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A gene homologous to plasminogen located on human chromosome 2q11–p11
GENOMICS
4,4&I-451
(1989)
SHORT COMMUNICATION A Gene Homologous to Plasminogen located Chromosome 2qll -pl 1 SUSAN LYNN FRANK,* *Department
IVANA KLISAK, t ROBERT S. SPARKES, t AND ALDONS of Microbiology and tDepartment of Medicine, University of California, Los Angeles, California Received
August
11, 1988;
revised
Academic
Prws,
December
J. hJsa**t
School of Medicine, 90024
7, 1988
the amino-terminal half of kringle 4, and the other corresponding to sequences 3’ of kringle 4. We recently assigned the apolipoprotein(a) (APOA) gene to the same region (human chromosome 6q26-27) (Frank et al., 1988). Recent studies by McLean et al. (1987) demonstrated extraordinary sequence homology between the two genes. APOA contains 37 copies of a plasminogen-like kringle 4 domain followed by domains that resemble kringle 5 and the protease domain of plasminogen. The homology between these two genes ranges from 78 to lOO%, raising the possibility that the original PLG assignment to chromosome 6q26-27 resulted from cross-hybridization to APOA sequences. To confirm the PLG assignment, we used a PLG gene probe corresponding to kringles l-3 for Southern analysis of a somatic cell hybrid panel. This probe detected human bands of 7.5 and 5.7 kb as well as some smaller, weakly hybridizing bands that segregated with chromosome 6 and a 4.2-kb band that segregated with chromosome 2 (Frank et al., 1988). We now report the results of in situ hybridization using the PLG probe and present data showing the selective removal of the 4.2-kb band by high-stringency washing. Regional localization of the PLG gene was examined by in situ hybridization of an 863-base PLG cDNA probe encoding kringles l-3. Figure 1 shows the distribution of 200 silver grains over metaphase chromosomes scored for 162 cells. Significant accumulations of grains occur over the long arm of chromosome 6 and over the centromeric region of chromosome 2. Figure 2a shows the grain density of individual bands on chromosome 6, with peak grain density occurring at q26-27, the same region reported for APOA. Of the 162 cells examined, 92 (56%) exhibited grains, and 14% of the cells exhibiting grains showed specific labeling in the 6q26-qter region. This represents 9% of the total grains and a ninefold enrichment of grains in this region (P < 0.005 using Pearson-X2 analysis). The 6q2627 peak detected with the PLG gene probe is not a
A plasminogen probe encoding kringles l-3 detects homologous loci on human chromosomes 6 and 2 by somatic cell hybrid analysis. Regional localization by in situ hybridization places the loci at 6q26-27 and 2pl l-q1 1. Further analysis by varying washing stringencies of hybridization Biters reveals a greater homology with the chromosome 6 locus than with the chromosome 2 locus. These results confirm localization of the plasminogen gene to human chromosome 6q2627 and indicate that a homologous sequence of unknown identity resides on chromosome 2pll-qll. Q 1089
on Human
Inc.
Plasminogen is a member of the superfamily of regulatory proteases involved in the fibrinolytic and blood coagulation systems. The structural regions of plasminogen consist of five homologous tandemly repeated, amino-terminal domains called kringles and a single trypsin-like, carboxyl-terminal proteolytic domain (see Cast&no, 1981, for review). Kringles act as autonomous structural and functional units that mediate the binding of multidomain proteins to other proteins (Vali and Patthy, 1982; Trexler and Patthy, 1983; Patthy et al., 1984). Prothrombin, tissue-type plasminogen activator, coagulation factor XII, and urokinase are among the other plasma proteases known to contain homologous structures (see Patthy, 1985, for review). All kringle structures identified thus far occur in the amino-terminal extensions of trypsin-type proteases. Apolipoprotein(a), and fibronectin to a lesser extent, has also been shown to contain kringle-like structures but has no known proteolytic activity (Banyai et al., 1983; McLean et al., 1987). Murray et al. (1987) assigned the human plasminogen (PLG) gene to chromosome 6q26-27 by somatic cell hybrid analysis and in situ hybridization. Analysis was performed using two probes, one corresponding to 449
All
Copyright 8 1989 rights of reproduction
O&38-7543/89 $3.00 by Academic Press, Inc. in any form reserved.
