To clarify the position of the TNFA and TNFB genes on the HLA map, we have assigned TNFA to large DNA restriction fragments separated by field inversion gel electrophoresis, which hybridize with either class III- or class I-specific probes as well. These results prove that the TNFA locus is localized between the HLA class III region and the HLA-B locus

Bloemer, Katharina

Ragoussis, Jiannis

Weiss, Elisabeth H.

Ziegler, Andreas

English

Ragoussis, J

Bloemer, K

Weiss, EH

Ziegler, A

Oxford University Research Archive (ORA)

Immunogenetics

Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class I and III regions by field inversion gel electrophoresis.

Oxford University Research Archive

Open Access LMU

Immunogenetics 27: 66-69, 1988 Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class field inversion gel electrophoresis 11HlllUllO- genetics © Springer-Verlag 1988 I and III regions by J. Ragoussis 1, K. Bloemer 2, E.H. Weiss z, and A. Ziegler 1 1 Medizinische Klinik und Medizinisch-Naturwissenschaflliches Forschungszentrum, University of TiJbingen, Ob dem Himmelreich 7, Federal Republic of Germany 2 Institute of Immunology, University of Munich, Goethestrasse 31, D-8000 Munich 2, Federal Republic of Germany The human major histocompatibility (HLA) complex is lo- cated on the short arm of chromosome 6 in the 6p21.31-6p21.33 region (Spring et al. 1985, Ziegler et al. 1985a). The physical length of the entire HLA complex is unknown so far, but our estimate based on the separation of DNA fragments containing HLA genes by pulsed-field gel electrophoresis (Schwartz and Cantor 1984, Carle and Olson 1984) indicates that it encompasses at least 2500 kb pairs (Ragoussis et al. 1986). This estimate has recently been confirmed (Lawrance t al. 1987). Apart from the highly polymorphic class I and II loci, the HLA complex contains the genes for several complement components and 21-steroid hydroxylase (class III region) (Lamm and Olaisen 1985). In addition, the loci for tumor necrosis fac- tor (TNFA) as well as lymphotoxin (TNFB) are also near or even within the HLA region (Nedwin et al. 1985). Spies and co-workers (1986) demonstrated that the TNF genes map either centromeric to HLA-DP or telomeric to the class I1 region, although apparently not in the vicinity of any known class I or III genes. The recent demonstration that the TNF loci are situated 70 kb upstream of the H-2D gene in the BALB/c mouse between the class III and class I regions (Mfiller et al. 1987a, b) suggested an analogous location in man, because the genetic organization of the major histocompatibility complexes (MHC) of both spe- cies is very similar. To clarify the position of the TNFA and TNFB genes on the HLA map, we have assigned TNFA to large DNA restriction fragments eparated by field inversion gel electrophoresis (FIGE) (Carle et al. 1986), which hybridize with either class 1II- or class I- specific probes as well. These results prove that he TNFA locus is localized between the HLA class III region and the HLA-B locus. To avoid interpretative difficulties which might arise from haplotype-specific restriction fragment length poly- morphisms, mutant human cell lines with monosomy 6or HLA hemizygosity were employed. All mutants were derived from BJAB-B95.8.6 lymphoma cells with the HLA haplotypes A1, Cw4,B35 and A2, C-,B13 (Spring et al. 1985). Mutant BM 19.7 is a monosomy 6 mutant cell line retaining the A2 haplotype (Ziegler et al. 1985b). BM 28.7 also exhibits monosomy 6, but with loss of the chro- mosome bearing the A2 haplotype (Ragoussis et al. 1986). In the interstitial deletion mutant BM 2.2.3, the class I region of the A2 haplotype has been deleted, but the class II and III regions of this haplotype are still present, as is the chromosome 6 carrying the A1 haplotype (Ziegler et al. 1985a). Large genomic DNA fragments were generated with various restriction enzymes, separated by FIGE, and ana- lyzed by hybridization to Southern blots (Southern 1975) under stringent conditions. In the Bss HII digests (Fig. 1), a 370 kb DNA fragment was detected which hybri- dized both to the HLA-B locus-specific probe that had been derived 29 kb 5' of the HLA-B gene (Fig. lb) and to the TNFA probe (Fig. lc), indicating linkage of TNFA with the HLA-B locus. The class III loci were present on another Bss HII fragment 125 kb in length (Fig. ld). The 370 kb Bss HII fragment did not contain any class I or class II genes (Fig. le and not shown). Hybridizations with the same probes to blots of Nru I-digested DNA (Fig. 2) gave evidence for the linkage