- Email: [email protected]
Genetic mapping of the gene for a novel tyrosine kinase, Blk, to mouse chromosome 14
GENOMICS
9, 762-764
(1991)
SHORT COMMUNICATION Genetic Mapping of the Gene for a Novel Tyrosine Kinase, B/k, to Mouse Chromosome 14 CHRISTINE
A.
KOZAK,
*,’ SUSAN
M.
DYMECKI,
t,*
JOHN
E. NIEDERHUBER,
t,g
AND STEPHEN
V.
DESIDERlot,S
*Laboratory of Molecular MIcrobiology, National institute of Allergy and /nfect/ous Diseases, National lnstltutes of Health, Bethesda, Maryland 20892; and TDepartment of Molecular Biology and Genetics, $Howard Hughes Medical Institute, and SDepartment of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ReceivedAugust20,
1990,
The gene Blk, which encodes a novel tyrosine kinase expressed preferentially in B-lymphoid cells, was mapped by Southern blot analysis of DNA from the progeny of an intersubspecific backcross. Blk maps to the proximal region of chromosome 14 with the gene order centromere-(Np-1, Tcra)-Blk-sys-Es-IO. 8~ 1991 .4rademir Press. Inc.
The Src family of protooncogenes encodes a set. of homologous, membrane-associated protein tyrosine kinases that are likely to function in the regulation of cellular growth and differentiation (Hanks et al., 1988; Cooper, 1990). Seven members of the Src family have been described: Src, Lyn, Yes, Fyn, Fgr, Hck, and Lck (Hanks et al., 1988; Cooper, 1990). We recently isolated from mouse lymphoid cells a cDNA that represents an additional member of the Src family, Rlk. The Blk cDNA encodes a 55kDa protein-tyrosine kinase (~55”‘~); expression of Blk is restricted to cells of the B-lymphoid lineage (Dymecki et al., 1990). Within the B lineage, Blk is expressed in some pro-B cells and in most pre-B and mature B cells, but not in plasma cells (Dymecki et al., 1990; P. Zwollo, S.M.D., and S.V.D., unpublished). On the basis of its preferential expression in B-lymphoid cells, it is likely that Blk functions in a signal transductory pathway specific to this lineage. Because tyrosine phosphorylation has been implicated in t,ransduction of signals that effect B-cell activation, including engagement of surface immunoglobulin (Gold et al., 1990; Campbell
’ To whom correspondence should be addressed at National Inst.itutes of Health, LMM, NIAID. Building3. Room 324. Rethesda, MD 20892.
rewed
November
28.
1990
and Sefton, 19901, an attractive hypot,hesis is that Blk plays a direct role in B-cell activation. As part of our effort to distinguish Blk from other members of the Src family, and because of the potential association between mutations at. Rlk and disorders of immune function, we have positioned t.his gene on the mouse linkage map. Here we show that Blk resides on Chromosome 14. DNA samples from t,he progeny of an intersuhspeties backcross were examined for inheritance of a restriction enzyme length variant (RFLV) of Rlk by Southern blot analysis, using a hybridization probe (designated the Blk probe) containing the first 1292 bp of Blk cDNA (GenBank Accession No. M30903). For this cross, NFS/N mice were obtained from the Division of Natural Resources, NIH (Bethesda, MD). AKR/J and C58/d mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Wild-derived Mus musculus musculus (Skive) were obtained from Dr. M. Potter, NCI? NIH, from his colony at, Hazelton Laboratories (Rockville, MD; NC1 Contract NO1 CR-71085). M. m. musculus