SHORT COMMUNICATION Assignment
of the Gene for Intercellular Adhesion (/cam-l) to Proximal Mouse Chromosome
CHRISTIE M. BALLANTYNE,**’
CHRISTINE A. KOZAK,t
WILLIAM
E. O’BRIEN,?
Molecule-l 9
AND ARTHUR L. BEAUDET*
‘Department of Medicine and #Howard Hughes Medical Institute and Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030; and tNationa/ Institute of Allergy and infectious Diseases, National Institutes of Health, Bethesda, Maryland 20814 Received
May
18,
1990;
Intercellular adhesion molecule-l (ICAM-1) is an integral membrane protein, a member of the immunoglobulin superfamily, and a ligand for LFA-1. a p2 leukocyte integrin. ICAMhas a tissue distribution similar to that of the major histocompatability complex class II antigens and is likely to play a role in inflammatory responses. We have mapped this gene to proximal mouse chromosome 9 by using mouse-hamster somatic cell hybrids and an interspeties backcross. Since human ICAM- 1 maps to chromosome 19, it joins the LDL receptor to establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Murine Icam-l maps between Cbl-2 and the centromere in the same region as one of the susceptibility genes for insulin-dependent diabetes mellitus (I&-2) that is postulated to play a role in immune function and inflammation leading to insulitis. The mapping of Icam-l to the region known to contain the Zdd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic ,c 1991 Academic Press. Inc. variation in Icam-1.
Intercellular adhesion molecule-l (ICAM-1) is a transmembrane protein that plays a key role in the generation of an inflammatory response (Staunton et al., 1988). The adhesion of leukocytes to microvascular endothelium is essential for their migration into inflamed tissue. ICAM-1. a member of the immunoglobulin superfamily, is the ligand for leukocyte function-associated antigen-l (LFA-l), a member of the 82 leukocyte integrin group. ICAMin humans was initially noted to have a tissue distribution similar to that of the major histocompatibility complex (MHC) class II antigens, i.e., vascular endothelium, germinal ’ To whom corrrspondence lege of Medicine, 6565 Fnnnin
should he addressed at Baylor CalSt., M.S. A601, Houston, TX 770:~).
rwsed
September
25,
1990
center cells, interdigitating reticulum cells, macrophages, lymphoid tissue, and epithelial cells in tonsil and thymus (Dustin et al., 1986). Additional studies have shown that ICAMis also expressed on keratinocytes and bronchial epithelium when inflammatory cytokines are present (Wegner et al., 1990). ICAMexpression on cultured endothelial cells is enhanced by interleukin-1, interferon-y, and lipopolysaccharide. Increased expression of ICAMhas been noted on the endothelium of glomeruli, arteries, and capillaries in kidney allografts undergoing rejection. Furthermore, treatment with monoclonal antibodies to ICAMdelayed the onset of acute cellular allograft rejection and reversed established acute rejection (Cosimi et al., 1989). ICAMhas been shown to play an important role in T-cell interaction and activation (Dougherty et al., 1988). Increased expression of ICAMon bronchial epithelium has been postulated to play a role in acute bronchial asthma (Wegner et al., 1990). ICAMhas been shown in vitro t,o function as a receptor for the rhinoviruses (Staunton et al., 1989b) and for Plasmodium falciparum (Berendt et al., 1989). Although ICAMis a major ligand for LFA-1 and appears essential for transendothelial migration of leukocytes in uitro (Smith et al., 1988), other adhesion molecules are also important in endothelium-leukocyte interactions. Endothelial-leukocyte adhesion molecule-l (ELAM-1) (Bevilacquaetal., 1989),vascular cell adhesion molecule-l (VCAM-1) (Osborn et al., 1989), intercellular adhesion molecule-2 (ICAM-2) (Staunton et al., 1989a), and platelet-endothelial cell adhesion molecule-l (PECAM-1) (Newman et al.. 1990) are all expressed on endothelium and may play important roles in the regulation of leukocyte trafficking. Whereas the human genetic disorder leukocyte adhesion deficiency (LAD) has clarified the bio-
548
SHORT
Ha
MO
1
2
3
4
COMM~JNI(‘ATION
5
- .9.4 --b -6.5 --b --w
-4.3
FIG. 1. Autoradiogram of’ a Southern blot 01’ hamster-mouse somatic cell hybrid DNAs digested with HnmHT and hybridized with the mouse ICAMcDNA probe. Parental (Chinese hamster DNA is in lane Ha and parental mouse DNA is lane Mo. Five representative somatic cell hybrid DNAs are shown in lanes 1-5. The top two arrows point to the 7.0. and 5.2.kb hamster bands. The bottom arrow points to t.he mouse doublet at 1.3 kh. The numbers on the right (in kh) mark the position of’ X DNA fragments produced by digestion with HindlII.
