© 1991 S. Kargcr AG. Basel 0301-0171/91/0573-0117S2.75/0

Cytogenet Cell G enet 57:117-118 ( 1991 )

Chromosomal location of the human tumor necrosis factor receptor genes E. Baker,1 L.Z. Chen.1C.A. Smith,2 D.F. Callen,1 R. Goodwin,2 and G.R. Sutherland 1 1Departm ent o f Cytogenetics and Molecular Genetics, Adelaide Children's Hospital. North Adelaide (Australia) and 2 Immunex Research and Development Corporation, Seattle. WA (USA)

Abstract. TNFR1 and TNFR2, the genes encoding the two forms of the human tumor necrosis factor receptor, were localized to normal human chromosomes by in situ hybridization

and Southern blot analysis of a series of human x mouse hybrid cell lines. T N F R 1 maps to 12p 13 and TNFR2 maps to 1p36.

Tumor necrosis factor alpha (TNFA) and beta (TNF'B. lymphotoxin) are structurally-related cytokines involved in a wide range of biological activities (for review, see Beutler and Cerami, 1988). These activities are initiated by the binding of these ligands to specific cell surface receptors present on a variety of different cells and for which these ligands cross-compete for binding (Aggarwal et al., 1985). Recently, two distinct receptors for human TNF were cloned and found to have similar extracel­ lular ligand-binding domains which define a new family of receptors (Loetscheret al., 1990: Schall et al., 1990: Smith et al., 1990). Utilizing these recombinantly-expressed receptors it was further shown that both forms are capable of binding both TNFA and TNFB with high affinity. We report the use of these cloned cDNAs to determine the chromosomal location of the genes for these receptors.

Figure 1 shows the distribution of silver grains over 40 metaphases hybridized with the TNFR2 probe. On the short arm of chromosome 1 there were 31 (23%) of the 134 silver grains detected on these metaphases. This localizes TNFR2 to 1p35-> p36, but most probably to lq36.3. The in situ hybridization results for TNFR I showed (Fig. 2) that from 35 metaphases on which there were 107 silver grains. 24 (22.4% of all silver grains) were on the short arm of chromosome 12. This result localizes TNFR1 to the region 12p 13. Both these locations were con­ firmed by the results of the hybridizations to the second normal male (data not shown). Neither probe showed any evidence of specific hybridization to other locations in the genome. Southern blot analysis of the hybrid cell panel showed (Ta­ ble 1) that hybrid cell lines CY7. CY 12, C Y 14. and CY 175 con­ tained the TNFR1 sequence as shown by 3.7, 1.4. and 0.7-kb bands in lanes containing /fcoRI-digested DNA from these lines and from the human DNA controls. Similar results were obtained using the ///«dill and Ta^I-digested DNAs where the band sizes were 4.5 and 10.8 kb, respectively. The only human

Received 22 January 1991: accepted 2 April 1991. Request reprints from Dr. G.R. Sutherland. Department of Cytogenetics and Molecu­ lar Genetics, Adelaide Children’s Hospital. North Adelaide, SA 5006 (Australia).

Table I. Assignment o f TNFR I to hum an chromosome 12 Line

Human chromosome content

CY2 CY3 CY5 CY6 CY7 CY12 CY13 CY14 CY150 CY175

der (X) t (X; 16) (q26:q24) der ( 16) t (X; 16) (q26:q24) der ( 10) t ( 10: ! 6) (q26:q22).22 der ( 10) t ( 10; 16) (q24.3;q22.1),8 der (3) t (3; 16) (q 13.2;q 13), 10.12 der ( 16) t ( 12; 16) (q24:p 11.21,2,7.8.12.21 ± 1 der ( 16) t ( 1:16) (q44;p 13.11), 3.11,14,17,20.21,22 der ( 16) t (4; 16) (q31.1;p 13.3),4.5,12,14,20.21 der (9) t (9; 16) (q 12:q 11.2).4or5.18 der ( 16) t ( 16;18) (pi 3.1 ;q 11.2),4,6,7,11,12,18.19.20.22.X

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Probes. The probe for TNFR2, p80, was a 640-bp NotVBylU fragment cloned into Nol\/Bam H l-cul Bluescript SK. (Stralegene). The probe for T N F R I.p 6 0 , was a !-kb£coR l insert o f cDNA cloned into the £coRI site of a Gemble-based plasmid. Cell lines. Construction of the hum an x mouse hybrid cell lines has been described (Callen et al.. 1990) and their hum an chromosome content is shown in Table I. Southern blot hybridization. The probes were radiolabeled with -,2P-dCTP by the m ultiprim e reaction and hybridized using standard methods to South­ ern blots of/tcoR I, //tw ill I. and /'«//I-digested DNA from the hybrid cells and from normal human leukocytes as controls. In situ hybridization. This was as previously described (Sutherland et al., 1988). The probes were labeled using three tritiated nucleotides, hybridized to metaphases from two normal males at a concentration of 0.05-0.1 pg/ml. and exposed for 12-13 d. All silver grains touching chromosomes were counted to determ ine the pattern of hybridization of the probe.