males were mated with C58/J, NFS/N, or AKR/J females, and the F, females were mated with musculus males to produce first backcross mice. In HglII digests of DNA from the laboratory strain progenit.ors of this cross, the BEk probe hybridized to two DNA fragments of 23 and 9.2 kb; in a similar digest of DNA from musculus. the Blk probe detect,ed fragments of >28 and 14.8 kb (Fig. 1). Of 105 backcross progeny, 45 inherited bot,h inbred strain bands, consistent with the expected 1:l segregation ration for a single autosomal gene. Backcross progeny were also screened for inheritance of RFLVs for over 130 DNA markers and for 13 isozyme markers on the 19
SHORT
autosomal linkage groups in the mouse. Comparison of the inheritance of the inbred strain Blk variant with these markers demonstrated linkage with markers on proximal Chromosome 14. These markers included: (1.) the isozyme markers Np-I and Es-IO, typed by histochemical staining following electrophoresis on starch gels (Harris and Hopkinson, 1976); (2) the DNA marker sys (symplastic spermatids), typed using as hybridization probe a 1.7-kb fragment to identify an EcoRI RFLV as described previously (MacGregor et al., 1990); and (3) Tcra, the a-subunit of the T-cell receptor, typed for a BstEI RFLV following hybridization with a 570-bp NcoI fragment including most of C,, and part of the 3’ untranslated region (Pardoll et al., 1987). Comparison of the inheritance pattern of the Blk RFLV with these markers indicates that Blk lies between sys and Np-I (Table 1). Backcross mice derived from the NFS/N cross were also typed for Tcra, which also lies in this interval (Doolittle et al., 1990), to determine the proximity of these markers. No recombinants were detected between Tcra and Np-1 in 71 mice, confirming the close linkage of these markers. Taken together, these data suggest the following gene order: centromere-(Np-1, Tcra)-Blk-sys-Es-IO. In addition to Blk, three other members of the Src family show preferential expression in hematopoietic cells-Lck, Hck, and Fgr. Of these genes, only Blk is expressed specifically in B-lymphoid cells. In the
a
b
c
- >28 -23
-14.8 -
763
COMMUNICATION
9.2
FIG. 1. Southern blot analysis of DNAs from an intersuhspeties hackcross using the Rlfi hyhridization probe. DNA was extracted from the livers of inhred mice, ?t4. m. musculus. and the hackcross progeny. DNA samples were digested with &/II, fractionated by electrophoresis through 0.4% agarose gels, and transferred to nylon memhranes (Hyhond N+, Amersham). The Blh prohe was labeled by t.he random primer method (6). Hyhridization and wash condirions were as described previously (12). Lanes a-c, individual hackcross mice. Fragment sizes in kilohase pairs are indicated to the right.
TABLE
1
Segregation of the Blk Hybridizing Restriction Fragment with Alleles of Es-IO, Np-1 and sys in 105 Progeny of an Intersubspecies Backcross Inheritance NFS/N Mice Parentals
of the allele
NJ,- I
Blk
,s,vs
+
+
t
I!;.+ 10
Number of mice
t i
Single recomhinants
+
t
i
+
+ I +
t
t
I
+ +
:14 51 2 0 t5 4 4 .5
Recombination” Locus
pair
Np-I.Blk Blk.sys s,w.Ew- 10
“( Recombination (il SE)
rln 9/105 9/1os 2/l 05
8.6 t 2.7 8.6 t 2.7 1.9 + 1.3
a Percentage recombination between restriction fragments standard error were calculated according to Green (8) from number of recomhinants (r) in a sample size of n.