logical importance of CD& there have been no human diseasesattributable to an inherited disorder of any of the endothelial adhesion molecules. We have in the mouse in the search for any mapped Icam-l previously described mutant phenotypes that might be due to a mutation in this gene. (Human gene symbol is ICAM-1, mouse gene symbol is Icam-I, and human or mouse gene product is ICAM-1.) The murine cDNA for ICAMwas cloned by screening a murine thymus cDNA library using the human cDNA as a probe (Ballantyne et al., 1989). The sequence of murine ICAMcDNA covers 2522 bp, with an open reading frame encoding 537 amino acids beginning with ATG at base 23 and ending with TGA at bp 1634. The cDNA probe, 2OA-I, contains bp 1 through 1897. Labeling of 2OA-1 was accomplished using [3”P]dCTP by the random priming method (Feinberg et al., 1983). A panel of 18 Chinese hamster-mouse somatic cell hybrid DNAs previously characterized for the retention of mouse chromosomes was used for mapping Icam(Hoggan et al., 1988). The Chinese hamstermouse hybrid DNAs and the parental DNAs were prepared by standard procedures (Hoggan et al., 1988) and digested with BamHI; 6 pg of DNA was electrophoresed in 0.9% agarose gels and transferred to nylon membranes. Membranes were washed and hybridized as previously described (Zoghbi et al., 1988). The digestion of Chinese hamster and mouse DNAs with BamHI produced cross-reactive bands of 7.0 and 5.2 kb in the hamster (Fig. 1, lane 1) and a doublet of 4.3 kb in the mouse (Fig. 1, lane 2). Analysis of DNA from hybrid cells demonstrated that 9 of 18 hybrids con-
tained the mouse bands. Analysis of the distribution of mouse chromosomes and the presence or absence of these fragments in the hybrids showed no discordances for chromosome 9 and at least four discordances for all other chromosomes (Table l), t.herefore indicating that the gene for Icamis present on mouse chromosome 9. To define a more precise map location for Ivan-I, genomic DNAs from the inbred mouse NFS/N and from the wild mouse Mus musculus musculus (Skive) were examined for restriction fragment length polymorphisms detected by the murine cDNA probe 20A1. NFS/N strain mice were obtained from the Division of Natural Resources, NIH (Bethesda, MD). M.m. musdus mice were obtained from a laboratory colony derived from mice originally trapped in Skive, Denmark, and maint,ained by Dr. M. Potter (NCI, NIH, Contract l-CBC-5584) at Hazelton Laboratories (Rockville, MD). NFSjN females were mated with M.m. muscu1u.s males, and the F, females were backcrossed with M.m. musculus males to produce the experimental animals. DNAs were extracted from TABLE
1
Analysis of Concordance between Specific Mouse Chromosomes and Icam-l in 18 Chinese Hamster y Mouse Somatic Cell Hybrids .___ Numher 01’ hybrids” ! hvhridization/chrornosome)
-
u Symbols represent the presence (+/) or absence ( m/j ut’ the mouse Icnm-l restrict,ion fragment as related to the presence (/+j or absence (/- ) of a particular mouse chromosome. The number 01 discordant observat.ions is the sum of’ the i /and /+ observations. Seven oft.he hybrids were karyotyprd: the remainder were typed f’or the presenre or absence of markers on specific mouse chromosomes.