450
SHORT
COMMUNICATION
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PLG probe and the chromosome 6 locus than between the PLG probe and the chromosome 2 locus, consistent with the assignment of the structural locus of PLG to human chromosome 6q26-27. This assignment is further substantiated by results from Weitkamp et al. (1988) demonstrating linkage between a locus controlling quantitative variation of Lp(a) lipoprotein phenotypes and PLG phenotypes. Quantitative variations of Lp(a) appear to be controlled by polymorphisms of the APOA gene at 6q26-27, and co-inheritance of the two phenotypes indicates close linkage. Although the identity of the 4.2-kb band on chromosome 2 is unknown, it appears that the homologous sequence corresponds to kringles 1, 2, and/or 3, and not kringles 4 or 5, since the gene was not detected
IA,
qPqPPP(IPllPqP
13
14
15
16
17
18
19
20
21 22
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CHROMOSOMES
FIG. 1. In situ hybridization of plasminogen cDNA to human chromosomes. The cDNA corresponda to kringles l-3 of plasminogen and was a gift from John McLean, Department of Cardiovascular Research at Genentech (6). This schematic representation of human metaphase chromosomes depicts grains observed over the entire genome in 162 cells. The probe was labeled with *H to a specific activity of about 4 X 10’ cpm/l.cg ae described by Rigby et al. (10) and was hybridized to chromosomes from normal human lymphocytes by a method developed by Harper and Saunders (5) and modified by Cannizzaro and Emanuel (2).
result of cross-hybridization to APOA gene sequences because kringles l-3 are absent in the APOA gene. Unfortunately, the resolution of in situ hybridization is not great enough to order the 6q26-27 peaks obtained with the APOA and PLG probes. We can therefore conclude only that the APOA and PLG loci are physically very close. Figure 2b shows the grain density of individual bands on chromosome 2, with peak gram density at 2pl l-q1 1. Of the 92 metaphases with grains, 35% show labeling on chromosome 2, and 15% of the total grains were located in the 2p12-2q14 region. This represents a fivefold enrichment of grams compared to background levels (P < 0.005 using Pearson-x2 analysis). These results are consistent with our somatic cell hybrid studies showing two loci homologous to the PLG probe (Frank et al., 1966). To confirm that the PLG gene resides on human chromosome 6q26-27 and not on 2pll-qll, Southern hybridization filters containing EcoRl-digested human DNA were hybridized at 6B°C and washed at varying temperatures. Figure 3a shows bands of 7.5, 5.7, and 4.2 kb detected after washing (in 0.1X SSC) at 65°C. Figure 3b shows the results using the same PLG probe after hybridization at 65°C but washing (in 0.1X SSC) at 7O’C. Only the 7.5-H and 6.7-kb bands are present. These results indicate a greater homology between the
a
6
2
FIG. 2. In situ hybridization of plasminogen vidual bands of chromosome 6 (a) and chromosome
cDNA over 2 (b).
indi-
SHORT
a
COMMUNICATION
b
2. CANNIZZARO,C. A., AND EMANUEL, B. S. (1984). An improved method for G banding chromosomes after in situ hybridization. Cytogenet.
7.5 * 5.7
*
4.2
+
FIG. 3. Southern hybridization of plasminogen sequences in EcoRI-digested DNA. The probe was labeled to a specific activity of about 1 X lo* cpm/pg. Filters were hybridized at 65°C and washed iu 0.1X SSC at 65°C (a) or 7O’C (b). The methods used were described previously (4).
using probes that correspond to kringle 4 or sequences 3’ or kringle 4 (Murray et al., 1987). This sequence may represent a pseudogene, or it may be an unidentified kringle-containing protein. An increasing number of proteins with kringle-like structures have been isolated. Comparisons of these proteins will be of interest for understanding their evolutionary and functional relationships. REFERENCES 1. BANYAI, L., VAF~ADI,A., AND PATI’HY, L. (1983). Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEES L.&t. 163: 3741.
451
Cell Genet.
38: 308-309.