and the
mouse, Lck and Fgr reside on chromosome 4 (Ceci et al., 1989), while Hck has been mapped to Chromosome 2 (Siracusa et al., 1990). The map location ofBlk on Chromosome 14 confirms that Blk is a distinct member of the Src family. While the human homolog of Blk has not yet been mapped, the proximal half of mouse Chromosome 14 contains regions of linkage homology with human chromosomes 10, 13, and 14 (Nadeau, 1989). Our localization of Blk wit,hin this mouse linkage group indicates that it lies between the regions with known homology to human chromosomes13 and 14 and suggeststhat its genetic mapping in man may help define the extent of these regions. If the Blk product plays an essential role in normal B-cell development, we would expect mutations in Blk to impair immune function. Chromosome 14 carries a previously described immunodeficiency locus, hairless locus (hr), mutations at which are associated with increased incidence of leukemia, high titers of ecotropic and xenotropic retroviruses, and impaired cellular immune responses (reviewed by Green, 1989). Our observations suggest that Blk maps proximal to hr and, therefore, that these genes are distinct. ACKNOWLEDGMENTS We thank for preparing
M. Peyser for technical the manuscript. This
assistance and H. R. Marshall work was supported in part hy
764
SHORT
COMMUNICATION
grants to S.V.D. from the Howard Hughes Medical Institute and the National Cancer Institute (CA16519). S.M.D. is a trainee of the Medical Scientist Training Program of the National Institutes of Health.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
CAMPBELL, M., AND SEFTON, B. (1990). Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin. EMBOJ. 9: 2125-2131. CECI, J. D., SIRACUSA, L. D., JENKINS, N. A., AND COPELAND. N. G. (1989). A molecular genetic linkage map of mouse chromosome 4 including the localization of several proto-oncogenes. Genomics 5: 699-709. COOPER, J. A. (1990). The Src-family of protein-tyrosine kinases. In “Peptides and Protein Phosphorylation” (B. Kemp and P. F. Alewood, Eds.), pp. 85-J 15, CRC Press, Boca Raton, FL. DOOLITTLE, D. P., DAVISON, M. T., RODERICK, T. H., AND HILLYARD, A. L. (1990). Locus map of mouse. Mouse Genume 87: 14-27. DYMECKI, S., NIEDERHLJBER, J.. AND DESIDERIO, Y. (1990). Specific expression of a novel tyrosine kinase gene, Blk. in B lymphoid cells. Science 247: 332-336. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. GOLD, M. R., LAW, D. A., AND DEFRANCO, A. L. (1990). Stimulation of protein tyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature fLondonl 345: 810-813.
8. GREEN, 9.
10.
E. L. (1981). Linkage, recombination and mapping. In “Genetics and Probability in Animal Breeding Experiments,” pp. 77-113. MacMillan, New York. GREEN, M. C. (1989). Catalog of mutant genes and polymorphic loci. In “Genetic Variants and Strains of the Laboratory Mouse” (Lyon and Searle, Eds.), pp. 12-403, Oxford Univ. Press, New York. HANKS, S. K., QUINN, A. M., AND HUNTER, 7’. 11988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52.
II.
HARRIS, H.. AND HOPKINSON, D. A. (1976). “Handbook Enzyme Electrophoresis in Human Genetics,” North land, Amsterdam.
12.
HOGGAN, M.D., HALLXN, N. F.. BUCKLER, C. E., AND I~OZAK, C. A. (1988). Genetic mapping of the mouse c-fms proto-oncclgene to chromosome 18. J. Viral. 62: 1055-1056. MACGREGOR, G. R.. RUSSEI.L, L. D., VAN BEEK, M. E. A. H.. HANTEN. G. R., KOVAK, M. .J., KOZAK, C. A., MEISTRXH, M. I,.. AND OVERBEEK, P. A. (1990). Symplastic spermatids (s,w), a recessive insertional mutation in mice causing a defect in spermatogenesis. Proc. Natl. Acad. Sci. (ISA 87: X)16-5020. NADEAU, .J. H. (1989). Maps of linkage and synteny homologies between mouse and man. 7’rends Genet. 5: 82-86. PAHDOI,L, D., FOWI.KES, B. J., LECHLER, R. I., GERMAIN. R.. AND SCHWARTZ, R. H. (1987). Early genetic events in T cell development analyzed by in situ hybridization. J. EQ. Med. 165: 1624-16:38. QIRAWSA, I,. D., SILAN, C. M., JUSTICE, M. J.. MERCEH, .J. A., BAUSKIN. A. R., BEN-NERIAH, Y., L)UBOULE, D., HASTIE. N. D., COPELAND, N. G., AND JENKINS, N. A. (1990). A molecular genetic linkage map of mouse chromosome 2. Genomics 6: 491-504.