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chromosome material unique to the lines hybridizing to the TNFR l probe is chromosome 12, which is consistent with the in situ hybridization result. The probe to TNFR2 gave a complex pattern of mouse and human bands with the hybrid cell panel and confirmation of the in situ hybridization result could not be made by this method. The genes TNFA and TNFB have both been localized to the short arm of chromosome 6 at p 2 1.3. The receptors for these

Aggarwal BB. Et-ssalu TE, Hass PE: Characterization of receptors for tumor necrosis factor and their regula­ tion by gamma-interferon. Nature 318:665-667 (1985). Bcutler B, Cerami A: Tumor necrosis, cachexia, shock and inflammation: a common mediator. A Rev Biochem 57:505-518(1988). Bruns GAP. Sherman SL: Report of the committee on the genetic constitution of chromosome 1. Tenth International Workshop on Human Gene Mapping. Cytogenet Cell Genet 51:67—90 (1989). Callen DF. Baker E, Eyre HJ, Lane SA: An expanded mouse-human hybrid cell panel for mapping hu­ man chromosome 16. Annls G init 33:190-195 (1990).

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Fig. 2. Distribution of silver grains over all chromosomes in 35 metaphases after in situ hybridization with the T N FR l probe.

ligands are now shown to lie on separate chromosomes at 12p 13 and I p36. It is of interest that these are two regions of the human genome known to be involved in nonrandom chromosome change in neoplastic disorders (Trent et al.. 1989). The localiza­ tion ofTN FR2 and TNFRl to lp36 and 12p 13 may be of inter­ est in terms of genome evolution as other related genes, ENOl and EN02. have also been assigned to these two regions (Bruns and Sherman. 1989; Ropers and Craig, 1989).

Loetscher H. Pan YCE. Lahm HW, Gentz R. Brockhaus M.Tabuchi H. Lesslauer W: Molecular cloning and expression of the human 55 kd tumor necrosis fac­ tor receptor. Cell 61:351-359 (1990). Ropers H-H, Craig IW: Report of the committee on the genetic constitution of chromosomes 12 and 13. Tenth International Workshop on Human Gene Mapping. Cytogcnet Cell Genet 51:259-279 (1989). Schall TJ. Lewis M. Koller KJ. Lee A. Rice GC. Wong GHW. Gatanaga T. Granger GA. Lentz R. Raab H, Kohr WJ, Goeddel DV: Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61:361-370(1990). Smith CA. Davis T. Anderson D. Solam !.. Beckmann

MP, Jerzy R. Dower SK, Cosman D. Goodwin RG: A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248:1019-1023 (1990). Sutherland GR. Baker E. Callen DF. Hyland VJ, May BK. Bawden MJ. Hcaly HM. Borthwick IA: 5-Aminolevulinate synthase is at 3p21 and thus not the primary defect in X-linkcd sideroblastic anemia. Am J hum Genet 43:331-335 ( 1988). Trent JM. Kaneko Y. Mitelman F: Report of the com­ mittee on structural chromosome changes in neo­ plasia. Tenth International Workshop on Human Gene Mapping. Cytogenel Cell Genet 51:533-562 (1989).

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Fig. I. I he sublocaluation of ihc membrane skeletal protein 4.1 gene on human chromosome by fluorescence in situ hybridization. The positive sig­ nals are shown in yellow-green fluorescence against a background of redstained UN A tpropidium iodide) Vletapluisc spread of a normal diploid cell, (b) Partial metaphase spread, (c) Idiogram o f chromosome I. I he vertical bar indicates the position ol the protein 4.1 gene. I wo fluorescence spots are seen because both sister chromatids were labeled.

Chromosomal location of the human tumor necrosis factor receptor genes.

TNFR1 and TNFR2, the genes encoding the two forms of the human tumor necrosis factor receptor, were localized to normal human chromosomes by in situ h...
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