Immunogenetics 35: 58-61, 1992
II111 UllO-
genetics
© Springer-Verlag 1992
Chromosomal locations of the genes coding for the integrin #6 and subunits Geoffrey W. Krissansen 1, Qian Yuan t, David Jenkins l, Wei-Meng Jiang 1, Lesley Rooke 2, Nigel K. Spurr 2, Michael Eccles 3, Euphemia Leung 1, and James D. Watson t 1 Department of Molecular Medicine, School of Medicine, University of Auckland, Auckland, New Zealand a Human Genetic Resources Laboratory, Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, UK 3 Department of Biochemistry, University of Otago, Dunedin, New Zealand Received March 26, 1991; revised version received June 6, 1991
Integrins are a large family of a/3 subunit receptors, expressed at the cell surface, which mediate cell-cell and cell-matrix interactions during many biological processes including embryogenesis, wound healing, tumor metastasis, and immunity (reviewed by Springer 1990; Hemler 1990). Recently, investigators have exploited the high/3 gene sequence homology with the aid of the polymerase chain reaction (PCR) to amplify and isolate four new integrin/3 subunits, termed integrin/34 (Suzuki and Naitoh 1990),/35 (Suzuki et al. 1990),/36 (Sheppard et al. 1990), and/37 (Yuan et al. 1990). Integrins /34 and/35 are expressed on epithelial cells in association with the V L A a 6 (Hemler et al. 1989) and VnRo~ (Ramaswamy and Hemler 1990; Smith et al. 1990) subunits, respectively. Integrin /36 is also found on epithelial cells (Sheppard et al. 1990) but its associated a subunit(s) has not yet been identified. In contrast, the /37 subunit apears to be restricted to leukocytes and thus may be a new member of the LeuCAM subset with an as yet undefined immunological role (Yuan et al. 1990). Several of the most functionally related integrins are organized in gene clusters. The genes coding for the three a snbunits of the Leu-CAM subset are clustered on the short arm of human chromosome 16 (Corbi et al. 1988) and the V L A al and a 2 genes are both located on human chromosome 5 (Rettig et al. 1984; Hemler 1990). The genes encoding the GPIIb and GPIIIa subunits comprising the GPIIblIIa complex are both contained on a 260 kilobase (kb) fragment that maps to human chromosome bands 17q21-22 (Bray et al. 1988). The coordinate expression of the GPIIb and GPIIIa genes may depend on their close physical proximity. The chromosomal locations of the /36 and /37 genes were determined to investigate their possible linkage with other members of the integrin supergene family. The insert of clone 1 (137) (Yuan et at. 1990) Was used to detect the human/37 gene in Hind III digests of human genomic Address correspondence and offprint requests to: G.W. Krissansen.
D N A from a variety of cell lines and somatic cell hybrids. Even under conditions of high-stringency washing [0.1 × standard sodium citrate (SSC), 50 °C] used in these
Fig. 1. Hybridization of the human /37 probe with DNA from the human-rodentsomaticcell hybrids. GenomicDNA (20 ~tg)was digested with Hind III and the fragments were separated by electrophoresis in a 0.6 % agarose gel and transferred to GeneScreen Plus (NEN Research Products, Boston, MA). Integrin ~37-specificsequences were detected with the 3zP-labeled insert of clone 1 (/37). The sizes, in kb, of the hybridizing restriction fragments are indicated in the right margin.
G.W. Krissansen et al.: Chromosomal assignment of integrin/36 and/37 genes
experiments the human cDNA probe detected bands in all four human, mouse, rat, and hamster DNAs (Fig. 1). Sequence obtained from cDNA clones encoding mouse/37 revealed that the human and mouse/37 genes are highly similar (70%; unpublished data). The two fragments of 20 and 2.6 kb detected in human DNA proved to be diagnostic, as no fragments of these sizes were detected in rodent DNAs. A 5' 0.39 kb Pst 1-Pst 1 fragment (nucleotides 83-473) of the insert of clone 1 (/37) detected the 20 kb fragment only (data not shown). The human probe was then used in hybridization experiments with DNA from a panel of 15 somatic cell hybrids. As summarized in Table 1 complete concordance was observed between the presence of the two Hind III fragments and chromosome 12. All other chromosomes showed discordancy in four or more of the 15 cell lines. In particular the cell hybrid lc~A9, which contains chromosomes 12, 21, and the X chromosome only, was positive (Fig. 1). Chromosomes 21 and X and 19 of the remaining chromosomes were excluded by one or more examples of discordance (see hybrids FIR5R3 and DT1.2.4; Fig. 1 and Table 1). However, the presence of additional, highly related, sequences on chromosome 9 was not directly excluded since no hybrid contained chromosome 9. A Dde I-Sau 3A fragment of a genomic clone H.A/36 encoding amino acid residues 115-176 (Sheppard et al. 1990) and containing 36 base pair (bp) of 5' intronic sequence was used to detect the integrin/36 gene in Hind III digests of human genomic DNA from cell lines and somatic cell hybrids as detailed above for the assignment of integrin/37. A single fragment of 3.6 kb was detected in the human cell line, and this was distinguishable from fragments detected in rodent DNAs (Fig. 2). A human band of identical size was also detected with an 135 bp DNA probe which had been generated by PCR amplification of a region of /36 genomic DNA encoding amino acid residues 136-180 (Sheppard et al. 1990; data not shown). Analysis for the presence of the human 3.6 kb fragment in the somatic cell hybrids revealed a strong concordance with chromosome 2 (Table 2). All other chromosomes were discordant in five or more of the 18 cell lines. The most informative hybrid was CTP 41A2 which contained a relatively small number of chromosomes in addition to chromosome 2. The additional chromosomes 6, 7, 14, 17, and X in this hybrid were excluded by discordance in other hybrids (for example see hybrids MOG34A4 and PCTBA1.8). The presence of highly related sequences on chromosome 9 was not excluded since no hybrid contained chromosome 9. The assignment of the integrin /36 gene to chromosome 2 is notable because the VnRc~ gene also resides on chromosome 2 (Sosnoski et al. 1989). In some cell types the VnRc~ subunit is associated with multiple/3 subunits (Krissansen et al. 1990). VnR~-associated /3 subunits include /3t (Bodary and McClean 1990; Vogel
59
Fig. 2. Hybridization of the human/36 probe with DNA from the humanrodent somatic cell hybrids. Genomic DNA was processed as described in Fig. 1. Integrin /36-specific sequences were detected with the 32p_ labeled insert of clone H.A. /36- The clone had been isolated from a human fetal liver genomic library constructed in the Charon 4A vector (Lawn et al. 1978; American Type Culture Collection, Rockville, MD, unpublished data). The size, in kb, of the hybridizing restriction fragments are indicated in the right margin.