of TNFA to the class III genes. Two Nru I fragments of 710 and 690 kb were detected which contained the class III genes (Fig. 2a and not shown) and TNFA (Fig. 2b). It is likely that partial digestion of an Nru I site is responsible for the generation of two fragments of similar size carry- ing the class III and TNFA genes. The class I region probes hybridized to a distinct fragment of about 800 kb (Fig. 2c). An even larger Nru I fragment of more than 1000 kb carried class II sequences (not shown). To prove linkage, double digests with Nru I and Bss HII were performed as well (Fig. 3). The hybridization pattern obtained with the class lII probes howed that the 125 kb Bss HII fragment carrying the class III region is contained within the Nru I fragment (Fig. 3a). However, the Bss HII fragment hybridizing with the TNF probe is reduced to a length of only about 50-90 kb (Fig. 3b). Con- J. Ragoussis el al. : Physical inkage of TNFA and TNFB genes to HLA-B 67 gig. la-e.  Southern blot hybridization of human DNA digested with Bss HII. Low melting point (LMP)-agarose blocks containing cells from the cell lines BM19.7, BJAB-B95.8.6, BM28.7, and BM2.2.3 (106 cells per block) were prepared according to the method of van der Bliek and co- workers (1986), slightly modified for FIGE: 2 x 107 cultured cells were pelleted and resuspended in PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 8), pelleted again, and resuspended in 1 ml PBS. One percent of LMP agarose (BRL ultra pure) in PBS was melted, equilibrated at 37 °C, and mixed well with an equal volume of cell suspension. The agarose/cell mixture was poured into a series of slots in a perpex mold. After solidification, the agarose blocks were deposited in a lysis mix [0.5 M ethylenediaminetetraacetate (EDTA), pH 9.5, 1% sodium lauryl sarcosine, 1 mg/ml proteinase K] at 1 ml per block and incubated for 48 h at 50 °C. The lysis mix was washed off overnight with TE buffer [10 mM Tris/HC1, pH 8, 10 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)], 20 nil/block. Prior to digestion, the blocks were washed for 1 h in restriction enzyme buffer containing 0.1 mM PMSF (RE) (10 ml/block). One hundred microliters RE buffer was mixed with 25 units of enzyme in an Eppendorf tube; a 1/2 block was added and incubated for 6 h at 37 °C. The reaction was stopped with 1 ml TE containing 0.1% NaDoSO 4. Half a block was loaded in a 17 x 15 x0.8 cm 1% agarose gel (Seakem GTG) in 0.25 xTBE (22.5 mM Tris borate, 0.5 mM EDTA, pH 8) and placed in an electrophoresis chamber. FIGE was carried out for 24 h at 150 V or 189 V constant, 15 °C. The voltage gradient was inverted periodically with a time ratio of 3 : 1 and the longer time increasing from 10 to 60 or 120 s. We used a computer-controlled power supply (CS 130, version 2.1) supplied by the EMBL, Heidelberg. After a run, the gel was stained in ethidium bromide (1 gg/ml) for 30 rain, destained for 30 rnin in H20, and photographed under 302 nm UV light (a). The DNA was then blotted to Amersham Hybond membranes, baked for 2 h at 80 °C, and hybridized sequentially with various DNA probes. Prehybridization and hybridization solutions were as follows: 3 X SSC (1 × SSC is 0.15 M NaC1/0.015 M sodium citrate, pH 7.4) containing 5 × Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 10 % dextran sulfate, 0.5 % NaDodSO, and 100 gg/ml salmon sperm DNA. Prehybridization was performed for 3 h at 65 °C and hybridization overnight at 65 °C with 32p dCTP-labeled DNA probe (Feinberg and Vogelstein 1983) at 106 cpm/ml. Washing was performed in four steps: 10 rain in 2 x SSC, 0.1% NaDodSO 4 at room temperature; 30 min in 1 x SSC, 0.1% NaDodSO 4 at 65 °C; 30 rain in 0.3 x SSC, 0. i % NaDodSO 4 at 65 °C and 30 min in 0. i x SSC, 0.1% NaDodSO 4at 65 °C. The filters were exposed for 2-7 days to Kodak XAR-5 film at -70  °C. Before rehybridization, the hybrid- ized DNA probe was removed in bidistilled H20 at 70 °C and the result checked by autoradiography. An HLA-B locus-specific probe, a 5.0 kb Kpn I/Hind III fragment, was isolated which is located 29 kb 5' of the HLA-B gene (Weiss et al. 1987). Another B locus-specific probe, a 2.2 kb Pst I/Barn HI fragment, was isolated 26 kb 3' of the B gene. Probes for C4 and C2 (class III region) were kindly provided by D. Campbell (Belt et al. 1984) and P. Schneider. The TNFA probe was a Hind III/Sal I fragment derived 20 kb 5' of the TNFA gene (Kioussis et al. 1987). As molecular size markers, the yeast strain way 5-4A prepared as described (Schwartz and Cantor 1984) and X concatemers prepared as described (Van Ommen and Verkerk 1986) were used. The HLA-B 5' locus probe detected a 370 kb fragment (b) which was also recognized by the TNF probe (e) but not by either the C4 (d) or the C2 probe (not shown), which hybridize to a 125 kb Bss HII fragment, or by the HLA-B 3' probe, which hybridized to a 270 kb fragment (e) 68 J. Ragoussis et al.: Physical inkage of TNFA and TNFB genes to HLA-B Fig. 3a-c. Southern blot hybridization of human DNA doubly digested with Nru I and Bss HII. DNA from the cell line BM19.7 was doubly digested with Nru I and Bss HII (left) or Bss HII alone (right). FIGE conditions were as in Figure 1 except hat the longer time increased linearly from 10 to 120 s. Hybridization was carried out sequentially (as described in Fig. 1) with C4 (a), TNFA (b), and the HLA-B5 ' locus probe (e) Fig. 2a-c. Southern blot hybridization of human DNA digested with Nru I. DNA from the cell line BM28.7 was digested with Nru I. The conditions for FIGE were as in Figure l, except hat the voltage was increased to 189 V. Southern blotting and hybridization conditions were identical with those employed for Bss HII-digested DNA. The C2 probe detected two bands of slightly larger or smaller sizes than 700 kb (a) and these were also hybridizing to the TNFA probe (b). The HLA-B5' locus-specific probe detected another fragment of 800 kb (e) which con- rains also the HLA-B gene (not shown) sequently, the HLA-B locus-specific probe was expected to hybridize with a different band of about 300 kb, and indeed, a new band of this approximate size (280 kb) was found (Fig. 3c). This result proves that the Nru I and Bss HII fragments overlap by 50-90 kb, and the TNF genes must be localized within this region (Fig. 4). Since an HLA-B gene probe itself and a fragment, iso- lated 26 kb 3' of this gene, hybridized to a different Bss HII fragment (270 kb) (Fig. le), it is possible to give a rather precise stimate of the distance between TNFA and HLA-B. The TNFA locus and the closely linked (Nedospasov etal. 1986) TNFB gene are therefore located about 300 kb centromeric tothe HLA-B gene. Preliminary experiments employing double digests with Nru I and Not I indicate a distance of TNF to the class III region of not more than 300-400 kb. Since the TNFA/class III region- carrying Nru I fragment does not contain class II genes, the minimal distance between the class II loci and the class I region must be about 1000 kb. These data are in agreement with the mapping of the TNF genes in the mouse, where they are located 70 kb proximal to the H-2D gene. However, it is very likely that the physical distance between HLA-B and TNFis consider- ably larger in the human genome than in the mouse. Since the association of particular HLA class I and II alleles with a variety of diseases (Tiwari and Terasaki 1985) is still an enigma, the mapping of loci in the vicinity of these genes might provide further insight into the under- lying mechanisms. Therefore the location of TNFA and TNFB by FIGE or related techniques in the HLA region may contribute to an understanding of HLA-disease as- sociations. Since the region occupied by the TNFA and TNFB genes is only about 7 kb (Nedospasov etal. 1986), Class l l  C lass l l l  C lass l  DIR C4 C2 TN FIA/B HLA- -B  I I / /  I t  1000 700 800 r125 t , ' ~ 370 270 Nru l  k~ BssHI I  kb Fig. 4. Physical map of the central part of the HLA complex based on separation of large DNA restriction fragments by FIGE. -4/--, the existence of additional Nru I fragments between the ones de- tected cannot be excluded J. Ragoussis et al.: Physical inkage of TNFA and TNFB genes to HLA-B 69 it is tO be expected that several  addit ional  loci wil l  be found between the class I and I I I  regions.  The preparat ive appli- cation of  F IGE  in conjunct ion with c loning techniques (Michiels  et al. 1987) wil l  help to br idge the long chro- mosomal  distance between the di f ferent class I genes and the class II I  region.  Acknowledgments. This work was supported by the Deutsche For- schnngsgemeinschaft t rough SFB 120 (project A4) and the Genzentrum Mtinchen. We are grateful to Ms. Andrea Schenk for her valuable techni- cal assistance and to Ms. Gudrun Szalay for her help in the preparation of the molecular size markers. We also thank Dr. Denise Barlow, Mr. Carol Stettner, and Mr. Alfons Riedinger (EMBL, Heidelberg) for help in setting up field inversion gel electrophoresis. Note added in proof: Improved separation conditions in FIGE suggest that he Nru I fragment containing class I genes may be even larger than 800 kb (Fig. 2c), possi- bly about 1000 kb. References  Belt, K. T., Carroll, M. C., and Porter, R. R. : Multiple forms of human complement component. Cell 36: 907-914, 1984 Carle, G.F. and Olson, M.V.: Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel elec- trophoresis. Nucleic Acids Res. 12: 5647-5664, 1984 Carle, G. F., Frank, M., and Olson, M. V.: Electrophoretic separations of large DNA molecules in one-dimensional electric fields that are periodically inverted. Science 232: 65-68, 1986 Feinberg, A. P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments o high specific activity. Anal. Biochem. 