Immunogenetics 28: 439-444, 1988 1111101111,0- genetics

© Springer-Verlag 1988

Proximity of the CTLA-1 serine esterase and Tcr loci in mouse and man

Katherine Harper 1, Marie-Genevieve Matt~i 2, Dominique Simon 3, Marie Suzan 1, Jean-Louis Gu~net 3, Patrick Haddad 4, Marilyne Sasportes 4, and Pierre Golstein 1

1 Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, F-13288 Marseille C6dex 9, France z INSERM U242, CHU Timone, F-13385 Marseille C&tex 5, France 3 Institut Pasteur, Unit6 de G6n6tique des Mammif'eres, 25 Rue du Dr. Roux, F-75724 Paris C6dex 15, France 4 Institut de Recherches sur les Maladies du Sang, Hopital Saint-Louis, 2 Place du Dr. Fournier, F-75475 Paris Cddex 10, France

Abstract. The serine esterase CTLA-1 gene was shown by in situ hybridization to map to the D segment of mouse chromosome 14, the same localization as a member of the immunoglobulin super family, Tcr~. To further demon- strate the proximity of CTLA-1 and Tcr~, genetic linkage was tested in mouse using restriction fragment length polymorphisms and a backcross progeny, and no recom- bination was observed in the 100 backcross products studied. Recombination events between TcrJCTLA-1 and the markers Gdh-X and NP-1 show that the most probable order of these loci in the mouse 14D region is NP-1- TcrJCtla-l-Gdh-X. In man, the human homo- logue of CTLA-1 was shown by in situ hybridization to map on chromosome 14, at 14qll-q12, where Tcr~ also maps. Using the human cell line SUP-T1, bearing the inversion inv(14) (qll; q32), we further demonstrated the loci order in man to be centromere-NP-1-Tcr~- CTLA-1. To complement the cytogenetic and genetic map- ping data, we tried to determine the physical distance be- tween the two genes by pulsed field gel electrophoresis (PFGE). DNA prepared from various cell types, both mouse and human, were digested with a panel of rare cut- ter enzymes and hybridized first with CTLA-1, then with Tcr~ probes. None of the bands identified hybridized with both Tc L and CTLA-1 probes for either mouse or human cells. Although the physical mapping by PFGE is inconclusive, the cytogenetic and genetic data support close linkage of the Tcr~ and CTLA-1 genes in both mouse and man, suggesting homology between the D region of mouse chromosome 14 and the ql l -q l2 region of human chromosome 14, encompassing the Tcr~ and CTLA-1 loci. These findings also provide another example of proximity of genes coding for a member of the Ig super- family and a serine esterase.

Address correspondence and offprint requests to?" P. Golstein

Introduction

Sequence analysis shows a close structural relationship between the various chains of the T-cell receptor (Tcr) (Collins and Owen 1985) and class I and class II major histocompatibility complex (MHC) molecules, all mem- bers of the immunoglobulin superfamily. There also exists a functional relationship between the Tcr genes and the MHC complex (Hood et al. 1985), the T-cell receptor molecule only recognizing antigen on the presenting target cell in conjunction with the appropriate surface glyco- proteins encoded by the MHC. More generally, many members of this family are engaged in heterophilic recog- nition with other members of the same family, mostly at the surface of cells belonging to the immune or the nervous system.

The serine esterases constitute another large family of molecules with sequence homologies in certain regions, exhibiting the same protease activity but with very diverse specificities. They serve as sophisticated, regulatory ele- ments of the cell, with functions ranging from regulation of metabolic pathways to activation of preexisting factors such as in complement and coagulation cascade systems.

Two components of the complement cascade, C2 and factor B which possess serine protease domains, map close to the MHC class I and class II loci (Mtiller et al. 1987). Also, the serine esterase trypsin gene maps close to the Tcr~ locus (Bucan et al. 1986). What now proves to be another example of proximity between genes of these two families is the case of the serine esterase CTLA-1 gene, found to map (by in situ hybridization) to mouse chromosome 14 (Brunet et al. 1986) close to the Tcr~ chain gene (Kranz et al. 1985). CTLA-1 transcripts have been shown to be preferentially expressed in activated T cells and to be coinduced with cytotoxicity. Furthermore, it has been suggested that the Ctla-1 product has a role parallel to that of some complement components possibly within a cascade system related to T cell-mediated

440 K. Harper et al.: Proximity of Tcr and CTLA-1 loci

cytotoxicity. The purpose of the present work was to con- firm and extend the information on the chromosomal proximity and order of the Tcr~ and Ctla-1 loci in mouse, and to obtain similar information for the Tcr~ and CTLA-1 homologous genes in man.