3. CASTELLINO, F. J. (1981). Recent advances in the chemistry of fibrinolytic systems. C&m. Rev. 81: 431-446. 4. FRANK, S. L., KLISAK, I., SPARKES, R. S., MOHANDAS, T., TOMLINSON, J. E., MCLEAN. J. W., LAWN, R. M., AND LUSIS, A. J. (1988). The apolipoprotein(a) gene resides on human chromosome 6q26-27, in close proximity to the homologous gene for plasminogen. Hum. Genet. 79: 352-356. 5. HARPER, M. E., AND SAUNDERS, G. S. (1981). Localization of single copy DNA sequences on G-banded chromosomes by in situ hybridization. Chromasoma 83: 431-439. 6. MCLEAN, J. W., TOMLINSON, J. E., KUANG, W. J., EATON, D. L., CHEN, E. Y., MESS, G. M., SCANU, A. M., AND LAWN, R. M. (1987). cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature (London,J 330: 132-137. 7. MURRAY, J. C., BUETOW, K. H., DONOVAN, M., HORNUNY, S., MOTULSKY, A. G., DISTECHE, C., DYER, K., SWISSHELM, K., ANDERSON,J., GIBLE~T, E., SADLER, E., EDDY, R., AND SHOWS, T. B. (1987). Linkage disequilibrium of plasminogen polymorphisms and assignment of the gene to human chromosome 6q266q27. Amer. J. Hum. Genet. 40: 338-350. a. PATTHY, L., TREXLER, M., VALI, Z., BANYAI, L., AND VARADI, A. (1984). Kringles: Modules specialized for protein bindingHomology of the gelatin-binding region of fibronectin with the kringle structures of proteases. FEBS Z&t. 1’71: 131-136. 9. PA?THY, L. (1985). Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 41: 657-663. 10. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., AND BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113: 237-251. 11. TREXLER, M., AND PATTHY, L. (1983). Folding autonomy of the hringle 4 fragment of human plasmiuogen. Proc. Natl. Acad. Sci. USA 80: 2457-2461. 12. VALI, M., AND PA?THY, L. (1982). Location of the intermediate and high affinity o-aminocarbozylic acid-binding sites in humau plasminogen. J. Biol. Chcm. 267: 2104-2110. 13. WEITKAMP, L. R., GUTTORMSEN, S. A., AND SCHULTZ, J. S. (1988). Linkage between the loci for the Lp(a) lipoprotein (LP) and plasminogen (PLG). Hum. Genet. 79: 80-82.
4,4&I-451
(1989)
SHORT COMMUNICATION A Gene Homologous to Plasminogen located Chromosome 2qll -pl 1 SUSAN LYNN FRANK,* *Department
IVANA KLISAK, t ROBERT S. SPARKES, t AND ALDONS of Microbiology and tDepartment of Medicine, University of California, Los Angeles, California Received
August
11, 1988;
revised
Academic
Prws,
December
J. hJsa**t
School of Medicine, 90024
7, 1988
the amino-terminal half of kringle 4, and the other corresponding to sequences 3’ of kringle 4. We recently assigned the apolipoprotein(a) (APOA) gene to the same region (human chromosome 6q26-27) (Frank et al., 1988). Recent studies by McLean et al. (1987) demonstrated extraordinary sequence homology between the two genes. APOA contains 37 copies of a plasminogen-like kringle 4 domain followed by domains that resemble kringle 5 and the protease domain of plasminogen. The homology between these two genes ranges from 78 to lOO%, raising the possibility that the original PLG assignment to chromosome 6q26-27 resulted from cross-hybridization to APOA sequences. To confirm the PLG assignment, we used a PLG gene probe corresponding to kringles l-3 for Southern analysis of a somatic cell hybrid panel. This probe detected human bands of 7.5 and 5.7 kb as well as some smaller, weakly hybridizing bands that segregated with chromosome 6 and a 4.2-kb band that segregated with chromosome 2 (Frank et al., 1988). We now report the results of in situ hybridization using the PLG probe and present data showing the selective removal of the 4.2-kb band by high-stringency washing. Regional localization of the PLG gene was examined by in situ hybridization of an 863-base PLG cDNA probe encoding kringles l-3. Figure 1 shows the distribution of 200 silver grains over metaphase chromosomes scored for 162 cells. Significant accumulations of grains occur over the long arm of chromosome 6 and over the centromeric region of chromosome 2. Figure 2a shows the grain density of individual bands on chromosome 6, with peak grain density occurring at q26-27, the same region reported for APOA. Of the 162 cells examined, 92 (56%) exhibited grains, and 14% of the cells exhibiting grains showed specific labeling in the 6q26-qter region. This represents 9% of the total grains and a ninefold enrichment of grains in this region (P < 0.005 using Pearson-X2 analysis). The 6q2627 peak detected with the PLG gene probe is not a
A plasminogen probe encoding kringles l-3 detects homologous loci on human chromosomes 6 and 2 by somatic cell hybrid analysis. Regional localization by in situ hybridization places the loci at 6q26-27 and 2pl l-q1 1. Further analysis by varying washing stringencies of hybridization Biters reveals a greater homology with the chromosome 6 locus than with the chromosome 2 locus. These results confirm localization of the plasminogen gene to human chromosome 6q2627 and indicate that a homologous sequence of unknown identity resides on chromosome 2pll-qll. Q 1089
on Human
Inc.