SHORT
mouse livers, cleaved with PstI, run on 0.4% agarose gels for 48 h at 24 V, and transferred to nylon menibranes (Hybond N+, Amersham). Membranes were washed and hybridized as previously described (Hoggan et al., 1988). Southern blot hybridization of DNAs from NFS/N M.m. musculus mice with the ICAMcDNA probe showed that NFS/N mice produced cross-reactive bands of 6.3, 1.0, and 0.5 kb, and that M.m. musculus DNA produced bands of 6.1, 1.0, and 0.5 kb (Fig. 2). Analysis of progeny of the backcross [NFS/N X M.m. musculus) F, x M.m. musculus] showed that 36 of 83 inherited the NFS fragment. We compared the segregation pattern of this RFLP in 83 mice with other chromosome 9 markers, including Cbl-2 and Mpi-1. Cbl-2 was typed by Southern blot analysis using the v-cbl probe (Regnier et al., 1989) which cross-hybridized with PuuII fragments of 8.2,2.3, and 1.4 kb in the parental NFS/N mouse DNA and 4.7, 2.7, and 1.0 kb in M.m. musculus. Cbl-2 was scored by segregation of the 2.3. and 1.4.kb NFS/N fragments. Isozymes of Mpi-1 were typed by histochemical staining aft.er electrophoresis of kidney ext,racts on starch gels. A total of nine recombinants were observed between Icam-l and Cbl-2 for a recombination distance of 10.8 + 3.4 CM. Nineteen recombinants were observed between Icam- and Mpi-1 for a recombination fraction of 22.9 + 4.6 CM, and an additional 20 mice were typed for Icam- and Mpi-I t.o produce a final recombination distance between Icnm-l and Mpi-1 of 23 + 4.2 CM (Table 2). These data establish t,hat Icam-l is at the centromeric end of chromosome 9 proximal to Cbl-2. 12
3
549
COMMLINICATION
4
-1-o
-0-5
FIG. 2. Autl,radiogram 01 a Southern blot ofDNAs from interspecies hackcross and parents digested with I’stl and hybridized with the mouse ICAMcDNA probe. The 6.Skh fragment is unique to NFS/K (lane 4) and the (i.l-kh fragment is unique to M m. murcu1ct.s (he 31. Ilanes 1 and 2 are hackcross mice which are hrterozygws ~‘or the 6% and 6.1 -kh fragments.
TABLE
2
Segregation of Icam-l Hybridizing with Cbl-2 and Mpi-1 among 83 Progeny species Backcross Inheritance
Mice
Icnm-
1
Fragments of an Inter-
of NFS/N allele” C‘hl-2
Nonrecomhinants
+
+
Single recomhinants
+
+ I
Number of progeny
Mpl-l
:12 32 2 8 7 2
I
t +
+ “r, Recomhinat Locus
pair
Icnm-I. C’hl-2 t’hl-2, Mpi- I Icnm- 1, Mpi- 1
ion’
r/n 9/x3
I o/s:3 19/w
*SF: 10.8
i
3.4
I2.0 5 3.6 22.9 f 4.6
a A t indicates presence of NSF/N allele; indicates ILL. m. musculu.\ allele. h Percentage recombination for each pair of loci and standard error were calculated (8) from the numher ofrecombinants (r) in a sample size of n. ’ Twenty additional mice were typed for Icam-l and hfpi-I li)r total percentage recombination of ?4/103 r 3.3 i 1.2.