Materials and methods

Nucleic aeidprobes. C1.1 (Brunet et al. 1986) and C1SC4 were cDNA and genomic DNA clones, respectively, of mouse CTLA-1. HuCTLA-1 was a genomic DNA clone corresponding to the human homologue of CTLA-1 (Haddad et al. 1988). Tcr~ cDNA clone (Winoto et al. 1985) was derived from the constant region of the mouse Tcr~ gene. P~A5 (Sire et al. 1984) was a cDNA clone derived from the human Tcr~ gene. NP-1 was a genomic clone of the human nucleoside phosphorylase gene (Goddard et al. 1983). Gdh-X was derived from a sequence on the human X chromosome which has 89 % sequence homology with the glutamate dehydrogenase gene (Hanauer et al. 1987).

In situ hybridization. Details of the chromosomal localization of mouse CTLA-1 gene by in situ hybridization are presented elsewhere (Brunet et al. 1986). Mapping of HuCTLA-1 by in situ hybridization was carried out on chromosome preparations obtained from phytohemagglutanin- stimulated human lymphocytes cultured for 72 h. 5-Bromodeoxyuridine was added (60 gg/ml medium) for the final 7 h of culture to obtain high quality chromosomal R-banding. The probe HuCTLA-1 was tritium- labeled by nick translation to a specific activity of 9.4 x 107 dpm gg - 1. The radiolabeled probe was then hybridized to metaphase spreads at a final concentration of 10 ng per ml of hybridization solution as previous- ly described (Mattdi et al. 1985). Chromosomal localization of the HuCTLA-1 and NP-1 genes was carried out by the aforementioned tech- nique on the human chromosome 14 inversion cell line SUP-T1, inv(14) (q l l ; q32) (Bear et al. 1985). The break points in this inversion occur in the Tcr~ joining region (14qll) and proximally to the immunoglobu- lin heavy chain JH region (14q32), the Tcr~ constant region being trans- located to the (14q32) region. Only one of the two chromosomes 14 per diploid genome bears the inversion, the other one having the normaI arrangement.

Linkage analysis using interspecific mouse backcrosses. For the chro- mosomal localization of CTLA-1 and Tcr~ genes, we used the method described by Benoit and co-workers (1985) and Avner and co-workers (1988). DNAs of the inbred mouse strains C57BL/6-Pas, BALB/c-Pas, CBA/Pas (three classical laboratory strains), and SPE/Pas (an inbred mouse strain derived from trapped wild specimens of the Mus spretus species) were digested with several restriction enzymes and probed with C1.1, Tcr~, and Gdh-X probes to detect informative restriction frag- ment length polymorphisms (RFLPs). DNA samples prepared from var- ious backcrosses of the type (C57BL/6 × SPE)FI female xC57BL/6 male were then hybridized with all three probes under the same condi- tions using the technique of Southern (1975). The presence of the SPE (Mus spretus)-specific fragment was then scored for each case, and its pattern of distribution among the backcross animals matched with an already established pattern concerning various genetic markers. In the case of nucleoside phosphorylase (NP), protein variants existing be- tween the species C57BL/6, CBA or BALB/c, and the SPE/Pas animals, exhibiting different electrochemical properties, were used to type the backcross individuals. The presence of the SPE (Mus spretus)-specific protein variant was scored in the same way as the RFLPs.