132: 6-13, 1983 Kioussis, D., Wilson, F., Daniels, C., Leveton, C., Taverne, J., and Playfair, H. L.: Expression and rescuing of a cloned human tumor necrosis factor gene using an EBV-based shuttle cosmid vector. EMBO J. 6: 355-361, 1987 Lamm, L. U. and Olaisen, B.: Report of the Committee on the Genetic Constitution ofChromosomes 5 and 6. Eighth International Work- shop on Human Gene Mapping. Cytogenet. Cell Genet. 40: 128-155, 1985 Lawrance, S.K., Smith, C.L., Srivastava, R., Cantor, C.R., and Weissman, S. M.: Megabase-scale mapping of the HLA gene com- plex by pulsed-field gel electrophoresis. Science 235: 1387-1390, 1987 Michiels, F., Burmeister, M., and Lehrach, H.: Derivation of clones close to met by preparative field-inversion gel electrophoresis. Science 236: 1305-1308, 1987 Mtiller, U., Jongeneel, V., Nedospasov, S.A., Fischer Lindahl, K., and Steinmetz, M.: Tumor necrosis factor and lymphotoxin map close to H-2D in the mouse major histocompatibility complex. Na- ture 325: 265-267, 1987a MiJller, U., Stephan, D., Philippsen, P., and Steinmetz, M.: Orientation and molecular map position of the complement genes in the mouse MHC. EMBO J. 6: 369-373, 1987b Nedospasov, S.A., Shakov, A.N., Turetskaya, R.L., Mett, V.A., Azizov, M.M., Georgiev, G.P., Korobko, V.G., Dobrynin, V. N., Filippov, S. A., Bystrov, E. F., Boldyreva, E. F., Chuvpilo, S.A., Chumakov, A.M., Shingarova, L.N., and Ovchinnikov, Y. A.: Tandem arrangement of genes coding for tumor necrosis fac- tor (TNF-c 0 and lymphotoxin (TNF-/3) in the human genome. CoM Spring Harbor Symp. Quant. Biol. 51: 611-624, 1986 Nedwin, G.E., Naylor, S.L., Sakaguchi, A.Y., Smith, D., Jarrett- Nedwin, J., Pennica, D., Goeddel, D. V., and Gray, P. W. : Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localisation. Nucleic Acids Res. 13: 6361-6373, 1985 Ragoussis, J., van der Bliek, A., Trowsdale, J., and Ziegler, A.: Map- ping of HLA genes using pulsed field gradient electrophoresis. FEBS Lett. 204: 1-4, 1986 Schwartz, D.C. and Cantor, C.R.: Separation of yeast chromosome- sized DNAs by pulsed field gradient gel electrophoresis. Cell 37: 67-75, 1984 Southern, E.M.: Detection of specific sequences among DNA frag- ments eparated by gel electrophoresis. J. Mol. Biol. 98: 503-517, 1975 Spies, T., Morton, C.C., Nedospasov, S.A., Fiers, W., Pious, D., and Strominger, J. L.: Genes for the tumor necrosis factors c~ and ~3 are linked to the human major histocompatibility complex. Proc. Natl. Acad. Sci. U.S.A. 83: 8699-8702, 1986 Spring, B., Fonatsch, C., Mtiller, C., Pawelec, G., K6mpf, J., Wernet, P., and Ziegler, A.: Refinement ofHLA gene mapping with induced B-cell line mutants. Immunogenetics 21: 277-291, 1985 Tiwari, L.J. and Terasaki, P.I.: HIM and Disease Associations. Springer Verlag, Berlin, 1985 van der Bliek, A. M., van der Velde-Koerts, T., Ling, V., and Borst, P. : Overexpression a d amplification of five genes in a multi-drug resistant Chinese hamster ovary cell line. Mol. Cell. Biol. 6: 1671-1678, 1986 van Ommen, G.J.B. and Verkerk, J. M. H.: Restriction analysis of chromosomal DNA in a size range up to two million base pairs by pulse field gradient electrophoresis. In K. E. Davies (ed.): Human Genetic Diseases: A Practical Approach, pp. 113-133, IRL Press, Oxford, 1986 Weiss, E. H., Bloemer, K., Doerner, C., Kuon, W., Lang, M., Pohla, H., Schattenkirchner, M. and Riethmiiller, G.: Molecular biology of the HLA-B27 locus. Brit. J. Rheumatol., in press, 1987 Ziegler, A., Zimmer, F.-J., Fonatsch, C., So, A., and Trowsdale, J.: Mapping of HLA class I and class II genes using HLA deletion mu- tant cell lines. Eighth International Workshop on Human Gene Mapping. Cytogenet. Cell Genet. 40: 788, 1985a Ziegler, A., Mtiller, C., Heinig, J., Radka, S.F., K6mpf, J., and Fonatsch, C.: Monosomy 6in a human lymphoma cell line induced by selection with a monoclonal antibody. Immunobiology 169: 455-460, 1985b Received July 17, 1987; revised version received September 1, 1987 