Three genes (Ldlr, Idd-2, and Ets-1) have been mapped previously in this region of mouse chromosome 9. The LDL receptor gene had previously been the only gene mapped to human chromosome 19 (~13.2-13.1) and proximal mouse 9 (Wang et a/., 1988). ICAMhas been mapped to human chromosome 19 by analysis of somatic cell hybrids (Katz et al., 1985). The data in this study establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Ets-1 maps t.o human chromosome llq. The genetic mapping of Icam-l to this region of chromosome 9 is of particular interest because of another locus mapped t,o this region, Idd-2. Zdd-2 is one of three recessive genes involved in the development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice (Prochazka et al., 1987). Both cellular autoimmunity and humoral autoimmunity against pancreatic @cells appear to be cent,ral features in the pathogenesis of insulin-dependent diabetes in NOD mice. The key histopathological lesion is insulitis, a leukocytic infiltration of the pancreatic islets. In addition to the MHC-associated Idd-1 locus on chromosome 17, there is evidence that a second locus, Idd2. is locat.ed on chromosome 9 bet.ween the centromere and Thy-l (Prochazka et al., 1987). Thy-1 and
550
SHORT
COMMIINI(‘ATION
Cbl-2 are tightly linked, and our data suggest that Zcam-1, like Idd-2, maps proximal to the Thy-l,/CbE-2 region on mouse chromosome 9. Icam-I has been shown to have an important function both in T-cell interaction and in the development of an inflammatory response, and therefore a mutation in Icam-l could be relevant to the development of insulitis in this disease. The mapping of lcam-l to the region known to contain the Idd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic variation in Icam-1.
9.
10. KATZ,
from the American Grace Watson for
Il.
1”.
:3.
D. L.. Intercellular
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E. I,.
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(ICAM-1). B. (1983).
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J., WHITE, G. (:.. W. A. (1990). PE-
p.,
TIZARD, (i., AND
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PROCHAZKA, M.. LEITER, MAN, D. I,. (19X7). Three
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and Probability in Animal Univ. Press, New York.
KOZAK,
D. V., AND
C. A.. KINGXEY.
D. E., MARLIN, SPRINGER. T.
ICAMdemonstrates immunoglobulin and 92-933. 17.
1X.
A.
D. M..
S. D., STRA’I’OWA, (1988). Primary
(:oLF,-
.JENKINS,
C.. D~s~IN, structure of
interaction between members of the integrin supergene families. (‘ell 52:
I). E.. DIJSTIN, M. L,., AND SPRINGER, 7'. A. ( 1989a). Functional cloning of ICAM-2, a cell adhesion ligand for I,FA-I homologous to IC’AM-1. Naturr (I,ondon) 339: 6164.
STAUNTON,
S~.AIINTON,
D. E.. MERLUZZI,
K., MARLIN,
S. D., AND
V. d.. RWFHLEIN.
SPRINGER,
T. A. (1989h).
sion molecule, ICAM-1, is the major noviruses. (‘cl/ 56: 849-853.
surface
R., BARTON, A cell
receptor
adhe-
for rhi-
19.
WANG, LALLEY,
1,-M., KILLARY, P. A., BELL,
A. M., FANG, X-E.. PARRIO’I”I’, S. K.. G. I., AND SAKAGUCHI, A. Y. (1988). Chromosome assignment of mouse insulin. colony stimulating factor 1, and low-density lipoprotein receptors. Genomics 3: 17%l’i:,.
“0.
WEGNER, LE’I.TS,
c‘. D.. GIJNDEL. R. I,. G., AND ROTHLEIN,
sion molecule-l (ICAMScicnw 47: 456-459.
for
to
1). (:.,
QEHREZE,
recessive loci required for insulinin nonobese diabetic mice. Scirnw 237:
diabetes
REGNIER,
E. H..
STAUNTON. M. L., AND
137:
restriction endonuclease fragments Anal. Riochem. 132: 6-l 3. “Genetics
L., HESSION, S., CHI-RUSSO,
R
16.
The
intercellular adhesion molecule-l (ICAMof an immune response. Eur. J. Zmmunol.
adherence
on activated
SM~~‘H, (1. W., RO’~HLEIN, R., HUGHES. B. ,J., MARIY(:ALC‘O, M. M., KUDLOFF. H. E.. SCHMALSTIEG, F. C., AND ANDERSON, L). C. (1988). Recognition of an endothelial determinant for (‘D-IX dependent human neutrophil adherence and transendothelial migration. J. C’lin. Invest. 82: 1746-1756.
fal-
T., CONTI, Immunosuppres-
DUSTIN, M. L., ROTHLEIN, R., BHAN, A. Ii., C. A., AND SPRINGER, T. A. (1986). Induction interferon-y: Tissue distribution, biochemistry,
of a natural 245-254.
antigens expressed 15: 10%106.