et al. 1990),/33 (Fitzgerald et al. 1987), /35 (Smith et al. 1990), and /33b (Krissansen et al. 1990); and the/36 sequence is highly similar to the/31, /33, and/35 sequences (Argraves et al. 1987; Fitzgerald et al. 1987; Rosa et al. 1988; Ramaswamy and Hemler 1990). However the/33 gene is clustered on chromosome 17 with the gene encoding its other a chain partner GPIIb (Bray et al. 1988), and the/31 gene maps to chromosome 10 (Rettig et al. 1984). The assignment of the human/37 gene to chromosome 12 is significant because the gene encoding the VLA 5 subunit resides on chromosome 12 at bands 12q1-13 (Sosnoski et al. 1988). VLA-5 serves an immunological function on T cells in synergizing with the T-cell receptor during T-cell activation (Shimizu et al. 1990). Similarly, a potential immunological role for/37 is suggested by the finding that/37 shares identity with a molecule highly expressed on mouse intraepithelial lymphocytes (IEL; Kilshaw and Murant 1990; Yuan et al. 1991). It was suggested that the IEL molecule may perform an adhesive function which modulates or focuses effector cell activity
60
G . W . Krissansen et al.: C h r o m o s o m a l assignment of integrin #6 and/37 genes
T a b l e 1. Correlation between specific h u m a n chromosomes and the integrin #7 gene in 15 somatic cell hybrids. Somatic cell hybrids
Human chromosome
Reference*
1
2
3
4
5
6
7
8
CTP34B4 3W4CL5 MOG34A4 DUR4.3 SIR74ii T W I N 19-D 12 lenA9
+
+
+
-
+
+
+
+
CTP41A2 DT1.2.4 FG10 SIF4A31 F4Scl3C112t PCTBA1.8 THYB1.3 FIR5R3
--
+ +
+ +
+
+
+ + +
+ + +
-
+
+
-
+
-
+
-
+ +
10
11
12
13
14
15
16
17
18
-
+ + +
+ + + + +
+ + +
+ + + + +
+ + +
+ + -
+ + + +
+
+ + +
+ + +
+
-
+
-
+
+
+
+
19
20
21
22
X
+ -
+
+ + + +
+ + +
+ + + + +
-
+
+ +
+
+
+
+ --
+
+
+
+ +
+
--
+ +
+
+
+
*
-
+
-
I
--
+ +
+ --
+ + + + + +
+
-
+ +
1 1 1 1 2 1 3 4 1
5 5 3 2 33
4 7 1 3 27
6 1 7 1 53
+
+ + --
--
+
+ +
--
+
+ +
+
4 7 1 3 27
2 6 2 5 47
(number of hybrid clones) 5 3 3 3 3 3 0 6 7 6 6 7 7 8 2 1 2 2 1 1 0 2 4 4 4 4 4 7 27 33 40 40 33 33 47
3 6 2 4 40
4 7 1 3 27
7 8 0 0 0
3 8 0 4 27
6 4 4 1 33
+
3 6 2 4 40
3 7 1 4 33
5 5 3 2 33
5 5 3 2 33
1 8 0 6 40
2 7 1 5 40
1 1 1 1 1
+
+
#7 g e n e / c h r o m o s o m e retention +/+ -/+/-/+ Percent discordant
+
9
* 1. Davies et al. 1989; 2. Edwards et al. 1985; 3. Bai et al. 1982; 4. Goodfellow et al. 1980. * The hybrid F 4 S c 1 3 C l 1 2 contains the short a r m of c h r o m o s o m e 1 only.