Pulsed field electrophoresis. We used orthogonal-field-alternation gel electrophoresis, as described by Carle and Olson (1985). The two units used in the study were supplied by the European MolecuIar Biology

Laboratory (EMBL, Heidelberg, Federal Republic of Germany) and LKB (Broma, Sweden). The conditions employed for electrophoresis and preparation of high relative mass DNA are described by Nguyen and co-workers (1987). The gels were run for 44 h or 18 h at 350 V, 70 mA with a switching interval of 1.5 rain in the case of the EMBL apparatus and 330 V, 100 mA with a switching interval of 1 min for the LKB unit with a constant temperature of 14 °C. Yeast chromosomes were used as size markers. DNA was prepared from mouse (C57BL/6) testicular cells, sperm cells, and the cell line KB5C20 (Albert et al. 1982) and from human peripheral blood lymphocytes and cell line 49,XXXXY (M. Fellous, Institut Pasteur). The rare-cutter restriction endonucleases used in this study are listed in Table 2. The blots prepared from mouse DNA digests were hybridized with the probes C1SC4 and Tcr,, and the blots prepared from human DNA digests were hybridized with the probes HuCTLA-1 and PGA5 and oligo-labeled (Feinberg and Vogel- stein 1984) to a specific activity of 1 x 108 cpm/I.tg. Hybridization and posthybridization washes were carried out according to the protocol described by Nguyen and co-workers (1987). X-ray films were exposed to the membranes for 4-7 days. Between hybridizations the membranes were stripped in 2 mM ethylenediaminetetraacetate, 0.1% sodium dodecyl sulfate for 15 min at 80 °C, and X-ray film was exposed to them overnight to confirm the absence of residual signal.

Results

Earlier experiments (Brunet et al. 1986) using in situ hybridization and the cDNA clone C 1.1 indicated that the CTLA-1 gene was localized to the D segment of mouse chromosome 14, the same localization as the Tcr~ gene (Dembic et al. 1985). We found no recombinant for the Tcr~ and Ctla-1 loci among a sample of 100 interspecific backcross individuals, indicating a tight genetic linkage between these two loci (see Table 1).

To determine if the proximity of these two loci also existed in man, cloned HuCTLA-1 genomic DNA was used as a probe in in situ hybridization on metaphases from PHA-activated human peripheral lymphoblasts. In the 120 metaphase cells examined, 47 silver grains were located on chromosome 14, 80% of these mapped to the 14, qll-ql3 region with a maximum in the 14q12 band (see Fig. 1). The Tcr~ gene had been previously shown (Cac- cia et al. 1985, Croce et al. 1985) to map to this same region.

Table 1. Recombination events between markers in the D segment of mouse chromosome 14

Interval Recombination Meioses Approximate events distance

TcrJ CTLA-1 0 100 TcrJNP-1 1 51 2 .0+ 1.9 TcrJGdh-X 5 65 7.7 + 3.3 Gdh-X/NP-1 4 51 7.8 +- 3.8

Number of recombination events detected between the markers Tcr~, CTLA-1, Gdh-X (using RFLPs), and NP-1 (by different electrophoretic properties of protein variants). Genetic distance measured in centi- morgans

K. Harper et al. : Proximity of Tc G and CTLA-1 loci 441

Fig. la. Two partial human metaphases showing the specific sites of hybridization to chromosome 14. Top." arrowheads indicate silver grains on Giemsa-stained chromosomes, following autoradiography. Bottom." chromosomes with silver grains subsequently identified by R-banding (F. P. G. technique), b Idiogram of the human G-banded chromosome 14 illustrating the distribution of labeled sites for HuCTLA-1

On the basis of the cytogenetic and genetic evidence in the mouse and of the cytogenetic evidence in man show- ing proximity of Tc G and CTLA-1 genes, we decided to determine the physical distances involved by pulsed field electrophoresis. DNA prepared from mouse testicular cells and sperm cells gave bands when probed with C1SC4. The Tc G probe, however, gave no clear hybridization patterns on blots prepared from these cells. DNA prepared from the cell line KB5C20 gave hybridiza- tion patterns for both C1SC4 and TcG probes using the enzymes Sal I, Sfi I, and Xho I, with fragment sizes rang- ing from 50 kb to 700 kb. None of the bands, however, showed hybridization with both probes.