I,. .J., map-
15.
leukocyte adhesion molefor neutrophils related to cornand lectins. Science 243: 1160-
COSIMI, A. B., GEOFFRIAN, ROTHLEIN, R., AND COLVIN,
STANLEY, K., MURRAY, M. F. (1985). Chromosome
P. J., BERNDT, M. C., GORSKI, S., PADDOCK, C., AND MULLER.
OSBORN, HOWSKYL,
KOZAK,
N. A.. COPELAND, N. G., LANGDON, W. Y.. AND MORSE, H. C.. III (1989). Identification of two murine loci homologous to the v-&l oncogene. J. Viral. 63: 3678-3682.
NEWBOLD, molecule-
sion of’ cynomolgus recipients of renal allografts by R6.5, a monoclonal antibody to ICAM-I. fn “Leukocyte Adhesion Molecules” (T. A. Springer. D. C. Anderson, A. S. Rosenthal, and R. Rothlein, Eds.), pp. 274-281. Springer-Verlag, New York. 5.
1:1.
14.
5853.
1 is an endothelial ciparum. Nature
NEWMAN, LYMAN,
(‘. E., AND
CAM-1 (CD31) cloning and relation to adhesion the immunoglohulin gene superfamily. Science 1.2”‘.
C. M.. O’BRIEN, W. E.. AND BEAUDET. A. I,. (1989). Nucleotide sequence ofthe cDNA for murine intercellular adhesion molecule-l (ICAM). Nucleic Acids Res. 17: SIMMONS. K. (1989).
PARKER, M.. E. A.. AND GREAVES,
ping of cell membrane cells. Eur. .J. Immunol.
BALLANTYNE,
A. R., MARSH,
F. E.,
dependent “X6-289.
REFERENCES
BERENDT, C. I., AND
N. I?., BUCKLER,
mapping of the mouse c-fms proto-oncoIA. J. Viroi. 62: 10X-1056.
expression cloning of vascular cell adhesion molecule 1. a cytokine-induced endothelial protein that hinds to lymphocytes. Cell 59: 1209-1311.
This wurk was supported by a grant-in-aid Heart Association, Texas Affiliate. We thank the preparation of this manuscript.
2.
M. I>., HAI,DEN,
CI.ARK,
ACKNOWLEDGMENTS
1.
HOGGAN,
(‘. A. (1988). Genetic gene lo chromosome
21.
H.,
REILLY,
P., HAYNES,
N.,
R. (1990). Intercellular adhein the pathogenesis of asthma.
ZOGHBI, H. Y., DAIGER, S. P., MCCALL. A., O’BRIEN, W. IX., AND BEAUDET, A. I,. (1988). Extensive DNA polymorphism at the factor SIIIa (FIXA) locus and linkage to HLA. Amer. J.
Hum.
Grnet.
42: 877-883.
of the Gene for Intercellular Adhesion (/cam-l) to Proximal Mouse Chromosome
CHRISTIE M. BALLANTYNE,**’
CHRISTINE A. KOZAK,t
WILLIAM
E. O’BRIEN,?
Molecule-l 9
AND ARTHUR L. BEAUDET*
‘Department of Medicine and #Howard Hughes Medical Institute and Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030; and tNationa/ Institute of Allergy and infectious Diseases, National Institutes of Health, Bethesda, Maryland 20814 Received
May
18,
1990;
Intercellular adhesion molecule-l (ICAM-1) is an integral membrane protein, a member of the immunoglobulin superfamily, and a ligand for LFA-1. a p2 leukocyte integrin. ICAMhas a tissue distribution similar to that of the major histocompatability complex class II antigens and is likely to play a role in inflammatory responses. We have mapped this gene to proximal mouse chromosome 9 by using mouse-hamster somatic cell hybrids and an interspeties backcross. Since human ICAM- 1 maps to chromosome 19, it joins the LDL receptor to establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Murine Icam-l maps between Cbl-2 and the centromere in the same region as one of the susceptibility genes for insulin-dependent diabetes mellitus (I&-2) that is postulated to play a role in immune function and inflammation leading to insulitis. The mapping of Icam-l to the region known to contain the Zdd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic ,c 1991 Academic Press. Inc. variation in Icam-1.