T a b l e 2. Correlation between specific h u m a n c h r o m o s o m e s and the integrin Somatic cell
#6 gene
in 18 somatic cell hybrids. Reference*
Human chromosome
hybrids
CTP34B4 CTP41A2 FG10 SIR4ii PLTI.S LSR34S49 3W4CL5 MOG34A4 DUR4.3 TWINI9-D12 DT1.2.4 SIF4A31 F 4 S c l 3 C 112' FIR5R3 PCTBA1.8 THYB1.3 1 ~A9 HORL9X
1
2
3
4
5
6
7
8
+ + +
+
+
-
+
+ +
+ +
+
+ + + +
+ +
+ + -
+
+
-
+
-
+
+ + + + +
+
-
+
+
+
+
+ +
+ + -
+
+ +
+
-
+
+
+
9
-
10
+
+ + + + +
/36 g e n e / c h r o m o s o m e retention (number +/+ 3 6 3 2 -/9 12 7 9 +/3 0 5 3 -/+ 3 0 3 4 Percent discordant 33 0 44 39
13
14
15
16
17
18
+
-
+ +
-
+
+ +
+
+ +
+ + + + -
+ + + + + + +
+ +
--
12
+
+
+
11
+ + + + +
+ + + +
+ + +
+ + + + + -
+ -+ -+ -+
+ + + + + +
19
20
+ + + +
+
+
+ + + +
+ -
+ + +
+
21
22
X
+ + + + + + + + + + + +
+ + + + + + + +
+ + + + + +
+ +
-
+ + +
4 6 6 2 44
2 8 4 4 44
5 2 10 1 61
+ +
of hybrid clones) 3 4 3 3 0 9 9 10 10 12 3 3 2 2 0 3 2 3 3 6 33 28 28 28 33
2 8 4 4 44
1 7 5 5 56
3 7 5 3 44
2 7 2 4 33
5 5 7 1 44
2 8 4 4 44
2 9 3 4 39
4 7 5 2 39
5 7 5 1 33
1 11 1 5 33
2 9 3 4 39
1 1 1 1 2 3 1 1 1 1 1 4 1 1 5 6 1 1
* 1. Davies et al. 1989; 2. Martin et al. 1987; 3. Spurr et ai. 1986; 4. E d w a r d s et al. 1985; 5. Bai et al. 1982; 6. Goodfellow et al. 1980. t The hybrid F 4 S c 1 3 C l 1 2 contains the short a r m of c h r o m o s o m e 1 only.
G.W. Krissansen et al.: Chromosomal assignment of integrm/36 and during IEL defence of the intestinal mucosa (Kilshaw and M u r a n t 1990). W h i l s t t h e s e q u e n c e o f t h e /37 s u b u n i t s h o w e d h i g h e s t s i m i l a r i t y to/32 (46 %; Y u a n et al. 1990) t h e g e n e e n c o d i n g the /32 s u b u n i t h a s b e e n a s s i g n e d to c h r o m o s o m e 21 ( C o r b i et al. 1988). T o date, i n c l u d i n g t h e r e s u l t s o f this c o m m u n i c a t i o n , 14 o f 19 i n t e g r i n g e n e s h a v e b e e n a s s i g n e d , a n d o n l y t h e /31 a n d / 3 2 g e n e s a r e s e g r e g a t e d a l o n e o n t h e i r r e s p e c t i v e chromosomes. The extent and evolutionary significance o f l i n k a g e s b e t w e e n i n t e g r i n g e n e s s u c h as t h a t b e t w e e n t h e ~6 a n d VnRo~ g e n e s , a n d the/37 a n d V L A 5 ~ g e n e s rem a i n s to b e d e t e r m i n e d . Acknowledgments. This work was supported by grants from the Health
Research Council of New Zealand, the Auckland Medical Research Foundation and the Cancer Society of New Zealand Inc. We thank Lois Duncan and Gae Thompson for excellent secretarial help in preparing the manuscript.
References Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M.D., and Ruoslahti, E. : Amino acid sequence of the human fibronectin receptor. J Cell Biol 105:1183-1190, 1987 Bai, Y., Sheer, D., Hiorns, L., Knowles, R.W., and Tunnacliffe, A.: A monoclonal antibody recognising a cell surface antigen coded for by a gene on human chromosome 17. Ann Hum Genet 46: 337-347, 1982 Bodary, S. and McClean, J. S.: The integrin beta 1 subunit associates with the vitronectin receptor alpha V subunit to form a novel vitronectin receptor in a human embryonic kidney cell line. J Biol Chem 265: 5938-5941, 1990 Bray, P.F., Barsh, G., Rosa, J.-P., Luo, X.Y., Magenis, E., and Shuman, M. A. : Physical linkage of the genes for platelet membrane glycoproteins IIb and Ilia. Proc Natl Acad Sci USA 85: 8683-8687, 1988 Corbi, A.L., Larson, R.S., Kishimoto, T.K., Springer, T.A., and Morton, C. C. : Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac-1 and p150,95. J Exp Med 167. 1597-1607, 1988 Davies, A.A., Moss, S.E., Crompton, M.R., Jones, T.A., Spurr, N.K., Sheer, D., Kozak, C., and Crumpton, M.J.: The gene coding for the p68 calcium-binding protein is localized to bands q32-q34 of human chromosome 5 and to mouse chromosome 11. Hum Genet 82: 234-238, 1989 Edwards, Y. H., Parkar, M., Povey, S., West, L. F., Farrington, F. M., and Solomon, E.: Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe. Ann Hum Genet 49: 101-109, 1985 Fitzgerald, L. A., Steiner, B., Rall, S. C., Lo, S.-S., and Phillips, D. R.: Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. J Biol Chem 262: 3936-3939, 1987 Goodfellow, P., Banting, G., Levy, R., Povey, S., and McMichael, A. : A human X-linked antigen defined by a monoclonal antibody. Somatic Cell Genet 6: 777-787, 1980 Hemler, M.E.: VLA proteins in the integrin family: Structures, functions, and their role on leukocytes. Annu Rev Immunol 8: 365-400, 1990 Hemler, M.E., Crouse, C., and Sonnenberg, A.: Association of the VLAot6 subunit with a novel protein. J Biol Chem 264: 6529-6535, 1989 Kilshaw, P. J. and Murant, S. J. : A new surface antigen on intraepithelial lymphocytes in the intestine. Eur J lmmuno120: 2201-2207, 1990
/37 genes
61