The same panel of enzymes was tested on the human cell line 49,XXXXY, human peripheral blood lympho- cytes (PBLs) and fibroblasts with the probes HuCTLA-1 and PGA5 (TcG) (see Table 2). The clearest results were obtained with DNA prepared from the 49,XXXXY cell line (see Fig. 2). Bands were ident i fed with both HuCT- LA-1 and PGA5 probes using the enzymes B s s H I, Bgl I, Sst II, Not I, Sal I, Nru I, Sfi I, and Xho I. As with the mouse cells and probes, none of the PGA5-1abeled bands corresponded to the HuCTLA-1 positive bands. To determine the minimum distance between Tc G and Ctla-1 loci, we sought to obtain a restriction map of the

Table 2. Resulting fragment lengths of DNA prepared from the cell line 49,XXXXY digested with a panel of enzymes and probed with PGA5 (TcG) and HuCTLA-1

Restriction Probes enzymes

PGA5 HuCTLA- 1 (kb) (kb)

BssH I 50 600 Bgl I 160 30

50 Nar I 600 Not I 50 620 Nru I 620 610

10 Sal I 35 420

2OO Sfi I 200 430

30 Sst II 120 700

70 590 Xho I 70 100

30

DNA prepared from the cell line 49,XXXXY digested with the listed restriction endonucleases was hybridized with the probes PGA5 and HuCTLA-1. The resulting bands corresponded to DNA fragments rang- ing in size from less than 30 kb to 700 kb. No fragment showed cross hybridization between the probes for any enzyme tested

442 K. Harper et al.: Proximity of Tcr, and CTLA-1 loci

Fig. 2a and b. Photograph of a gel showing DNA prepared from human 49,XXXXY cells digested with a panel of "rare-cutter" restriction en- donucleases, BssH I, Bgl II, Nae I, Not I, Nru I, Pvu I, Sal I, Sfi I, and Xho I. a The autoradiograph of the corresponding blot showing the band pattern following hybridization with HuCTLA1. b The autoradiograph following subsequent hybridization with the human Tcr, probe PGA5

region involved. DNA prepared from the cell l~e 49,XXXXY was double-digested with various combina- tions of the endonucleases Not I, Nar I, BssH I, Sst II, Nae I, and Nru I and the blots hybridized with the HuCT- LA-1 probe. All combinations of double digests produced a single band ranging in size from 590 kb to 600 kb (results not shown). While making restriction enzyme mapping and determination of minimum distance between the mar- kers very difficult, these results did however demonstrate that Ctla-1 and Tcr~ loci are separated by a minimum of one Hpa II tiny fragment (HTF) island (Bird et al. 1985, Bird 1986) and that Ctla-1 is most probably flanked by HTF islands about 600 kb apart.

To. determine the chrl)mosomal order of T cr~, CTLA-1, and NP-1 on human chromcfsome 1~, we tested metaphase preparations of the human chromosome 14 in- version cell line SUP-T1 by in situ hybridization using the probes HuCTLA-1 and NP-1. In normal human metaphase preparations all three NP-1, Tcr,, and CTLA-1 genes have been shown to map to 14qll-q12 (Croce et al. 1985, Caccia et al. 1985, this communica- tion, Remes et al. 1984, and Nomach et al. 1977). Using

HuCTLA-1 as a probe for in situ hybridization on the SUP-T1 cell line, 39 silver grains were associated with the normal chromosome 14; of these, 33 (85%) mapped to the q11-q12 region (see Fig. 3a). Thirty-six silver grains were associated with the inv(14) (qll; q32) chro- mosome, and 29 (81%) mapped to the telomeric break point in the q32 region.

Using the NP-1 clone as a probe, 26 silver grains were found to be associated with the normal chromosome 14; of these, 18 (72%) were located in the q11-q12 region (see Fig. 3b). Of the 25 silver grains associated with the inv(14) (q11; q32) chromosome, 17 (68%) also mapped

to the q l l - q l 2 region. The HuCTLA-1 gene was thus shown to be translocated to 14q32 in the SUP-T1 inver- sion, with NP-1 remaining at 14ql 1. From these data we conclude that NP-1 lies centromeric to Tcr~, and since the SUP-T1 inversion break-point is known to be within the Tcr, gene (Baer et al. 1985, Mengle-Gaw et al. 1987), CTLA-1 must lie distal to the site corresponding to the Tcr~ probe used in these experiments.

