GENOMICS
11, 770-772
(1991)
SHORT COMMUNICATION Chromosomal TERESA L. JOHNSON,* *Department
Assignment in Mouse of Matrix and Bone GLA Protein Genes ALAN Y. SAKAGUCHI,*
PETER A. LAuw,t
GLA Protein
AND ROBIN J. LEACH*
of Celiuiar and Structural Bioiogy, University of Texas Health Science Center, San Antonio, and t Wayne State University School of Medicine, Detroit, Michigan 48201 Received
February
25, 1991;revised
The formation of calcified bone is preceded by the sequential accumulation of noncollagenous proteins in the extracellular bone matrix. These noncollagenous proteins, produced by cells of the osteoblastic lineage, include matrix Gla protein (MGLAP) and bone Gla protein (BGLAP; osteocalcin). MGLAP and BGLAP are vitamin K-dependent, calcium-binding proteins that contain residues of y-carboxyglutamic acid (Gla). The COOH-terminal domain of MGLAP and the secreted form of BGLAP share approximately 20% sequence homology in rat (Price and Williamson, 1985). Conserved amino acids in BGLAP isolated from various species are also present in MGLAP, suggesting that these proteins arose from gene duplication followed by divergent evolution (Price and Williamson, 1985). Studies were initiated in our laboratory to determine the chromosomal localization of these genes in the mouse genome using somatic whole cell hybrids and karyotypically simple microcell hybrids. To map mouse Mglap, a cDNA probe was constructed using reverse transcription and polymerase
770 Inc. reserved.
June 13.1991
chain reaction (PCR) amplification (Saiki et al., 1988) of mouse mRNA. Total RNA was isolated (Chomczynski and Sacchi, 1987) from 12- to 14-day mouse embryos. At this time in development, chondrification and ossification centers are visible. The mouse RNA was reverse transcribed and subsequently PCR amplified with MGLAP-specific oligonucleotide primers constructed from the published rat cDNA sequence (Price et al., 1987). The sequences of the two primers, 5’-TGGCAGCCCTGTGCTATGAATCT-3’ and $-GCTGCCTGAAGTAGCGGTTGTA-8, correspond to nucleotide positions 44-66 and 274-295, respectively. The sequence of the resulting PCR product shared greater than 90% homology with the rat MGLAP cDNA sequence (Johnson and Leach, unpublished results). Mapping of mouse Bghp was accomplished with the use of the rat BGLAP cDNA clone, pR22-11 (Celeste et aZ., 1986). The chromosomal locations of Mglup and Bghp were determined by Southern blot analysis of DNA from a panel of 12 mouse X Chinese hamster somatic whole cell hybrids (Francke et al., 1977) known to contain the mouse chromosomes indicated in Table 1. Hybridization of the mouse Mghp probe to DNA isolated from the hybrids revealed a Chinese hamsterspecific 14-kb BamHI fragment and an 8-kb mousespecific BamHI fragment (Fig. la). Correlation of the mouse-specific fragment with the mouse DNA content of the hybrid clones suggested that the gene for Mglup resides on mouse Chromosome 6, with the next most likely location being mouse Chromosome 8 (Table 1). Since complete concordance was not observed for cosegregation of MgZup and Chromosome 6, we utilized rat X mouse microcell hybrids to confirm the chromosomal location. Microcell-mediated chromosome transfer is a method by which a single chromosome or subset of intact chromosomes is transferred from one mamma-
Matrix Gla protein (MGLAP) and bone Gla protein (BGLAP) are calcium-binding, vitamin K-dependent proteins produced by cells of the osteoblastic lineage. Sequence homology suggests that the genes for these proteins evolved from a common ancestor. Somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map Mglap to mouse Chromosome 6 and Bglap to mouse Chromosome 3. Human MGLAP has previously been mapped to chromosome 12p, a region with homology to mouse Chromosome 6, and human BGLAP has been mapped to chromosome lq, a region with homology to mouse Chromosome 3. It appears that BGLAP is the third calcium-binding protein that maps to human chromosome Iq and mouse Chromosome 3. Q 1991 Academic PWB, IUC.