Intercellular adhesion molecule-l (ICAM-1) is a transmembrane protein that plays a key role in the generation of an inflammatory response (Staunton et al., 1988). The adhesion of leukocytes to microvascular endothelium is essential for their migration into inflamed tissue. ICAM-1. a member of the immunoglobulin superfamily, is the ligand for leukocyte function-associated antigen-l (LFA-l), a member of the 82 leukocyte integrin group. ICAMin humans was initially noted to have a tissue distribution similar to that of the major histocompatibility complex (MHC) class II antigens, i.e., vascular endothelium, germinal ’ To whom corrrspondence lege of Medicine, 6565 Fnnnin
should he addressed at Baylor CalSt., M.S. A601, Houston, TX 770:~).
rwsed
September
25,
1990
center cells, interdigitating reticulum cells, macrophages, lymphoid tissue, and epithelial cells in tonsil and thymus (Dustin et al., 1986). Additional studies have shown that ICAMis also expressed on keratinocytes and bronchial epithelium when inflammatory cytokines are present (Wegner et al., 1990). ICAMexpression on cultured endothelial cells is enhanced by interleukin-1, interferon-y, and lipopolysaccharide. Increased expression of ICAMhas been noted on the endothelium of glomeruli, arteries, and capillaries in kidney allografts undergoing rejection. Furthermore, treatment with monoclonal antibodies to ICAMdelayed the onset of acute cellular allograft rejection and reversed established acute rejection (Cosimi et al., 1989). ICAMhas been shown to play an important role in T-cell interaction and activation (Dougherty et al., 1988). Increased expression of ICAMon bronchial epithelium has been postulated to play a role in acute bronchial asthma (Wegner et al., 1990). ICAMhas been shown in vitro t,o function as a receptor for the rhinoviruses (Staunton et al., 1989b) and for Plasmodium falciparum (Berendt et al., 1989). Although ICAMis a major ligand for LFA-1 and appears essential for transendothelial migration of leukocytes in uitro (Smith et al., 1988), other adhesion molecules are also important in endothelium-leukocyte interactions. Endothelial-leukocyte adhesion molecule-l (ELAM-1) (Bevilacquaetal., 1989),vascular cell adhesion molecule-l (VCAM-1) (Osborn et al., 1989), intercellular adhesion molecule-2 (ICAM-2) (Staunton et al., 1989a), and platelet-endothelial cell adhesion molecule-l (PECAM-1) (Newman et al.. 1990) are all expressed on endothelium and may play important roles in the regulation of leukocyte trafficking. Whereas the human genetic disorder leukocyte adhesion deficiency (LAD) has clarified the bio-
548
SHORT
Ha
MO
1
2
3
4
COMM~JNI(‘ATION
5
- .9.4 --b -6.5 --b --w
-4.3
FIG. 1. Autoradiogram of’ a Southern blot 01’ hamster-mouse somatic cell hybrid DNAs digested with HnmHT and hybridized with the mouse ICAMcDNA probe. Parental (Chinese hamster DNA is in lane Ha and parental mouse DNA is lane Mo. Five representative somatic cell hybrid DNAs are shown in lanes 1-5. The top two arrows point to the 7.0. and 5.2.kb hamster bands. The bottom arrow points to t.he mouse doublet at 1.3 kh. The numbers on the right (in kh) mark the position of’ X DNA fragments produced by digestion with HindlII.