Multiple forms of human complement component.

Overexpression and amplification of five genes in a multi-drug resistant Chinese hamster ovary cell line.

Refinement ofHLA gene mapping with induced B-cell line mutants.

Report of the Committee on the Genetic Constitution of Chromosomes 5 and 6. Eighth International Workshop on Human Gene Mapping.

Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis.

Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA classI and III regions by field inversion gel electrophoresis

Universität München: Elektronischen Publikationen

Immunogenetics 27: 66-69, 1988 
Localization of the genes for tumor necrosis 
factor and lymphotoxin between the HLA class 
field inversion gel electrophoresis 
11HlllUllO- 
genetics 
© Springer-Verlag 1988 
I and III regions by 
J. Ragoussis 1, K. Bloemer 2, E .H.  Weiss z, and A. Ziegler 1 
1 Medizinische Klinik und Medizinisch-Naturwissenschaflliches Forschungszentrum, University of TiJbingen, Ob dem Himmelreich 7, Federal 
Republic of Germany 
2 Institute of Immunology, University of Munich, Goethestrasse 31, D-8000 Munich 2, Federal Republic of Germany 
The human major histocompatibility (HLA) complex is lo- 
cated on the short arm of chromosome 6 in the 
6p21.31-6p21.33 region (Spring et al. 1985, Ziegler et 
al. 1985a). The physical length of the entire HLA complex 
is unknown so far, but our estimate based on the separation 
of DNA fragments containing HLA genes by pulsed-field 
gel electrophoresis (Schwartz and Cantor 1984, Carle and 
Olson 1984) indicates that it encompasses at least 2500 
kb pairs (Ragoussis et al. 1986). This estimate has recently 
been confirmed (Lawrance et al. 1987). Apart from the 
highly polymorphic class I and II loci, the HLA complex 
contains the genes for several complement components 
and 21-steroid hydroxylase (class III region) (Lamm and 
Olaisen 1985). In addition, the loci for tumor necrosis fac- 
tor (TNFA) as well as lymphotoxin (TNFB) are also near 
or even within the HLA region (Nedwin et al. 1985). Spies 
and co-workers (1986) demonstrated that the TNF genes 
map either centromeric to HLA-DP or telomeric to the 
class I1 region, although apparently not in the vicinity of 
any known class I or III genes. The recent demonstration 
that the TNF loci are situated 70 kb upstream of the H-2D 
gene in the BALB/c mouse between the class III and class 
I regions (Mfiller et al. 1987a, b) suggested an analogous 
location in man, because the genetic organization of the 
major histocompatibility complexes (MHC) of both spe- 
cies is very similar. To clarify the position of the TNFA 
and TNFB genes on the HLA map, we have assigned 
TNFA to large DNA restriction fragments separated by 
field inversion gel electrophoresis (FIGE) (Carle et al. 
1986), which hybridize with either class 1II- or class I- 
specific probes as well. These results prove that the TNFA 
locus is localized between the HLA class III region and 
the HLA-B locus. 
To avoid interpretative difficulties which might arise 
from haplotype-specific restriction fragment length poly- 
morphisms, mutant human cell lines with monosomy 6 
or HLA hemizygosity were employed. All mutants were 
derived from BJAB-B95.8.6 lymphoma cells with the 
HLA haplotypes A1, Cw4,B35 and A2, C-,B13 (Spring et 
al. 1985). Mutant BM 19.7 is a monosomy 6 mutant cell 
line retaining the A2 haplotype (Ziegler et al. 1985b). BM 
28.7 also exhibits monosomy 6, but with loss of the chro- 
mosome bearing the A2 haplotype (Ragoussis et al. 1986). 
In the interstitial deletion mutant BM 2.2.3, the class I 