088%7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Texas 78284;
SHORT
TABLE Segregation
of Mouse Chromosomes
with
771
COMMUNICATION
1
MgZap and BgZap in Whole Cell and Microcell Mouse
Cell hybrid
Mglnp
B&p
EBS-1 EBS-2 EBS-5 EBS-9 EBS-10 EBS-11
+ + + -
+ + +
EBS-15 EBS-17
+ -
EBS-51 EBS-5-CS EBS-9-CS EBS-3-CS
+ + -
+ + + +
-
% Discordant on whole cell hybrids F(ll)G FWD W8)E F(3.8)8-6 LI Not
-
M&p &lqp
1
2
3
4
++++-+++++-+++++++++ +++--+++++-++-+++-++ ++++++++-+-++++++-++ -+++-++++--++-++++++ ++---+--+-+++++-+-+ + ---+++-+++-+-++-+-+-++ + +--++-+++++--+--++++++ ++++-+++-+-++-+++-+-+++--++++-++-++++++----+----+-++++-+33 58
42 33
25 17
+
-
-----+----+-+++-----
N.A.” N.A.0
_ +
-
-
+
5
6
7
Hybrids
chromosomes
6
9
10
11
12
13
14
15
16
17
--+
-
-
+
-
+
-
-
+
+
---+
+-+
-
+--
-
+-+-+--+
50 42
33 25
42 50
8 17
50 42
17 8
-
-
-
_ _
+-----------+ + + -
18
19
X
33 42
50 58
42 33
42 33
50 75
50 42
50 42
33 25
58 50
25 17
42 50
-
-
-
-
-
-
-
-
-
-
_ -
analyzed.
lian cell to another (Fournier, 1981). The resulting microcell hybrids are obtained at high frequency following selection. Microcell hybrid F(ll)G selectively retains mouse Chromosome 11, plus mouse Chromosomes 6, 13,14, and 15 (Killary and Fournier, 1984). Microcell hybrid F(8)D selectively retains Chromosome 8 and no other mouse chromosomes (Peterson et oz., 1985). DNA from hybrids F(ll)G and F(8)D was hybridized with the Mglup probe and only hybrid F(ll)G contained MgZup sequences. Chromosomes 11,13,14, and 15, also present in F(ll)G, were eliminated as locations for Mglup by their discordancy
rates in the whole cell hybrids (Table 1). Results from this experiment localize MgZup to mouse Chromosome 6. Under the same hybridization conditions used to map Mglup, the rat BGLAP probe detected a Chinese hamster BamHI fragment of 3.9 kb and three mouse BamHI fragments of 4.5, 7.4, and 8.6 kb. Bglup could not be clearly localized in whole cell hybrids since mouse Chromosomes 3, 6, 8, and 19 had similar discordancy rates of 17,17,8, and 17%, respectively (Table 1). Figure lb shows hybridization of the BgZup probe to rat X mouse microcell hybrids F(ll)G,
FIG. 1. DNA isolated from the hybrid and parental cells was digested with the restriction enzyme BanHI, fractionated by electrophoresis through horizontal 0.8% agarose gels, and transferred to nylon membranes in 10X SSC buffer. The membranes were hybridized with “P-labeled probes for mouse Mglup (a) or mouse Bgkxp (b) in a solution of 50% formamide, 0.5 M NaHPO,, 1 mM EDTA, 1% BSA, and 5% SDS at 42°C. Lane designations: (a) (1) EBS 1, (2) EBS 2, (3) EBS 5, (4) EBS 9, (5) EBS 10, (6) EBS 11, (7) EBS 15, (8) EBS 17, (9) EBS 51, (10) EBS 5-CS, (11) EBS 9-CS, (12) EBS 13-CS, (13) RAG (mouse control), (14) RJK-36 (Chinese hamster control). BamHI fragments hybridizing to the Mglup probe (arrows) are 14 kb (Chinese hamster) and 8 kb (mouse). (b) (1) F(8)D, (2) FB(8)D, (3) F(8)E, (4) F(3.8)8-6, BamHI fragments hybridizing to the Eglap probe are 4.4 kb (rat) and 4.5, (5) FB(3.8)8-6, (6) MEF (mouse control), (7) FAO-1 ( ra t control). 7.4, and 8.6 kb (mouse) (arrows).