logical importance of CD& there have been no human diseasesattributable to an inherited disorder of any of the endothelial adhesion molecules. We have in the mouse in the search for any mapped Icam-l previously described mutant phenotypes that might be due to a mutation in this gene. (Human gene symbol is ICAM-1, mouse gene symbol is Icam-I, and human or mouse gene product is ICAM-1.) The murine cDNA for ICAMwas cloned by screening a murine thymus cDNA library using the human cDNA as a probe (Ballantyne et al., 1989). The sequence of murine ICAMcDNA covers 2522 bp, with an open reading frame encoding 537 amino acids beginning with ATG at base 23 and ending with TGA at bp 1634. The cDNA probe, 2OA-I, contains bp 1 through 1897. Labeling of 2OA-1 was accomplished using [3”P]dCTP by the random priming method (Feinberg et al., 1983). A panel of 18 Chinese hamster-mouse somatic cell hybrid DNAs previously characterized for the retention of mouse chromosomes was used for mapping Icam(Hoggan et al., 1988). The Chinese hamstermouse hybrid DNAs and the parental DNAs were prepared by standard procedures (Hoggan et al., 1988) and digested with BamHI; 6 pg of DNA was electrophoresed in 0.9% agarose gels and transferred to nylon membranes. Membranes were washed and hybridized as previously described (Zoghbi et al., 1988). The digestion of Chinese hamster and mouse DNAs with BamHI produced cross-reactive bands of 7.0 and 5.2 kb in the hamster (Fig. 1, lane 1) and a doublet of 4.3 kb in the mouse (Fig. 1, lane 2). Analysis of DNA from hybrid cells demonstrated that 9 of 18 hybrids con-
tained the mouse bands. Analysis of the distribution of mouse chromosomes and the presence or absence of these fragments in the hybrids showed no discordances for chromosome 9 and at least four discordances for all other chromosomes (Table l), t.herefore indicating that the gene for Icamis present on mouse chromosome 9. To define a more precise map location for Ivan-I, genomic DNAs from the inbred mouse NFS/N and from the wild mouse Mus musculus musculus (Skive) were examined for restriction fragment length polymorphisms detected by the murine cDNA probe 20A1. NFS/N strain mice were obtained from the Division of Natural Resources, NIH (Bethesda, MD). M.m. musdus mice were obtained from a laboratory colony derived from mice originally trapped in Skive, Denmark, and maint,ained by Dr. M. Potter (NCI, NIH, Contract l-CBC-5584) at Hazelton Laboratories (Rockville, MD). NFSjN females were mated with M.m. muscu1u.s males, and the F, females were backcrossed with M.m. musculus males to produce the experimental animals. DNAs were extracted from TABLE
1
Analysis of Concordance between Specific Mouse Chromosomes and Icam-l in 18 Chinese Hamster y Mouse Somatic Cell Hybrids .___ Numher 01’ hybrids” ! hvhridization/chrornosome)
-
u Symbols represent the presence (+/) or absence ( m/j ut’ the mouse Icnm-l restrict,ion fragment as related to the presence (/+j or absence (/- ) of a particular mouse chromosome. The number 01 discordant observat.ions is the sum of’ the i /and /+ observations. Seven oft.he hybrids were karyotyprd: the remainder were typed f’or the presenre or absence of markers on specific mouse chromosomes.
