divided into three equal-sized parts, and the number of grains found in each region is re¬corded on the chart. The translocation and marker chromosomes were originally scored indepen¬dently, but they are included with their normal counterparts for the purposes of this illustration.B. An idiogram of mouse chromosome 4,"6' detailing the distribution of labeled sites.

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REFERENCES

1. LENGYEL, P. (1982). Biochemistry of interferons and their actions. Ann. Rev. Biochem. 51, 251-282.2. PESTKA, S. (1983). The human interferons

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From protein purification and sequence to cloning and ex¬

pression in bacteria: Before, between and beyond. Arch. Biochem. Biophys. 221, 1-37.3. KELLY, K.A., KOZAK, CA., DANDOY, F., SOR, F., SKUP, D., WINDASS, J.D., DeMAEYER-

GUIGNARD, J., PITHA, P., and DeMAEYER, E. (1983). Mapping of murine interferon- genes to chro¬mosome 4. Gene 26, 181-188.

4. LOVETT, M., COX, D.R., YEE, D., BOLL, W., WEISSMANN, C, EPSTEIN, C.J., and EPSTEIN,L.B. (1984). The chromosomal location of mouse interferon- genes. EMBO J. 3, 1643-1646.

5. DEXTER, T.M., ALLEN, T.D., SCOTT, D., and TEICH, N.M. (1979). Isolation and characterization ofa bipotential haematopoietic cell line. Nature 277, 471-474.

6. CAHILLY, L.A., and GEORGE, D., Cytogenet. Cell Genet, (in press).7. DAUGHERTY, B., MARTIN-ZANCA, D., KELDER, B., COLLIER, K., SEAMANS, T.C., HOTTA,

K., and PESTKA, S. (1984). Isolation and bacterial expression of a murine alpha leukocyte interferon. J.Interferon Res. 4, 635-643.

8. SHAW, G.D., BOLL, W., TAIRA, H., MANTEI, ., LENGYEL, P., and WEISSMANN, C. (1983).Structure and expression of cloned murine IFN- genes. Nucleic Acids Res. 11, 555-573.

9. ZWARTHOFF, E.C., MOOREN, A.T.A., and TRAPMAN, J. (1985). Organization, structure and expres¬sion of murine interferon alpha genes. Nucleic Acids Res. 13, 791-804.

9a. KELLEY, K.A., and PITHA, P.M. (1985). Characterization of a mouse interferon gene locus I. Isolationof a cluster of four interferon genes. Nucleic Acids Res. 13, 805-823.

10. CANNIZZARRO, L.A., and EMANUEL, B.E. (1984). An improved method for G-banding chromosomesafter in situ hybridization. Cytogenet. Cell Genet. 38, 308-309.

11. HARPER, M.E., ULLRICH, ., and SAUNDERS, G.F. (1981). Localization of the human insulin gene to

the distal end of the short arm of chromosome 11. Proc. Nati. Acad. Sci. USA 78, 4458-4460.12. IKEUCHI, T. (1984). Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its

application to high resolution chromosome banding. Cytogenet. Cell Genet. 38, 56-61.13. VAN DER KORPUT, J.A.G.M., HILKENS, J., KROEZEN, V., ZWARTHOFF, E.C., and TRAPMAN,

J. J. Gen. Virol, (in press).14. BUCKLE, V.J., EVANS, E.P., JONASSON, J.A., LYON, M.F., PETERS, J., SEARLE, A.G., and

WEDD, N.S. (1984). Chromosome maps of man and mouse II. Clin. Genet. 26, 1-11.15. DANDOY, F., KELLEY, K.A., DeMAEYER-GUIGNARD, J., DeMAEYER, E., and PITHA, P.M.

(1984). Linkage analysis of the murine interferon- locus on chromosome 4. J. Exp. Med. 160, 294-302.16. NESBITT, M., and FRANCKE, U. (1973). A system of nomenclature for band patterns of mouse chromo¬

somes. Chromosoma 41, 145-158.

Address reprint requests to:Dr. Donna L. George

Department of Human Genetics195 Medical Laboratory Building

University of PennsylvaniaPhiladelphia, PA 19104

Received 18 December 1984/Accepted 11 February 1985

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