Krissansen, G. W., Elliot, M. J., Lucas, C. M., Stomski, F. C., Berndt, M. C., Cheresh, D. A., Lopez, A. F., and Burns, G. F. : Identification of a novel integrin/3 subunit expressed on cultured monocytes (macrophages). Evidence that one c~ subunit can associate with multiple/3 subunits. J Biol Chem 265: 823-830, 1990 Lawn, R.M., Fritsch, E.F., Parker, R. C., Blake, G., and Maniatis, T.: The isolation and characterisation of linked delta- and betaglobin genes from a cloned library of human DNA. Cell 15: 1157-1174, 1978 Martin, D., Tucker, D. F., Gorman, P., Sheer, D., Spnrr, N. K., and Trowsdale, J.: The human placental alkaline phosphatase gene and related sequences map to chromosome 2 band q37. Ann Hum Genet 51: 145-152, 1987 Ramaswamy, H. and Hemler, M.E.: Cloning, primary structure and properties of a novel human integrin /3 subunit. EMBO J 9: 1561-1568, 1990 Rettig, W.J., Dracopoli, N.C., Goetzger, T.A., Spengler, B.A., Biedler, J. L., Oettgen, H. F., and Old, L. J.: Somatic cell genetic analysis of human cell surface antigens: chromosomal assignments and regulation of expression in rodent-human hybrid cells. Proc Natl Acad Sei USA 81: 6437-6441, 1984 Rosa, J.P., Bray, P.F., Gayet, O., Johnston, G.I., Cook, R.G., Jackson, K.W., Shuman, M.A., and McEver, R. P.: Cloning of glycoprotein IIIa cDNA from human erythroleukaemia cells and iocalisation of the gene to chromosome 17. Blood 72: 593-600, 1988 Sheppard, D., Rozzo, C., Starr, L., Quaranta, V., Erle, D.J., and Pytela, R. : Complete amino acid sequence of a novel integrin/3 subunit (/36) identified in epithelial ceils using the polymerase chain reaction. J Biol Chem 265: 11502-11507, 1990 Shirmzu, Y., van Seventer, G.A., Horgan, K.J., and Shaw, S.: Costimulation of proliferative responses of resting CD4 + T cells by the interaction of VLA-4 and VLA-5 with fibronecfin or VLA-6 with laminin. J Immunol 145: 59-67, 1990 Smith, J. W., Vestal, D.J., Irwin, S.V., Burke, T.A., and Cheresh, D.A.: Purification and functional characterisation of integrin ~v/35. J Biol Chem 265: 11008-11013, 1990 Sosnoski, D.M., Emanuel, B.S., Hawkins, A.L., van Tuinen, P., Ledbetter, D.H., Nussbaum, R.L., Kaos, F.-T., Schwartz, E., Phillips, D., Bennett, J.S., Fitzgerald, L.A., and Poncz, M.: Chromosomal localization of the genes for the vitronectin and fibronectin receptors a subunits and for platelet glycoproteins IIb and IIIa. J Clin Invest 81: 1993-1998, 1988 Springer, T. A.: Adhesion receptors of the immune system. Nature 346: 425-434, 1990 Spurr, N.K., Durbin, H., Sheer, D., Parkar, M., Bobrow, L., and Bodmer, W. F.: Characterisation and chromosomal assignment of a human cell surface antigen defined by the monoclonal antibody AUAI. lnt J Cancer 38: 631-636, 1986 Suzuki, S., Huang, Z.-S., and Tanihara, H. : Cloning of an integrin/3 subunit exhibiting high homology with integrin/3s subunit. Proc Natl Acad Sci USA 87: 5354-5358, 1990 Suzuki, S. and Naitoh, Y. : Amino acid sequence of a novel integrin /34 subunit and primary expression of the mRNA in epithelial cells. EMBO J 9: 757-763, 1990 Vogel, B. E., Tarone, G., Giancotti, F. P., Galit, J., and Ruoslahti, E.: A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1). J Biol Chem 265: 5934-5937, 1990 Yuan, Q., Jiang, W.-M., Krissansen, G. W., and Watson, J. D. : Cloning and sequence analysis of a novel/32-related integrin transcript from T lymphocytes: homology of integrin cysteine-rich repeats to domain III of laminin B chains, lnt Immunol 2: 1097-1108, 1990 Yuan, Q., Jiang, W.-M., Hollander, D., Leung, E., Watson, J. D., and Krissansen, G. W.: Identity between the novel integrin/37 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine. Biochem Biophys Res Commun 176: 1443-1449, 1991
II111 UllO-
genetics
© Springer-Verlag 1992
Chromosomal locations of the genes coding for the integrin #6 and subunits Geoffrey W. Krissansen 1, Qian Yuan t, David Jenkins l, Wei-Meng Jiang 1, Lesley Rooke 2, Nigel K. Spurr 2, Michael Eccles 3, Euphemia Leung 1, and James D. Watson t 1 Department of Molecular Medicine, School of Medicine, University of Auckland, Auckland, New Zealand a Human Genetic Resources Laboratory, Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, UK 3 Department of Biochemistry, University of Otago, Dunedin, New Zealand Received March 26, 1991; revised version received June 6, 1991
Integrins are a large family of a/3 subunit receptors, expressed at the cell surface, which mediate cell-cell and cell-matrix interactions during many biological processes including embryogenesis, wound healing, tumor metastasis, and immunity (reviewed by Springer 1990; Hemler 1990). Recently, investigators have exploited the high/3 gene sequence homology with the aid of the polymerase chain reaction (PCR) to amplify and isolate four new integrin/3 subunits, termed integrin/34 (Suzuki and Naitoh 1990),/35 (Suzuki et al. 1990),/36 (Sheppard et al. 1990), and/37 (Yuan et al. 1990). Integrins /34 and/35 are expressed on epithelial cells in association with the V L A a 6 (Hemler et al. 1989) and VnRo~ (Ramaswamy and Hemler 1990; Smith et al. 1990) subunits, respectively. Integrin /36 is also found on epithelial cells (Sheppard et al. 1990) but its associated a subunit(s) has not yet been identified. In contrast, the /37 subunit apears to be restricted to leukocytes and thus may be a new member of the LeuCAM subset with an as yet undefined immunological role (Yuan et al. 1990). Several of the most