Plasminogen is a member of the superfamily of regulatory proteases involved in the fibrinolytic and blood coagulation systems. The structural regions of plasminogen consist of five homologous tandemly repeated, amino-terminal domains called kringles and a single trypsin-like, carboxyl-terminal proteolytic domain (see Cast&no, 1981, for review). Kringles act as autonomous structural and functional units that mediate the binding of multidomain proteins to other proteins (Vali and Patthy, 1982; Trexler and Patthy, 1983; Patthy et al., 1984). Prothrombin, tissue-type plasminogen activator, coagulation factor XII, and urokinase are among the other plasma proteases known to contain homologous structures (see Patthy, 1985, for review). All kringle structures identified thus far occur in the amino-terminal extensions of trypsin-type proteases. Apolipoprotein(a), and fibronectin to a lesser extent, has also been shown to contain kringle-like structures but has no known proteolytic activity (Banyai et al., 1983; McLean et al., 1987). Murray et al. (1987) assigned the human plasminogen (PLG) gene to chromosome 6q26-27 by somatic cell hybrid analysis and in situ hybridization. Analysis was performed using two probes, one corresponding to 449
All
Copyright 8 1989 rights of reproduction
O&38-7543/89 $3.00 by Academic Press, Inc. in any form reserved.
450
SHORT
COMMUNICATION
15105LllJlI~I P
r
I II
IIII ’ 1
q
I
q
I I
I
P’q
2
I II P’q
I
4
3
II
II
P’q
5
201
I 11,
3,.
**s*
I.
I,
I
PLG probe and the chromosome 6 locus than between the PLG probe and the chromosome 2 locus, consistent with the assignment of the structural locus of PLG to human chromosome 6q26-27. This assignment is further substantiated by results from Weitkamp et al. (1988) demonstrating linkage between a locus controlling quantitative variation of Lp(a) lipoprotein phenotypes and PLG phenotypes. Quantitative variations of Lp(a) appear to be controlled by polymorphisms of the APOA gene at 6q26-27, and co-inheritance of the two phenotypes indicates close linkage. Although the identity of the 4.2-kb band on chromosome 2 is unknown, it appears that the homologous sequence corresponds to kringles 1, 2, and/or 3, and not kringles 4 or 5, since the gene was not detected
IA,
qPqPPP(IPllPqP
13
14
15
16
17
18
19
20
21 22
X
CHROMOSOMES
FIG. 1. In situ hybridization of plasminogen cDNA to human chromosomes. The cDNA corresponda to kringles l-3 of plasminogen and was a gift from John McLean, Department of Cardiovascular Research at Genentech (6). This schematic representation of human metaphase chromosomes depicts grains observed over the entire genome in 162 cells. The probe was labeled with *H to a specific activity of about 4 X 10’ cpm/l.cg ae described by Rigby et al. (10) and was hybridized to chromosomes from normal human lymphocytes by a method developed by Harper and Saunders (5) and modified by Cannizzaro and Emanuel (2).