region of the A2 haplotype has been deleted, but the class 
II and III regions of this haplotype are still present, as 
is the chromosome 6 carrying the A1 haplotype (Ziegler 
et al. 1985a). 
Large genomic DNA fragments were generated with 
various restriction enzymes, separated by FIGE, and ana- 
lyzed by hybridization to Southern blots (Southern 1975) 
under stringent conditions. In the Bss HII digests (Fig. 
1), a 370 kb DNA fragment was detected which hybri- 
dized both to the HLA-B locus-specific probe that had 
been derived 29 kb 5' of the HLA-B gene (Fig. lb) and 
to the TNFA probe (Fig. lc), indicating linkage of TNFA 
with the HLA-B locus. The class III loci were present on 
another Bss HII fragment 125 kb in length (Fig. ld). The 
370 kb Bss HII fragment did not contain any class I or 
class II genes (Fig. le and not shown). 
Hybridizations with the same probes to blots of Nru 
I-digested DNA (Fig. 2) gave evidence for the linkage of 
TNFA to the class III genes. Two Nru I fragments of 710 
and 690 kb were detected which contained the class III 
genes (Fig. 2a and not shown) and TNFA (Fig. 2b). It is 
likely that partial digestion of an Nru I site is responsible 
for the generation of two fragments of similar size carry- 
ing the class III and TNFA genes. The class I region probes 
hybridized to a distinct fragment of about 800 kb (Fig. 
2c). An even larger Nru I fragment of more than 1000 
kb carried class II sequences (not shown). 
To prove linkage, double digests with Nru I and Bss 
HII were performed as well (Fig. 3). The hybridization 
pattern obtained with the class lII probes showed that the 
125 kb Bss HII fragment carrying the class III region is 
contained within the Nru I fragment (Fig. 3a). However, 
the Bss HII fragment hybridizing with the TNF probe is 
reduced to a length of only about 50-90 kb (Fig. 3b). Con- 
J. Ragoussis el al. : Physical linkage of TNFA and TNFB genes to HLA-B 67 
gig. l a - e .  Southern blot hybridization of human DNA digested with Bss HII. Low melting point (LMP)-agarose blocks containing cells from the 
cell lines BM19.7, BJAB-B95.8.6, BM28.7, and BM2.2.3 (106 cells per block) were prepared according to the method of van der Bliek and co- 
workers (1986), slightly modified for FIGE: 2 x 107 cultured cells were pelleted and resuspended in PBS (10 mM sodium phosphate, 150 mM sodium 
chloride, pH 8), pelleted again, and resuspended in 1 ml PBS. One percent of LMP agarose (BRL ultra pure) in PBS was melted, equilibrated 
at 37 °C, and mixed well with an equal volume of cell suspension. The agarose/cell mixture was poured into a series of  slots in a perpex mold. 
After solidification, the agarose blocks were deposited in a lysis mix [0.5 M ethylenediaminetetraacetate (EDTA), pH 9.5, 1% sodium lauryl sarcosine, 
1 mg/ml proteinase K] at 1 ml per block and incubated for 48 h at 50 °C. The lysis mix was washed off overnight with TE buffer [10 mM Tris/HC1, 
pH 8, 10 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)], 20 nil/block. Prior to digestion, the blocks were washed for 1 h in restriction 
enzyme buffer containing 0.1 mM PMSF (RE) (10 ml/block). One hundred microliters RE buffer was mixed with 25 units of enzyme in an Eppendorf 
tube; a 1/2 block was added and incubated for 6 h at 37 °C. The reaction was stopped with 1 ml TE containing 0.1% NaDoSO 4. Half a block 
was loaded in a 17 x 15 x 0 . 8  cm 1% agarose gel (Seakem GTG) in 0.25 xTBE (22.5 mM Tris borate, 0.5 mM EDTA, pH 8) and placed in an 