functionally related integrins are organized in gene clusters. The genes coding for the three a snbunits of the Leu-CAM subset are clustered on the short arm of human chromosome 16 (Corbi et al. 1988) and the V L A al and a 2 genes are both located on human chromosome 5 (Rettig et al. 1984; Hemler 1990). The genes encoding the GPIIb and GPIIIa subunits comprising the GPIIblIIa complex are both contained on a 260 kilobase (kb) fragment that maps to human chromosome bands 17q21-22 (Bray et al. 1988). The coordinate expression of the GPIIb and GPIIIa genes may depend on their close physical proximity. The chromosomal locations of the /36 and /37 genes were determined to investigate their possible linkage with other members of the integrin supergene family. The insert of clone 1 (137) (Yuan et at. 1990) Was used to detect the human/37 gene in Hind III digests of human genomic Address correspondence and offprint requests to: G.W. Krissansen.
D N A from a variety of cell lines and somatic cell hybrids. Even under conditions of high-stringency washing [0.1 × standard sodium citrate (SSC), 50 °C] used in these
Fig. 1. Hybridization of the human /37 probe with DNA from the human-rodentsomaticcell hybrids. GenomicDNA (20 ~tg)was digested with Hind III and the fragments were separated by electrophoresis in a 0.6 % agarose gel and transferred to GeneScreen Plus (NEN Research Products, Boston, MA). Integrin ~37-specificsequences were detected with the 3zP-labeled insert of clone 1 (/37). The sizes, in kb, of the hybridizing restriction fragments are indicated in the right margin.
G.W. Krissansen et al.: Chromosomal assignment of integrin/36 and/37 genes
experiments the human cDNA probe detected bands in all four human, mouse, rat, and hamster DNAs (Fig. 1). Sequence obtained from cDNA clones encoding mouse/37 revealed that the human and mouse/37 genes are highly similar (70%; unpublished data). The two fragments of 20 and 2.6 kb detected in human DNA proved to be diagnostic, as no fragments of these sizes were detected in rodent DNAs. A 5' 0.39 kb Pst 1-Pst 1 fragment (nucleotides 83-473) of the insert of clone 1 (/37) detected the 20 kb fragment only (data not shown). The human probe was then used in hybridization experiments with DNA from a panel of 15 somatic cell hybrids. As summarized in Table 1 complete concordance was observed between the presence of the two Hind III fragments and chromosome 12. All other chromosomes showed discordancy in four or more of the 15 cell lines. In particular the cell hybrid lc~A9, which contains chromosomes 12, 21, and the X chromosome only, was positive (Fig. 1). Chromosomes 21 and X and 19 of the remaining chromosomes were excluded by one or more examples of discordance (see hybrids FIR5R3 and DT1.2.4; Fig. 1 and Table 1). However, the presence of additional, highly related, sequences on chromosome 9 was not directly excluded since no hybrid contained chromosome 9. A Dde I-Sau 3A fragment of a genomic clone H.A/36 encoding amino acid residues 115-176 (Sheppard et al. 1990) and containing 36 base pair (bp) of 5' intronic sequence was used to detect the integrin/36 gene in Hind III digests of human genomic DNA from cell lines and somatic cell hybrids as detailed above for the assignment of integrin/37. A single fragment of 3.6 kb was detected in the human cell line, and this was distinguishable from fragments detected in rodent DNAs (Fig. 2). A human band of identical size was also detected with an 135 bp DNA probe which had been generated by PCR amplification of a region of /36 genomic DNA encoding amino acid residues 136-180 (Sheppard et al. 1990; data not shown). Analysis for the presence of the human 3.6 kb fragment in the somatic cell hybrids revealed a strong concordance with chromosome 2 (Table 2). All other chromosomes were discordant in five or more of the 18 cell lines. The most informative hybrid was CTP 41A2 which contained a relatively small number of chromosomes in addition to chromosome 2. The additional chromosomes 6, 7, 14, 17, and X in this hybrid were excluded by discordance in other hybrids (for example see hybrids MOG34A4 and PCTBA1.8). The presence of highly related sequences on chromosome 9 was not excluded since no hybrid contained chromosome 9. The assignment of the integrin /36 gene to chromosome 2 is notable because the VnRc~ gene also resides on chromosome 2 (Sosnoski et al. 1989). In some cell types the VnRc~ subunit is associated with multiple/3 subunits (Krissansen et al. 1990). VnR~-associated /3 subunits include /3t (Bodary and McClean 1990; Vogel
59
Fig. 2. Hybridization of the human/36 probe with DNA from the humanrodent somatic cell hybrids. Genomic DNA was processed as described in Fig. 1. Integrin /36-specific sequences were detected with the 32p_ labeled insert of clone H.A. /36- The clone had been isolated from a human fetal liver genomic library constructed in the Charon 4A vector (Lawn et al. 1978; American Type Culture Collection, Rockville, MD, unpublished data). The size, in kb, of the hybridizing restriction fragments are indicated in the right margin.