result of cross-hybridization to APOA gene sequences because kringles l-3 are absent in the APOA gene. Unfortunately, the resolution of in situ hybridization is not great enough to order the 6q26-27 peaks obtained with the APOA and PLG probes. We can therefore conclude only that the APOA and PLG loci are physically very close. Figure 2b shows the grain density of individual bands on chromosome 2, with peak gram density at 2pl l-q1 1. Of the 92 metaphases with grains, 35% show labeling on chromosome 2, and 15% of the total grains were located in the 2p12-2q14 region. This represents a fivefold enrichment of grams compared to background levels (P < 0.005 using Pearson-x2 analysis). These results are consistent with our somatic cell hybrid studies showing two loci homologous to the PLG probe (Frank et al., 1966). To confirm that the PLG gene resides on human chromosome 6q26-27 and not on 2pll-qll, Southern hybridization filters containing EcoRl-digested human DNA were hybridized at 6B°C and washed at varying temperatures. Figure 3a shows bands of 7.5, 5.7, and 4.2 kb detected after washing (in 0.1X SSC) at 65°C. Figure 3b shows the results using the same PLG probe after hybridization at 65°C but washing (in 0.1X SSC) at 7O’C. Only the 7.5-H and 6.7-kb bands are present. These results indicate a greater homology between the
a
6
2
FIG. 2. In situ hybridization of plasminogen vidual bands of chromosome 6 (a) and chromosome
cDNA over 2 (b).
indi-
SHORT
a
COMMUNICATION
b
2. CANNIZZARO,C. A., AND EMANUEL, B. S. (1984). An improved method for G banding chromosomes after in situ hybridization. Cytogenet.
7.5 * 5.7
*
4.2
+
FIG. 3. Southern hybridization of plasminogen sequences in EcoRI-digested DNA. The probe was labeled to a specific activity of about 1 X lo* cpm/pg. Filters were hybridized at 65°C and washed iu 0.1X SSC at 65°C (a) or 7O’C (b). The methods used were described previously (4).
using probes that correspond to kringle 4 or sequences 3’ or kringle 4 (Murray et al., 1987). This sequence may represent a pseudogene, or it may be an unidentified kringle-containing protein. An increasing number of proteins with kringle-like structures have been isolated. Comparisons of these proteins will be of interest for understanding their evolutionary and functional relationships. REFERENCES 1. BANYAI, L., VAF~ADI,A., AND PATI’HY, L. (1983). Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEES L.&t. 163: 3741.
451
Cell Genet.
38: 308-309.
3. CASTELLINO, F. J. (1981). Recent advances in the chemistry of fibrinolytic systems. C&m. Rev. 81: 431-446. 4. FRANK, S. L., KLISAK, I., SPARKES, R. S., MOHANDAS, T., TOMLINSON, J. E., MCLEAN. J. W., LAWN, R. M., AND LUSIS, A. J. (1988). The apolipoprotein(a) gene resides on human chromosome 6q26-27, in close proximity to the homologous gene for plasminogen. Hum. Genet. 79: 352-356. 5. HARPER, M. E., AND SAUNDERS, G. S. (1981). Localization of single copy DNA sequences on G-banded chromosomes by in situ hybridization. Chromasoma 83: 431-439. 6. MCLEAN, J. W., TOMLINSON, J. E., KUANG, W. J., EATON, D. L., CHEN, E. Y., MESS, G. M., SCANU, A. M., AND LAWN, R. M. (1987). cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature (London,J 330: 132-137. 7. MURRAY, J. C., BUETOW, K. H., DONOVAN, M., HORNUNY, S., MOTULSKY, A. G., DISTECHE, C., DYER, K., SWISSHELM, K., ANDERSON,J., GIBLE~T, E., SADLER, E., EDDY, R., AND SHOWS, T. B. (1987). Linkage disequilibrium of plasminogen polymorphisms and assignment of the gene to human chromosome 6q266q27. Amer. J. Hum. Genet. 40: 338-350. a. PATTHY, L., TREXLER, M., VALI, Z., BANYAI, L., AND VARADI, A. (1984). Kringles: Modules specialized for protein bindingHomology of the gelatin-binding region of fibronectin with the kringle structures of proteases. FEBS Z&t. 1’71: 131-136. 9. PA?THY, L. (1985). Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 41: 657-663. 10. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., AND BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113: 237-251. 11. TREXLER, M., AND PATTHY, L. (1983). Folding autonomy of the hringle 4 fragment of human plasmiuogen. Proc. Natl. Acad. Sci. USA 80: 2457-2461. 12. VALI, M., AND PA?THY, L. (1982). Location of the intermediate and high affinity o-aminocarbozylic acid-binding sites in humau plasminogen. J. Biol. Chcm. 267: 2104-2110. 13. WEITKAMP, L. R., GUTTORMSEN, S. A., AND SCHULTZ, J. S. (1988). Linkage between the loci for the Lp(a) lipoprotein (LP) and plasminogen (PLG). Hum. Genet. 79: 80-82.