electrophoresis chamber. FIGE was carried out for 24 h at 150 V or 189 V constant, 15 °C. The voltage gradient was inverted periodically with 
a time ratio of 3 : 1 and the longer time increasing from 10 to 60 or 120 s. We used a computer-controlled power supply (CS 130, version 2.1) 
supplied by the EMBL, Heidelberg. After a run, the gel was stained in ethidium bromide (1 gg/ml) for 30 rain, destained for 30 rnin in H20, 
and photographed under 302 nm UV light (a). The DNA was then blotted to Amersham Hybond membranes, baked for 2 h at 80 °C, and hybridized 
sequentially with various DNA probes. Prehybridization and hybridization solutions were as follows: 3 X SSC (1 × SSC is 0.15 M NaC1/0.015 M 
sodium citrate, pH 7.4) containing 5 × Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 10 % dextran 
sulfate, 0.5 % NaDodSO, and 100 gg/ml salmon sperm DNA. Prehybridization was performed for 3 h at 65 °C and hybridization overnight at 65 °C 
with 32p dCTP-labeled DNA probe (Feinberg and Vogelstein 1983) at 106 cpm/ml. Washing was performed in four steps: 10 rain in 2 x SSC, 0.1% 
NaDodSO 4 at room temperature; 30 min in 1 x SSC, 0.1% NaDodSO 4 at 65 °C; 30 rain in 0.3 x SSC, 0. i % NaDodSO 4 at 65 °C and 30 min in 
0. i x SSC, 0.1% NaDodSO 4 at 65 °C. The filters were exposed for 2-7 days to Kodak XAR-5 film at - 7 0  °C. Before rehybridization, the hybrid- 
ized DNA probe was removed in bidistilled H20 at 70 °C and the result checked by autoradiography. An HLA-B locus-specific probe, a 5.0 kb 
Kpn I/Hind III fragment, was isolated which is located 29 kb 5' of the HLA-B gene (Weiss et al. 1987). Another B locus-specific probe, a 2.2 
kb Pst I/Barn HI fragment, was isolated 26 kb 3' of the B gene. Probes for C4 and C2 (class III region) were kindly provided by D. Campbell 
(Belt et al. 1984) and P. Schneider. The TNFA probe was a Hind III/Sal I fragment derived 20 kb 5' of  the TNFA gene (Kioussis et al. 1987). 
As molecular size markers, the yeast strain way 5-4A prepared as described (Schwartz and Cantor 1984) and X concatemers prepared as described 
(Van Ommen and Verkerk 1986) were used. The HLA-B 5' locus probe detected a 370 kb fragment (b) which was also recognized by the TNF 
probe (e) but not by either the C4 (d) or the C2 probe (not shown), which hybridize to a 125 kb Bss HII fragment, or by the HLA-B 3' probe, 
which hybridized to a 270 kb fragment (e) 
68 J. Ragoussis et al.: Physical linkage of TNFA and TNFB genes to HLA-B 
Fig. 3a-c.  Southern blot hybridization of human DNA doubly digested 
with Nru I and Bss HII. DNA from the cell line BM19.7 was doubly 
digested with Nru I and Bss HII (left) or Bss HII alone (right). FIGE 
conditions were as in Figure 1 except that the longer time increased 
linearly from 10 to 120 s. Hybridization was carried out sequentially 
(as described in Fig. 1) with C4 (a), TNFA (b), and the HLA-B5 ' locus 
probe (e) 
Fig. 2a-c.  Southern blot hybridization of human DNA digested with 
Nru I. DNA from the cell line BM28.7 was digested with Nru I. The 
conditions for FIGE were as in Figure l ,  except that the voltage was 
increased to 189 V. Southern blotting and hybridization conditions were 
identical with those employed for Bss HII-digested DNA. The C2 probe 
detected two bands of  slightly larger or smaller sizes than 700 kb (a) 
and these were also hybridizing to the TNFA probe (b). The HLA-B5'  
locus-specific probe detected another fragment of  800 kb (e) which con- 
rains also the HLA-B gene (not shown) 
sequently, the HLA-B locus-specific probe was expected 
to hybridize with a different band of about 300 kb, and 