No suitable inversion or translocation-bearing cell line was available to assist in determining the order of CTLA-1, Tcr~, NP-1, and Gdh-X loci on mouse chromosome 14. However, from the recombinations observed between the markers (Table 1) in the linkage analysis study, we were able to determine the most probable order of these loci and give preliminary estimates of genetic distances. No recombination was observed between the Tcr~ loci and the Ctla-1 loci in 100 meiosis, one recombination event was detected between Tcr~ and NP-1 in 51 meiosis, and five were recorded between Tcr~ and Gdh-X. Since there was no recombination between Tcr~ and Ctla-1, their order in relation to NP-1 and Gdh-Xcannot be determined. In the one case of a recombination event occuring between TcrJCtla-1 and NP-1, Gdh-X cosegregated with TcrJCtla-1. Conversely, in the three cases showing a recombination between Tcr~ and Gdh-X (that were tested with NP-1), NP-! consistently cosegregated with TcrJCtla-1. From these data, crude estimates can be given for the genetic distance between TcrJCtla-1 and NP-1 (2 cM) and between TcrJCtla-1 and Gdh-X (8 cM). The most probable order of the loci is NP-1-TcrJCTLA-1-Gdh-X. However, which of the NP-1 or Gdh-X genes lies closest to the centromere on moug~ chromosome 14 cannot be deduced from'tgesg data.

K. Harper et al.: Proximity of Tcr~ and CTLA-1 loci 443

14 C12D 13

12

11

11

12

13

21

2 2

2 3

24

31

32

a inv(14)(q11;q32)

13 f---r7-) 12 :~---:

11 •

3 2

31 •

• • • • • • e • • • • • •

i i • • • • e • • e e e • • e • e

b 14

11

11 t 2

13

21

2 2

23

24

31

32

inv (14)(qll ;q32)

11 e e i B e • 3, . . . . . " "

2 4 •

2 3 ~ •

e e

Fig. 3a and b. Idiograms of G-banded normal human chromosomes 14 and the inversion (14) (q11; q32) of the cell line SUP-T1, illustrating the distribution of labeled sites for a, the probe Hu-CTLA1 and b, the probe NP-1

Discussion

We demonstrate in this study the proximity of the im- munoglobulin superfamily member Tcr, and the serine esterase family member CTLA-1 in both mouse and man. Accurate determination of the physical distance between these genes by PFGE was impaired, however, by inter- vening unmethylated-CpG-rich stretches (HTF islands). The presence of HTF islands, usually associated with the 5' region of expressed genes, is increasingly being found to be a limitation of the PFGE technique.

With the already high number of known im- munoglobulin superfamily and serine esterase family genes, it is possible that the proximity of members of these families occurs simply by chance. It is, however, remark- able that there should already be three known examples of proximity between an immunoglobulin superfamily gene and a serine esterase gene (see Introduction and this report). In two of these cases, association of the genes exists in mouse and man and must therefore have antedat- ed mammalian speciation. The other possibility of a chance occurrence of their association would be that prior to mammalian speciation there was a repeated 'block' duplication which included ancestral immunoglobulin and serine esterase genes. Again, there is no evidence to for- mally exclude this possibility.

An entirely different explanation for the proximity of these genes is that of linkage conservation. Linkage con- servation would be evidence that the chromosomal seg- ment is protected from rearrangements because of regula- tory or functional interactions between loci involved (Honey et al. 1984, Nadeau and Taylor 1984, Berman et al. 1986, Bucan et al. 1986, Watson et al. 1986). This possible new example of synteny, defined as linkage con- servation of several loci between species, becomes more striking when considering the structural homology and functional relationship between Tcr~ and MHC class I and II elements and structural homology between CTLA-1 and the C2 and factor B complement products. Also, the fact that CTLA-1 and Tcr~ transcripts show overlapping tissue distributions raises further questions as to possible functional and/or regulatory interactions between CTLA-1 and Tcr~.

Acknowledgments. We thank our CIML colleagues, especially B. Jordan and P. Pontarotti, for advice and help in the pulsed field electrophoresis, the Wellcome Trust for a grant to K. H., and INSERM and CNRS for institutional support.

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444 K. Harper et al.: Proximity of Tc G and CTLA-1 loci

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Received July 11, 1988