SHORT
772
COMMUNICATION
F(B)D, F(B)E, and F(3.8)8-6. F(8)E selectively retains mouse Chromosome 8 plus Chromosome 9 (Peterson et al., 1985) and F(3.8)8-6 selectively retains a Robertsonian translocation involving mouse Chromosomes 3 and 8 (Chin and Fournier, 1989). Of the four samples examined, only F(3.8)8-6 DNA revealed mouse Bglap sequences. Data obtained from the microcell hybrid experiment localize Bglap to mouse Chromosome 3 (Table 1). Sequence homology between the COOH-terminal domain of MGLAP and the secreted form of BGLAP suggests that they arose from a common ancestor and could be considered members of a gene family (Price and Williamson, 1985). Gene families can be found either clustered on a chromosome or dispersed throughout the genome. As shown here, the gene family that includes MGLAP and BGLAP has become dispersed during evolution. Recently, the gene for human MGLAP has been localized to chromosome 12p (Cancela et al., 1990). This is in agreement with our data, since the short arm of this chromosome is genetically homologous to mouse Chromosome 6 (summarized by Lalley et al., 1989). The gene for human BGLAP has been regionally localized to chromosome lq (Puchacz et al, 1989). A small region of human chromosome lq is known to be homologous to mouse Chromosome 3 (summarized by Lalley et al., 1989), which is consistent with our localization of BgZap to mouse Chromosome 3. Other genes that map to human chromosome lq and mouse Chromosome 3 include /?-glucosidase (Gba), calcyclin (Cacy), mouse placental protein (Capl), and T-lymphocyte antigen (~$38) (summarized by Lalley et al, 1989). Two of these genes, Cucy and Cap& are proteins that bind calcium. The three Gla residues of BGLAP also bind calcium. BGLAP is thus the third gene with a calcium-binding domain that maps to these homologous regions in man and mouse.
REFERENCES 1.
2.
3.
4.
CHOMCZYNSKI, method of RNA phenol-chloroform
5.
FOURNIER, R. E. K. (1981). A general high-efficiency proeedure for production of microcell hybrids. Proc. Natl. Acad. Sci. USA 78: 6349-6353.
6.
FRANCKE, U., LALLEY, P. A., Moss, W., Iw, J., AND MINNA, J. D. (1977). Gene mapping of Mus musccdus by interspecific cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 19, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-l to chromosome 2. Cytogenet. Cell Genet. 19: 57-84. KILLARY, A. M., AND FOURNIER, R. E. K. (1984). A genetic analysis of extinction: Trans-dominant loci regulate espression of liver-specific traits in hepatoma hybrid cells. Cell 38: 523-534. LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., WOMACK, J. E., RODERICK, T. H., CREAU-GOLDBERG, N., HILLYARD, A. L., DOOLI’MXE, D. P., AND ROGERS, J. A. (1989). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 51: 503-532.
I.
8.
9.
P., AND SACCHI, N. (1987). Single step isolation by acid guanidinium thiocyanateextraction. And. Biochem. 162: 156-159.
PETERSON, T. C., KILLARY, A. M., AND FOURNIER, R. E. K. (1985). Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells. Mol. Cell. Bid. 5: 2491-2494.
10.
PRICE, P. A., AND WILLIAMSON, M. K. (1985). Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. J. Biol. Chem. 260: 14,971-14,975.
11.
PRICE, P. A., FRASER, J. D., AND Mm-VIRCA, G. (1987). Molecular cloning of matrix Gla protein: Implications for substrate recognition by the vitamin K-dependent y-carboxylase. Proc. Natl. Acad. Sci. USA 84: 8335-8339.
12.
PUCHACZ, E., LIAN, J. B., STEIN, G. S., WOZNEY, J. M., HUEBNER, K., AND CROCE, C. (1989). Chromosomal leealization of the human osteocalcin gene. Endocrinology 124: 2648-2650. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
ACKNOWLEDGMENTS We thank Dr. A. M. Killary for generously providing DNA from microcell hybrids F(ll)G, F(8)D, FB(8)D, and F(8)E and Dr. R. E. K. Fournier for generously providing DNA from microcell hybrids F(3.8)8-6 and FB(3.8)8-6. We also thank Dr. J. M. Wozney of Genetics Institute for providing the rat BGLAP probe pR22-11.
CANCELA, L., HSIEH, C-L., FRANCKE, U., AND PRICE, P. A. (1990). Molecular structure, chromosome assignment, and promoter organization of the human matrix gla protein gene. J. Biol. Chem. 265: 15,040-15,048. CELESTE, A. J., ROSEN, V., BUECKER, J. L., KRIZ, R., WANG, E. A., AND WOZNEY, J. M. (1986). Isolation of the human gene for bone gla utilizing mouse and rat cDNA clones. EMBO J. 5: 1885-1886. CHIN, A. C., AND FOURNIER, R. E. K. (1989). Tse-2: A transdominant extinguisher of albumin gene expression in hepatoma hybrid cells. Mol. CeU. Biol. 9: 3736-3743.