1:j.
14. 15.
16.
01 Hol-
9, 762-764
(1991)
SHORT COMMUNICATION Genetic Mapping of the Gene for a Novel Tyrosine Kinase, B/k, to Mouse Chromosome 14 CHRISTINE
A.
KOZAK,
*,’ SUSAN
M.
DYMECKI,
t,*
JOHN
E. NIEDERHUBER,
t,g
AND STEPHEN
V.
DESIDERlot,S
*Laboratory of Molecular MIcrobiology, National institute of Allergy and /nfect/ous Diseases, National lnstltutes of Health, Bethesda, Maryland 20892; and TDepartment of Molecular Biology and Genetics, $Howard Hughes Medical Institute, and SDepartment of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ReceivedAugust20,
1990,
The gene Blk, which encodes a novel tyrosine kinase expressed preferentially in B-lymphoid cells, was mapped by Southern blot analysis of DNA from the progeny of an intersubspecific backcross. Blk maps to the proximal region of chromosome 14 with the gene order centromere-(Np-1, Tcra)-Blk-sys-Es-IO. 8~ 1991 .4rademir Press. Inc.
The Src family of protooncogenes encodes a set. of homologous, membrane-associated protein tyrosine kinases that are likely to function in the regulation of cellular growth and differentiation (Hanks et al., 1988; Cooper, 1990). Seven members of the Src family have been described: Src, Lyn, Yes, Fyn, Fgr, Hck, and Lck (Hanks et al., 1988; Cooper, 1990). We recently isolated from mouse lymphoid cells a cDNA that represents an additional member of the Src family, Rlk. The Blk cDNA encodes a 55kDa protein-tyrosine kinase (~55”‘~); expression of Blk is restricted to cells of the B-lymphoid lineage (Dymecki et al., 1990). Within the B lineage, Blk is expressed in some pro-B cells and in most pre-B and mature B cells, but not in plasma cells (Dymecki et al., 1990; P. Zwollo, S.M.D., and S.V.D., unpublished). On the basis of its preferential expression in B-lymphoid cells, it is likely that Blk functions in a signal transductory pathway specific to this lineage. Because tyrosine phosphorylation has been implicated in t,ransduction of signals that effect B-cell activation, including engagement of surface immunoglobulin (Gold et al., 1990; Campbell
’ To whom correspondence should be addressed at National Inst.itutes of Health, LMM, NIAID. Building3. Room 324. Rethesda, MD 20892.
rewed
November
28.
1990
and Sefton, 19901, an attractive hypot,hesis is that Blk plays a direct role in B-cell activation. As part of our effort to distinguish Blk from other members of the Src family, and because of the potential association between mutations at. Rlk and disorders of immune function, we have positioned t.his gene on the mouse linkage map. Here we show that Blk resides on Chromosome 14. DNA samples from t,he progeny of an intersuhspeties backcross were examined for inheritance of a restriction enzyme length variant (RFLV) of Rlk by Southern blot analysis, using a hybridization probe (designated the Blk probe) containing the first 1292 bp of Blk cDNA (GenBank Accession No. M30903). For this cross, NFS/N mice were obtained from the Division of Natural Resources, NIH (Bethesda, MD). AKR/J and C58/d mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Wild-derived Mus musculus musculus (Skive) were obtained from Dr. M. Potter, NCI? NIH, from his colony at, Hazelton Laboratories (Rockville, MD; NC1 Contract NO1 CR-71085). M. m. musculus males were mated with C58/J, NFS/N, or AKR/J