indeed, a new band of this approximate size (280 kb) was 
found (Fig. 3c). This result proves that the Nru I and Bss 
HII fragments overlap by 50-90 kb, and the TNF genes 
must be localized within this region (Fig. 4). 
Since an HLA-B gene probe itself and a fragment, iso- 
lated 26 kb 3' of  this gene, hybridized to a different Bss 
HII fragment (270 kb) (Fig. le), it is possible to give a 
rather precise estimate of the distance between TNFA and 
HLA-B. The TNFA locus and the closely linked 
(Nedospasov et al. 1986) TNFB gene are therefore located 
about 300 kb centromeric to the HLA-B gene. Preliminary 
experiments employing double digests with Nru I and Not 
I indicate a distance of TNF to the class III region of not 
more than 300-400 kb. Since the TNFA/class III region- 
carrying Nru I fragment does not contain class II genes, 
the minimal distance between the class II loci and the class 
I region must be about 1000 kb. 
These data are in agreement with the mapping of the 
TNF genes in the mouse, where they are located 70 kb 
proximal to the H-2D gene. However, it is very likely that 
the physical distance between HLA-B and TNFis consider- 
ably larger in the human genome than in the mouse. 
Since the association of particular HLA class I and II 
alleles with a variety of diseases (Tiwari and Terasaki 
1985) is still an enigma, the mapping of loci in the vicinity 
of these genes might provide further insight into the under- 
lying mechanisms. Therefore the location of TNFA and 
TNFB by FIGE or related techniques in the HLA region 
may contribute to an understanding of HLA-disease as- 
sociations. Since the region occupied by the TNFA and 
TNFB genes is only about 7 kb (Nedospasov et al. 1986), 
C l a s s l l  C l a s s l l l  C l a s s l  
DIR C4  C2  TN  FIA/B H L A - - B  
I I 
/ /  I t  
1 0 0 0  7 0 0  8 0 0  
r125  t , ' ~ 3 7 0  2 7 0  
N r u l  
k~ 
B s s H I I  
kb 
Fig. 4. Physical map of the central part 
of  the HLA complex based on separation 
of large DNA restriction fragments by 
FIGE. -4/--, the existence of additional 
Nru I fragments between the ones de- 
tected cannot be excluded 
J. Ragoussis et al.: Physical linkage of TNFA and TNFB genes to HLA-B 69 
it is tO b e  expec t ed  tha t  seve ra l  add i t iona l  loci  wil l  b e  found  
b e t w e e n  the  class I and  I I I  r eg ions .  T he  p r e p a r a t i v e  appl i -  
ca t ion  o f  F I G E  in con j unc t i on  wi th  c lon ing  t echn iques  
(Mich ie l s  et  al. 1987) wil l  he lp  to b r i d g e  the  l ong  chro-  
m o s o m a l  d i s t ance  b e t w e e n  the  d i f f e ren t  class  I genes  and  
the  class III  r eg ion .  
Acknowledgments. This work was supported by the Deutsche For- 
schnngsgemeinschaft through SFB 120 (project A4) and the Genzentrum 
Mtinchen. We are grateful to Ms. Andrea Schenk for her valuable techni- 
cal assistance and to Ms. Gudrun Szalay for her help in the preparation 
of the molecular size markers. We also thank Dr. Denise Barlow, Mr. 
Carol Stettner, and Mr. Alfons Riedinger (EMBL, Heidelberg) for help 
in setting up field inversion gel electrophoresis. 
Note added in proof: 
Improved separation conditions in FIGE suggest that the Nru I fragment 
containing class I genes may be even larger than 800 kb (Fig. 2c), possi- 
bly about 1000 kb. 
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Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA classI and III regions by field inversion gel electrophoresis

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