13.
11, 770-772
(1991)
SHORT COMMUNICATION Chromosomal TERESA L. JOHNSON,* *Department
Assignment in Mouse of Matrix and Bone GLA Protein Genes ALAN Y. SAKAGUCHI,*
PETER A. LAuw,t
GLA Protein
AND ROBIN J. LEACH*
of Celiuiar and Structural Bioiogy, University of Texas Health Science Center, San Antonio, and t Wayne State University School of Medicine, Detroit, Michigan 48201 Received
February
25, 1991;revised
The formation of calcified bone is preceded by the sequential accumulation of noncollagenous proteins in the extracellular bone matrix. These noncollagenous proteins, produced by cells of the osteoblastic lineage, include matrix Gla protein (MGLAP) and bone Gla protein (BGLAP; osteocalcin). MGLAP and BGLAP are vitamin K-dependent, calcium-binding proteins that contain residues of y-carboxyglutamic acid (Gla). The COOH-terminal domain of MGLAP and the secreted form of BGLAP share approximately 20% sequence homology in rat (Price and Williamson, 1985). Conserved amino acids in BGLAP isolated from various species are also present in MGLAP, suggesting that these proteins arose from gene duplication followed by divergent evolution (Price and Williamson, 1985). Studies were initiated in our laboratory to determine the chromosomal localization of these genes in the mouse genome using somatic whole cell hybrids and karyotypically simple microcell hybrids. To map mouse Mglap, a cDNA probe was constructed using reverse transcription and polymerase
770 Inc. reserved.
June 13.1991
chain reaction (PCR) amplification (Saiki et al., 1988) of mouse mRNA. Total RNA was isolated (Chomczynski and Sacchi, 1987) from 12- to 14-day mouse embryos. At this time in development, chondrification and ossification centers are visible. The mouse RNA was reverse transcribed and subsequently PCR amplified with MGLAP-specific oligonucleotide primers constructed from the published rat cDNA sequence (Price et al., 1987). The sequences of the two primers, 5’-TGGCAGCCCTGTGCTATGAATCT-3’ and $-GCTGCCTGAAGTAGCGGTTGTA-8, correspond to nucleotide positions 44-66 and 274-295, respectively. The sequence of the resulting PCR product shared greater than 90% homology with the rat MGLAP cDNA sequence (Johnson and Leach, unpublished results). Mapping of mouse Bghp was accomplished with the use of the rat BGLAP cDNA clone, pR22-11 (Celeste et aZ., 1986). The chromosomal locations of Mglup and Bghp were determined by Southern blot analysis of DNA from a panel of 12 mouse X Chinese hamster somatic whole cell hybrids (Francke et al., 1977) known to contain the mouse chromosomes indicated in Table 1. Hybridization of the mouse Mghp probe to DNA isolated from the hybrids revealed a Chinese hamsterspecific 14-kb BamHI fragment and an 8-kb mousespecific BamHI fragment (Fig. la). Correlation of the mouse-specific fragment with the mouse DNA content of the hybrid clones suggested that the gene for Mglup resides on mouse Chromosome 6, with the next most likely location being mouse Chromosome 8 (Table 1). Since complete concordance was not observed for cosegregation of MgZup and Chromosome 6, we utilized rat X mouse microcell hybrids to confirm the chromosomal location. Microcell-mediated chromosome transfer is a method by which a single chromosome or subset of intact chromosomes is transferred from one mamma-
Matrix Gla protein (MGLAP) and bone Gla protein (BGLAP) are calcium-binding, vitamin K-dependent proteins produced by cells of the osteoblastic lineage. Sequence homology suggests that the genes for these proteins evolved from a common ancestor. Somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map Mglap to mouse Chromosome 6 and Bglap to mouse Chromosome 3. Human MGLAP has previously been mapped to chromosome 12p, a region with homology to mouse Chromosome 6, and human BGLAP has been mapped to chromosome lq, a region with homology to mouse Chromosome 3. It appears that BGLAP is the third calcium-binding protein that maps to human chromosome Iq and mouse Chromosome 3. Q 1991 Academic PWB, IUC.