females, and the F, females were mated with musculus males to produce first backcross mice. In HglII digests of DNA from the laboratory strain progenit.ors of this cross, the BEk probe hybridized to two DNA fragments of 23 and 9.2 kb; in a similar digest of DNA from musculus. the Blk probe detect,ed fragments of >28 and 14.8 kb (Fig. 1). Of 105 backcross progeny, 45 inherited bot,h inbred strain bands, consistent with the expected 1:l segregation ration for a single autosomal gene. Backcross progeny were also screened for inheritance of RFLVs for over 130 DNA markers and for 13 isozyme markers on the 19
SHORT
autosomal linkage groups in the mouse. Comparison of the inheritance of the inbred strain Blk variant with these markers demonstrated linkage with markers on proximal Chromosome 14. These markers included: (1.) the isozyme markers Np-I and Es-IO, typed by histochemical staining following electrophoresis on starch gels (Harris and Hopkinson, 1976); (2) the DNA marker sys (symplastic spermatids), typed using as hybridization probe a 1.7-kb fragment to identify an EcoRI RFLV as described previously (MacGregor et al., 1990); and (3) Tcra, the a-subunit of the T-cell receptor, typed for a BstEI RFLV following hybridization with a 570-bp NcoI fragment including most of C,, and part of the 3’ untranslated region (Pardoll et al., 1987). Comparison of the inheritance pattern of the Blk RFLV with these markers indicates that Blk lies between sys and Np-I (Table 1). Backcross mice derived from the NFS/N cross were also typed for Tcra, which also lies in this interval (Doolittle et al., 1990), to determine the proximity of these markers. No recombinants were detected between Tcra and Np-1 in 71 mice, confirming the close linkage of these markers. Taken together, these data suggest the following gene order: centromere-(Np-1, Tcra)-Blk-sys-Es-IO. In addition to Blk, three other members of the Src family show preferential expression in hematopoietic cells-Lck, Hck, and Fgr. Of these genes, only Blk is expressed specifically in B-lymphoid cells. In the
a
b
c
- >28 -23
-14.8 -
763
COMMUNICATION
9.2
FIG. 1. Southern blot analysis of DNAs from an intersuhspeties hackcross using the Rlfi hyhridization probe. DNA was extracted from the livers of inhred mice, ?t4. m. musculus. and the hackcross progeny. DNA samples were digested with &/II, fractionated by electrophoresis through 0.4% agarose gels, and transferred to nylon memhranes (Hyhond N+, Amersham). The Blh prohe was labeled by t.he random primer method (6). Hyhridization and wash condirions were as described previously (12). Lanes a-c, individual hackcross mice. Fragment sizes in kilohase pairs are indicated to the right.
TABLE
1
Segregation of the Blk Hybridizing Restriction Fragment with Alleles of Es-IO, Np-1 and sys in 105 Progeny of an Intersubspecies Backcross Inheritance NFS/N Mice Parentals
of the allele
NJ,- I
Blk
,s,vs
+
+
t
I!;.+ 10
Number of mice
t i
Single recomhinants
+
t
i
+
+ I +
t
t
I
+ +
:14 51 2 0 t5 4 4 .5
Recombination” Locus
pair
Np-I.Blk Blk.sys s,w.Ew- 10
“( Recombination (il SE)
rln 9/105 9/1os 2/l 05
8.6 t 2.7 8.6 t 2.7 1.9 + 1.3
a Percentage recombination between restriction fragments standard error were calculated according to Green (8) from number of recomhinants (r) in a sample size of n.