et al. 1990),/33 (Fitzgerald et al. 1987), /35 (Smith et al. 1990), and /33b (Krissansen et al. 1990); and the/36 sequence is highly similar to the/31, /33, and/35 sequences (Argraves et al. 1987; Fitzgerald et al. 1987; Rosa et al. 1988; Ramaswamy and Hemler 1990). However the/33 gene is clustered on chromosome 17 with the gene encoding its other a chain partner GPIIb (Bray et al. 1988), and the/31 gene maps to chromosome 10 (Rettig et al. 1984). The assignment of the human/37 gene to chromosome 12 is significant because the gene encoding the VLA 5 subunit resides on chromosome 12 at bands 12q1-13 (Sosnoski et al. 1988). VLA-5 serves an immunological function on T cells in synergizing with the T-cell receptor during T-cell activation (Shimizu et al. 1990). Similarly, a potential immunological role for/37 is suggested by the finding that/37 shares identity with a molecule highly expressed on mouse intraepithelial lymphocytes (IEL; Kilshaw and Murant 1990; Yuan et al. 1991). It was suggested that the IEL molecule may perform an adhesive function which modulates or focuses effector cell activity
60
G . W . Krissansen et al.: C h r o m o s o m a l assignment of integrin #6 and/37 genes
T a b l e 1. Correlation between specific h u m a n chromosomes and the integrin #7 gene in 15 somatic cell hybrids. Somatic cell hybrids
Human chromosome
Reference*
1
2
3
4
5
6
7
8
CTP34B4 3W4CL5 MOG34A4 DUR4.3 SIR74ii T W I N 19-D 12 lenA9
+
+
+
-
+
+
+
+
CTP41A2 DT1.2.4 FG10 SIF4A31 F4Scl3C112t PCTBA1.8 THYB1.3 FIR5R3
--
+ +
+ +
+
+
+ + +
+ + +
-
+
+
-
+
-
+
-
+ +
10
11
12
13
14
15
16
17
18
-
+ + +
+ + + + +
+ + +
+ + + + +
+ + +
+ + -
+ + + +
+
+ + +
+ + +
+
-
+
-
+
+
+
+
19
20
21
22
X
+ -
+
+ + + +
+ + +
+ + + + +
-
+
+ +
+
+
+
+ --
+
+
+
+ +
+
--
+ +
+
+
+
*
-
+
-
I
--
+ +
+ --
+ + + + + +
+
-
+ +
1 1 1 1 2 1 3 4 1
5 5 3 2 33
4 7 1 3 27
6 1 7 1 53
+
+ + --
--
+
+ +
--
+
+ +
+
4 7 1 3 27
2 6 2 5 47
(number of hybrid clones) 5 3 3 3 3 3 0 6 7 6 6 7 7 8 2 1 2 2 1 1 0 2 4 4 4 4 4 7 27 33 40 40 33 33 47
3 6 2 4 40
4 7 1 3 27
7 8 0 0 0
3 8 0 4 27
6 4 4 1 33
+
3 6 2 4 40
3 7 1 4 33
5 5 3 2 33
5 5 3 2 33
1 8 0 6 40
2 7 1 5 40
1 1 1 1 1
+
+
#7 g e n e / c h r o m o s o m e retention +/+ -/+/-/+ Percent discordant
+
9
* 1. Davies et al. 1989; 2. Edwards et al. 1985; 3. Bai et al. 1982; 4. Goodfellow et al. 1980. * The hybrid F 4 S c 1 3 C l 1 2 contains the short a r m of c h r o m o s o m e 1 only.