SHORT
mouse livers, cleaved with PstI, run on 0.4% agarose gels for 48 h at 24 V, and transferred to nylon menibranes (Hybond N+, Amersham). Membranes were washed and hybridized as previously described (Hoggan et al., 1988). Southern blot hybridization of DNAs from NFS/N M.m. musculus mice with the ICAMcDNA probe showed that NFS/N mice produced cross-reactive bands of 6.3, 1.0, and 0.5 kb, and that M.m. musculus DNA produced bands of 6.1, 1.0, and 0.5 kb (Fig. 2). Analysis of progeny of the backcross [NFS/N X M.m. musculus) F, x M.m. musculus] showed that 36 of 83 inherited the NFS fragment. We compared the segregation pattern of this RFLP in 83 mice with other chromosome 9 markers, including Cbl-2 and Mpi-1. Cbl-2 was typed by Southern blot analysis using the v-cbl probe (Regnier et al., 1989) which cross-hybridized with PuuII fragments of 8.2,2.3, and 1.4 kb in the parental NFS/N mouse DNA and 4.7, 2.7, and 1.0 kb in M.m. musculus. Cbl-2 was scored by segregation of the 2.3. and 1.4.kb NFS/N fragments. Isozymes of Mpi-1 were typed by histochemical staining aft.er electrophoresis of kidney ext,racts on starch gels. A total of nine recombinants were observed between Icam-l and Cbl-2 for a recombination distance of 10.8 + 3.4 CM. Nineteen recombinants were observed between Icam- and Mpi-1 for a recombination fraction of 22.9 + 4.6 CM, and an additional 20 mice were typed for Icam- and Mpi-I t.o produce a final recombination distance between Icnm-l and Mpi-1 of 23 + 4.2 CM (Table 2). These data establish t,hat Icam-l is at the centromeric end of chromosome 9 proximal to Cbl-2. 12
3
549
COMMLINICATION
4
-1-o
-0-5
FIG. 2. Autl,radiogram 01 a Southern blot ofDNAs from interspecies hackcross and parents digested with I’stl and hybridized with the mouse ICAMcDNA probe. The 6.Skh fragment is unique to NFS/K (lane 4) and the (i.l-kh fragment is unique to M m. murcu1ct.s (he 31. Ilanes 1 and 2 are hackcross mice which are hrterozygws ~‘or the 6% and 6.1 -kh fragments.
TABLE
2
Segregation of Icam-l Hybridizing with Cbl-2 and Mpi-1 among 83 Progeny species Backcross Inheritance
Mice
Icnm-
1
Fragments of an Inter-
of NFS/N allele” C‘hl-2
Nonrecomhinants
+
+
Single recomhinants
+
+ I
Number of progeny
Mpl-l
:12 32 2 8 7 2
I
t +
+ “r, Recomhinat Locus
pair
Icnm-I. C’hl-2 t’hl-2, Mpi- I Icnm- 1, Mpi- 1
ion’
r/n 9/x3
I o/s:3 19/w
*SF: 10.8
i
3.4
I2.0 5 3.6 22.9 f 4.6
a A t indicates presence of NSF/N allele; indicates ILL. m. musculu.\ allele. h Percentage recombination for each pair of loci and standard error were calculated (8) from the numher ofrecombinants (r) in a sample size of n. ’ Twenty additional mice were typed for Icam-l and hfpi-I li)r total percentage recombination of ?4/103 r 3.3 i 1.2.
Three genes (Ldlr, Idd-2, and Ets-1) have been mapped previously in this region of mouse chromosome 9. The LDL receptor gene had previously been the only gene mapped to human chromosome 19 (~13.2-13.1) and proximal mouse 9 (Wang et a/., 1988). ICAMhas been mapped to human chromosome 19 by analysis of somatic cell hybrids (Katz et al., 1985). The data in this study establish a new conserved syntenic segment between human chromosome 19 and proximal mouse chromosome 9. Ets-1 maps t.o human chromosome llq. The genetic mapping of Icam-l to this region of chromosome 9 is of particular interest because of another locus mapped t,o this region, Idd-2. Zdd-2 is one of three recessive genes involved in the development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice (Prochazka et al., 1987). Both cellular autoimmunity and humoral autoimmunity against pancreatic @cells appear to be cent,ral features in the pathogenesis of insulin-dependent diabetes in NOD mice. The key histopathological lesion is insulitis, a leukocytic infiltration of the pancreatic islets. In addition to the MHC-associated Idd-1 locus on chromosome 17, there is evidence that a second locus, Idd2. is locat.ed on chromosome 9 bet.ween the centromere and Thy-l (Prochazka et al., 1987). Thy-1 and
550
SHORT
COMMIINI(‘ATION
Cbl-2 are tightly linked, and our data suggest that Zcam-1, like Idd-2, maps proximal to the Thy-l,/CbE-2 region on mouse chromosome 9. Icam-I has been shown to have an important function both in T-cell interaction and in the development of an inflammatory response, and therefore a mutation in Icam-l could be relevant to the development of insulitis in this disease. The mapping of lcam-l to the region known to contain the Idd-2 gene raises the question of whether the phenotypic differences attributed to the Idd-2 locus might be due to genetic variation in Icam-1.
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