088%7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Texas 78284;
SHORT
TABLE Segregation
of Mouse Chromosomes
with
771
COMMUNICATION
1
MgZap and BgZap in Whole Cell and Microcell Mouse
Cell hybrid
Mglnp
B&p
EBS-1 EBS-2 EBS-5 EBS-9 EBS-10 EBS-11
+ + + -
+ + +
EBS-15 EBS-17
+ -
EBS-51 EBS-5-CS EBS-9-CS EBS-3-CS
+ + -
+ + + +
-
% Discordant on whole cell hybrids F(ll)G FWD W8)E F(3.8)8-6 LI Not
-
M&p &lqp
1
2
3
4
++++-+++++-+++++++++ +++--+++++-++-+++-++ ++++++++-+-++++++-++ -+++-++++--++-++++++ ++---+--+-+++++-+-+ + ---+++-+++-+-++-+-+-++ + +--++-+++++--+--++++++ ++++-+++-+-++-+++-+-+++--++++-++-++++++----+----+-++++-+33 58
42 33
25 17
+
-
-----+----+-+++-----
N.A.” N.A.0
_ +
-
-
+
5
6
7
Hybrids
chromosomes
6
9
10
11
12
13
14
15
16
17
--+
-
-
+
-
+
-
-
+
+
---+
+-+
-
+--
-
+-+-+--+
50 42
33 25
42 50
8 17
50 42
17 8
-
-
-
_ _
+-----------+ + + -
18
19
X
33 42
50 58
42 33
42 33
50 75
50 42
50 42
33 25
58 50
25 17
42 50
-
-
-
-
-
-
-
-
-
-
_ -
analyzed.
lian cell to another (Fournier, 1981). The resulting microcell hybrids are obtained at high frequency following selection. Microcell hybrid F(ll)G selectively retains mouse Chromosome 11, plus mouse Chromosomes 6, 13,14, and 15 (Killary and Fournier, 1984). Microcell hybrid F(8)D selectively retains Chromosome 8 and no other mouse chromosomes (Peterson et oz., 1985). DNA from hybrids F(ll)G and F(8)D was hybridized with the Mglup probe and only hybrid F(ll)G contained MgZup sequences. Chromosomes 11,13,14, and 15, also present in F(ll)G, were eliminated as locations for Mglup by their discordancy
rates in the whole cell hybrids (Table 1). Results from this experiment localize MgZup to mouse Chromosome 6. Under the same hybridization conditions used to map Mglup, the rat BGLAP probe detected a Chinese hamster BamHI fragment of 3.9 kb and three mouse BamHI fragments of 4.5, 7.4, and 8.6 kb. Bglup could not be clearly localized in whole cell hybrids since mouse Chromosomes 3, 6, 8, and 19 had similar discordancy rates of 17,17,8, and 17%, respectively (Table 1). Figure lb shows hybridization of the BgZup probe to rat X mouse microcell hybrids F(ll)G,
FIG. 1. DNA isolated from the hybrid and parental cells was digested with the restriction enzyme BanHI, fractionated by electrophoresis through horizontal 0.8% agarose gels, and transferred to nylon membranes in 10X SSC buffer. The membranes were hybridized with “P-labeled probes for mouse Mglup (a) or mouse Bgkxp (b) in a solution of 50% formamide, 0.5 M NaHPO,, 1 mM EDTA, 1% BSA, and 5% SDS at 42°C. Lane designations: (a) (1) EBS 1, (2) EBS 2, (3) EBS 5, (4) EBS 9, (5) EBS 10, (6) EBS 11, (7) EBS 15, (8) EBS 17, (9) EBS 51, (10) EBS 5-CS, (11) EBS 9-CS, (12) EBS 13-CS, (13) RAG (mouse control), (14) RJK-36 (Chinese hamster control). BamHI fragments hybridizing to the Mglup probe (arrows) are 14 kb (Chinese hamster) and 8 kb (mouse). (b) (1) F(8)D, (2) FB(8)D, (3) F(8)E, (4) F(3.8)8-6, BamHI fragments hybridizing to the Eglap probe are 4.4 kb (rat) and 4.5, (5) FB(3.8)8-6, (6) MEF (mouse control), (7) FAO-1 ( ra t control). 7.4, and 8.6 kb (mouse) (arrows).