T a b l e 2. Correlation between specific h u m a n c h r o m o s o m e s and the integrin Somatic cell
#6 gene
in 18 somatic cell hybrids. Reference*
Human chromosome
hybrids
CTP34B4 CTP41A2 FG10 SIR4ii PLTI.S LSR34S49 3W4CL5 MOG34A4 DUR4.3 TWINI9-D12 DT1.2.4 SIF4A31 F 4 S c l 3 C 112' FIR5R3 PCTBA1.8 THYB1.3 1 ~A9 HORL9X
1
2
3
4
5
6
7
8
+ + +
+
+
-
+
+ +
+ +
+
+ + + +
+ +
+ + -
+
+
-
+
-
+
+ + + + +
+
-
+
+
+
+
+ +
+ + -
+
+ +
+
-
+
+
+
9
-
10
+
+ + + + +
/36 g e n e / c h r o m o s o m e retention (number +/+ 3 6 3 2 -/9 12 7 9 +/3 0 5 3 -/+ 3 0 3 4 Percent discordant 33 0 44 39
13
14
15
16
17
18
+
-
+ +
-
+
+ +
+
+ +
+ + + + -
+ + + + + + +
+ +
--
12
+
+
+
11
+ + + + +
+ + + +
+ + +
+ + + + + -
+ -+ -+ -+
+ + + + + +
19
20
+ + + +
+
+
+ + + +
+ -
+ + +
+
21
22
X
+ + + + + + + + + + + +
+ + + + + + + +
+ + + + + +
+ +
-
+ + +
4 6 6 2 44
2 8 4 4 44
5 2 10 1 61
+ +
of hybrid clones) 3 4 3 3 0 9 9 10 10 12 3 3 2 2 0 3 2 3 3 6 33 28 28 28 33
2 8 4 4 44
1 7 5 5 56
3 7 5 3 44
2 7 2 4 33
5 5 7 1 44
2 8 4 4 44
2 9 3 4 39
4 7 5 2 39
5 7 5 1 33
1 11 1 5 33
2 9 3 4 39
1 1 1 1 2 3 1 1 1 1 1 4 1 1 5 6 1 1
* 1. Davies et al. 1989; 2. Martin et al. 1987; 3. Spurr et ai. 1986; 4. E d w a r d s et al. 1985; 5. Bai et al. 1982; 6. Goodfellow et al. 1980. t The hybrid F 4 S c 1 3 C l 1 2 contains the short a r m of c h r o m o s o m e 1 only.
G.W. Krissansen et al.: Chromosomal assignment of integrm/36 and during IEL defence of the intestinal mucosa (Kilshaw and M u r a n t 1990). W h i l s t t h e s e q u e n c e o f t h e /37 s u b u n i t s h o w e d h i g h e s t s i m i l a r i t y to/32 (46 %; Y u a n et al. 1990) t h e g e n e e n c o d i n g the /32 s u b u n i t h a s b e e n a s s i g n e d to c h r o m o s o m e 21 ( C o r b i et al. 1988). T o date, i n c l u d i n g t h e r e s u l t s o f this c o m m u n i c a t i o n , 14 o f 19 i n t e g r i n g e n e s h a v e b e e n a s s i g n e d , a n d o n l y t h e /31 a n d / 3 2 g e n e s a r e s e g r e g a t e d a l o n e o n t h e i r r e s p e c t i v e chromosomes. The extent and evolutionary significance o f l i n k a g e s b e t w e e n i n t e g r i n g e n e s s u c h as t h a t b e t w e e n t h e ~6 a n d VnRo~ g e n e s , a n d the/37 a n d V L A 5 ~ g e n e s rem a i n s to b e d e t e r m i n e d . Acknowledgments. This work was supported by grants from the Health
Research Council of New Zealand, the Auckland Medical Research Foundation and the Cancer Society of New Zealand Inc. We thank Lois Duncan and Gae Thompson for excellent secretarial help in preparing the manuscript.
References Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M.D., and Ruoslahti, E. : Amino acid sequence of the human fibronectin receptor. J Cell Biol 105:1183-1190, 1987 Bai, Y., Sheer, D., Hiorns, L., Knowles, R.W., and Tunnacliffe, A.: A monoclonal antibody recognising a cell surface antigen coded for by a gene on human chromosome 17. Ann Hum Genet 46: 337-347, 1982 Bodary, S. and McClean, J. S.: The integrin beta 1 subunit associates with the vitronectin receptor alpha V subunit to form a novel vitronectin receptor in a human embryonic kidney cell line. J Biol Chem 265: 5938-5941, 1990 Bray, P.F., Barsh, G., Rosa, J.-P., Luo, X.Y., Magenis, E., and Shuman, M. A. : Physical linkage of the genes for platelet membrane glycoproteins IIb and Ilia. Proc Natl Acad Sci USA 85: 8683-8687, 1988 Corbi, A.L., Larson, R.S., Kishimoto, T.K., Springer, T.A., and Morton, C. C. : Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac-1 and p150,95. J Exp Med 167. 1597-1607, 1988 Davies, A.A., Moss, S.E., Crompton, M.R., Jones, T.A., Spurr, N.K., Sheer, D., Kozak, C., and Crumpton, M.J.: The gene coding for the p68 calcium-binding protein is localized to bands q32-q34 of human chromosome 5 and to mouse chromosome 11. Hum Genet 82: 234-238, 1989 Edwards, Y. H., Parkar, M., Povey, S., West, L. F., Farrington, F. M., and Solomon, E.: Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe. Ann Hum Genet 49: 101-109, 1985 Fitzgerald, L. A., Steiner, B., Rall, S. C., Lo, S.-S., and Phillips, D. R.: Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. J Biol Chem 262: 3936-3939, 1987 Goodfellow, P., Banting, G., Levy, R., Povey, S., and McMichael, A. : A human X-linked antigen defined by a monoclonal antibody. Somatic Cell Genet 6: 777-787, 1980 Hemler, M.E.: VLA proteins in the integrin family: Structures, functions, and their role on leukocytes. Annu Rev Immunol 8: 365-400, 1990 Hemler, M.E., Crouse, C., and Sonnenberg, A.: Association of the VLAot6 subunit with a novel protein. J Biol Chem 264: 6529-6535, 1989 Kilshaw, P. J. and Murant, S. J. : A new surface antigen on intraepithelial lymphocytes in the intestine. Eur J lmmuno120: 2201-2207, 1990