and the
mouse, Lck and Fgr reside on chromosome 4 (Ceci et al., 1989), while Hck has been mapped to Chromosome 2 (Siracusa et al., 1990). The map location ofBlk on Chromosome 14 confirms that Blk is a distinct member of the Src family. While the human homolog of Blk has not yet been mapped, the proximal half of mouse Chromosome 14 contains regions of linkage homology with human chromosomes 10, 13, and 14 (Nadeau, 1989). Our localization of Blk wit,hin this mouse linkage group indicates that it lies between the regions with known homology to human chromosomes13 and 14 and suggeststhat its genetic mapping in man may help define the extent of these regions. If the Blk product plays an essential role in normal B-cell development, we would expect mutations in Blk to impair immune function. Chromosome 14 carries a previously described immunodeficiency locus, hairless locus (hr), mutations at which are associated with increased incidence of leukemia, high titers of ecotropic and xenotropic retroviruses, and impaired cellular immune responses (reviewed by Green, 1989). Our observations suggest that Blk maps proximal to hr and, therefore, that these genes are distinct. ACKNOWLEDGMENTS We thank for preparing
M. Peyser for technical the manuscript. This
assistance and H. R. Marshall work was supported in part hy
764
SHORT
COMMUNICATION
grants to S.V.D. from the Howard Hughes Medical Institute and the National Cancer Institute (CA16519). S.M.D. is a trainee of the Medical Scientist Training Program of the National Institutes of Health.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
CAMPBELL, M., AND SEFTON, B. (1990). Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin. EMBOJ. 9: 2125-2131. CECI, J. D., SIRACUSA, L. D., JENKINS, N. A., AND COPELAND. N. G. (1989). A molecular genetic linkage map of mouse chromosome 4 including the localization of several proto-oncogenes. Genomics 5: 699-709. COOPER, J. A. (1990). The Src-family of protein-tyrosine kinases. In “Peptides and Protein Phosphorylation” (B. Kemp and P. F. Alewood, Eds.), pp. 85-J 15, CRC Press, Boca Raton, FL. DOOLITTLE, D. P., DAVISON, M. T., RODERICK, T. H., AND HILLYARD, A. L. (1990). Locus map of mouse. Mouse Genume 87: 14-27. DYMECKI, S., NIEDERHLJBER, J.. AND DESIDERIO, Y. (1990). Specific expression of a novel tyrosine kinase gene, Blk. in B lymphoid cells. Science 247: 332-336. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. GOLD, M. R., LAW, D. A., AND DEFRANCO, A. L. (1990). Stimulation of protein tyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature fLondonl 345: 810-813.
8. GREEN, 9.
10.
E. L. (1981). Linkage, recombination and mapping. In “Genetics and Probability in Animal Breeding Experiments,” pp. 77-113. MacMillan, New York. GREEN, M. C. (1989). Catalog of mutant genes and polymorphic loci. In “Genetic Variants and Strains of the Laboratory Mouse” (Lyon and Searle, Eds.), pp. 12-403, Oxford Univ. Press, New York. HANKS, S. K., QUINN, A. M., AND HUNTER, 7’. 11988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52.
II.
HARRIS, H.. AND HOPKINSON, D. A. (1976). “Handbook Enzyme Electrophoresis in Human Genetics,” North land, Amsterdam.
12.
HOGGAN, M.D., HALLXN, N. F.. BUCKLER, C. E., AND I~OZAK, C. A. (1988). Genetic mapping of the mouse c-fms proto-oncclgene to chromosome 18. J. Viral. 62: 1055-1056. MACGREGOR, G. R.. RUSSEI.L, L. D., VAN BEEK, M. E. A. H.. HANTEN. G. R., KOVAK, M. .J., KOZAK, C. A., MEISTRXH, M. I,.. AND OVERBEEK, P. A. (1990). Symplastic spermatids (s,w), a recessive insertional mutation in mice causing a defect in spermatogenesis. Proc. Natl. Acad. Sci. (ISA 87: X)16-5020. NADEAU, .J. H. (1989). Maps of linkage and synteny homologies between mouse and man. 7’rends Genet. 5: 82-86. PAHDOI,L, D., FOWI.KES, B. J., LECHLER, R. I., GERMAIN. R.. AND SCHWARTZ, R. H. (1987). Early genetic events in T cell development analyzed by in situ hybridization. J. EQ. Med. 165: 1624-16:38. QIRAWSA, I,. D., SILAN, C. M., JUSTICE, M. J.. MERCEH, .J. A., BAUSKIN. A. R., BEN-NERIAH, Y., L)UBOULE, D., HASTIE. N. D., COPELAND, N. G., AND JENKINS, N. A. (1990). A molecular genetic linkage map of mouse chromosome 2. Genomics 6: 491-504.
1:j.
14. 15.
16.
01 Hol-