SHORT
772
COMMUNICATION
F(B)D, F(B)E, and F(3.8)8-6. F(8)E selectively retains mouse Chromosome 8 plus Chromosome 9 (Peterson et al., 1985) and F(3.8)8-6 selectively retains a Robertsonian translocation involving mouse Chromosomes 3 and 8 (Chin and Fournier, 1989). Of the four samples examined, only F(3.8)8-6 DNA revealed mouse Bglap sequences. Data obtained from the microcell hybrid experiment localize Bglap to mouse Chromosome 3 (Table 1). Sequence homology between the COOH-terminal domain of MGLAP and the secreted form of BGLAP suggests that they arose from a common ancestor and could be considered members of a gene family (Price and Williamson, 1985). Gene families can be found either clustered on a chromosome or dispersed throughout the genome. As shown here, the gene family that includes MGLAP and BGLAP has become dispersed during evolution. Recently, the gene for human MGLAP has been localized to chromosome 12p (Cancela et al., 1990). This is in agreement with our data, since the short arm of this chromosome is genetically homologous to mouse Chromosome 6 (summarized by Lalley et al., 1989). The gene for human BGLAP has been regionally localized to chromosome lq (Puchacz et al, 1989). A small region of human chromosome lq is known to be homologous to mouse Chromosome 3 (summarized by Lalley et al., 1989), which is consistent with our localization of BgZap to mouse Chromosome 3. Other genes that map to human chromosome lq and mouse Chromosome 3 include /?-glucosidase (Gba), calcyclin (Cacy), mouse placental protein (Capl), and T-lymphocyte antigen (~$38) (summarized by Lalley et al, 1989). Two of these genes, Cucy and Cap& are proteins that bind calcium. The three Gla residues of BGLAP also bind calcium. BGLAP is thus the third gene with a calcium-binding domain that maps to these homologous regions in man and mouse.
REFERENCES 1.
2.
3.
4.
CHOMCZYNSKI, method of RNA phenol-chloroform
5.
FOURNIER, R. E. K. (1981). A general high-efficiency proeedure for production of microcell hybrids. Proc. Natl. Acad. Sci. USA 78: 6349-6353.
6.
FRANCKE, U., LALLEY, P. A., Moss, W., Iw, J., AND MINNA, J. D. (1977). Gene mapping of Mus musccdus by interspecific cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 19, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-l to chromosome 2. Cytogenet. Cell Genet. 19: 57-84. KILLARY, A. M., AND FOURNIER, R. E. K. (1984). A genetic analysis of extinction: Trans-dominant loci regulate espression of liver-specific traits in hepatoma hybrid cells. Cell 38: 523-534. LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., WOMACK, J. E., RODERICK, T. H., CREAU-GOLDBERG, N., HILLYARD, A. L., DOOLI’MXE, D. P., AND ROGERS, J. A. (1989). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 51: 503-532.
I.
8.
9.
P., AND SACCHI, N. (1987). Single step isolation by acid guanidinium thiocyanateextraction. And. Biochem. 162: 156-159.
PETERSON, T. C., KILLARY, A. M., AND FOURNIER, R. E. K. (1985). Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells. Mol. Cell. Bid. 5: 2491-2494.
10.
PRICE, P. A., AND WILLIAMSON, M. K. (1985). Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. J. Biol. Chem. 260: 14,971-14,975.
11.
PRICE, P. A., FRASER, J. D., AND Mm-VIRCA, G. (1987). Molecular cloning of matrix Gla protein: Implications for substrate recognition by the vitamin K-dependent y-carboxylase. Proc. Natl. Acad. Sci. USA 84: 8335-8339.
12.
PUCHACZ, E., LIAN, J. B., STEIN, G. S., WOZNEY, J. M., HUEBNER, K., AND CROCE, C. (1989). Chromosomal leealization of the human osteocalcin gene. Endocrinology 124: 2648-2650. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.
ACKNOWLEDGMENTS We thank Dr. A. M. Killary for generously providing DNA from microcell hybrids F(ll)G, F(8)D, FB(8)D, and F(8)E and Dr. R. E. K. Fournier for generously providing DNA from microcell hybrids F(3.8)8-6 and FB(3.8)8-6. We also thank Dr. J. M. Wozney of Genetics Institute for providing the rat BGLAP probe pR22-11.
CANCELA, L., HSIEH, C-L., FRANCKE, U., AND PRICE, P. A. (1990). Molecular structure, chromosome assignment, and promoter organization of the human matrix gla protein gene. J. Biol. Chem. 265: 15,040-15,048. CELESTE, A. J., ROSEN, V., BUECKER, J. L., KRIZ, R., WANG, E. A., AND WOZNEY, J. M. (1986). Isolation of the human gene for bone gla utilizing mouse and rat cDNA clones. EMBO J. 5: 1885-1886. CHIN, A. C., AND FOURNIER, R. E. K. (1989). Tse-2: A transdominant extinguisher of albumin gene expression in hepatoma hybrid cells. Mol. CeU. Biol. 9: 3736-3743.
13.