/37 genes
61
Krissansen, G. W., Elliot, M. J., Lucas, C. M., Stomski, F. C., Berndt, M. C., Cheresh, D. A., Lopez, A. F., and Burns, G. F. : Identification of a novel integrin/3 subunit expressed on cultured monocytes (macrophages). Evidence that one c~ subunit can associate with multiple/3 subunits. J Biol Chem 265: 823-830, 1990 Lawn, R.M., Fritsch, E.F., Parker, R. C., Blake, G., and Maniatis, T.: The isolation and characterisation of linked delta- and betaglobin genes from a cloned library of human DNA. Cell 15: 1157-1174, 1978 Martin, D., Tucker, D. F., Gorman, P., Sheer, D., Spnrr, N. K., and Trowsdale, J.: The human placental alkaline phosphatase gene and related sequences map to chromosome 2 band q37. Ann Hum Genet 51: 145-152, 1987 Ramaswamy, H. and Hemler, M.E.: Cloning, primary structure and properties of a novel human integrin /3 subunit. EMBO J 9: 1561-1568, 1990 Rettig, W.J., Dracopoli, N.C., Goetzger, T.A., Spengler, B.A., Biedler, J. L., Oettgen, H. F., and Old, L. J.: Somatic cell genetic analysis of human cell surface antigens: chromosomal assignments and regulation of expression in rodent-human hybrid cells. Proc Natl Acad Sei USA 81: 6437-6441, 1984 Rosa, J.P., Bray, P.F., Gayet, O., Johnston, G.I., Cook, R.G., Jackson, K.W., Shuman, M.A., and McEver, R. P.: Cloning of glycoprotein IIIa cDNA from human erythroleukaemia cells and iocalisation of the gene to chromosome 17. Blood 72: 593-600, 1988 Sheppard, D., Rozzo, C., Starr, L., Quaranta, V., Erle, D.J., and Pytela, R. : Complete amino acid sequence of a novel integrin/3 subunit (/36) identified in epithelial ceils using the polymerase chain reaction. J Biol Chem 265: 11502-11507, 1990 Shirmzu, Y., van Seventer, G.A., Horgan, K.J., and Shaw, S.: Costimulation of proliferative responses of resting CD4 + T cells by the interaction of VLA-4 and VLA-5 with fibronecfin or VLA-6 with laminin. J Immunol 145: 59-67, 1990 Smith, J. W., Vestal, D.J., Irwin, S.V., Burke, T.A., and Cheresh, D.A.: Purification and functional characterisation of integrin ~v/35. J Biol Chem 265: 11008-11013, 1990 Sosnoski, D.M., Emanuel, B.S., Hawkins, A.L., van Tuinen, P., Ledbetter, D.H., Nussbaum, R.L., Kaos, F.-T., Schwartz, E., Phillips, D., Bennett, J.S., Fitzgerald, L.A., and Poncz, M.: Chromosomal localization of the genes for the vitronectin and fibronectin receptors a subunits and for platelet glycoproteins IIb and IIIa. J Clin Invest 81: 1993-1998, 1988 Springer, T. A.: Adhesion receptors of the immune system. Nature 346: 425-434, 1990 Spurr, N.K., Durbin, H., Sheer, D., Parkar, M., Bobrow, L., and Bodmer, W. F.: Characterisation and chromosomal assignment of a human cell surface antigen defined by the monoclonal antibody AUAI. lnt J Cancer 38: 631-636, 1986 Suzuki, S., Huang, Z.-S., and Tanihara, H. : Cloning of an integrin/3 subunit exhibiting high homology with integrin/3s subunit. Proc Natl Acad Sci USA 87: 5354-5358, 1990 Suzuki, S. and Naitoh, Y. : Amino acid sequence of a novel integrin /34 subunit and primary expression of the mRNA in epithelial cells. EMBO J 9: 757-763, 1990 Vogel, B. E., Tarone, G., Giancotti, F. P., Galit, J., and Ruoslahti, E.: A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1). J Biol Chem 265: 5934-5937, 1990 Yuan, Q., Jiang, W.-M., Krissansen, G. W., and Watson, J. D. : Cloning and sequence analysis of a novel/32-related integrin transcript from T lymphocytes: homology of integrin cysteine-rich repeats to domain III of laminin B chains, lnt Immunol 2: 1097-1108, 1990 Yuan, Q., Jiang, W.-M., Hollander, D., Leung, E., Watson, J. D., and Krissansen, G. W.: Identity between the novel integrin/37 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine. Biochem Biophys Res Commun 176: 1443-1449, 1991
