GENOMICS
6,572-574
(1990)
BRIEF REPORT Amp&2 WALTON Departments
of Medicine
Maps to Distal Mouse Chromosome in Linkage with Ampd-I S. MOSELEY, TAKAYUKI MORISAKI, RICHARD L. SABINA, EDWARD W. HOLMES, AND MICHAEL F. SELDIN and Biochemistry, Received
Duke University Medical
July 18, 1989;
revised
November
Center, Durham, North Carolina 277 70 16, 1989
genome hybridize to the Ampd-1 and Ampd-2 cDNAs. Indirect evidence suggeststhat both genes are linked: L6 myoblasts resistant to coformycin coamplify both genes while expressing only Ampd-2 (Morisaki et al., manuscript submitted). Recently, Ampd-1 was localized by linkage analyses to a segment of mouse chromosome 3 that was shown by long-range restriction mapping to be conserved with a segment of human chromosome lp (Kingsmore et al., 1990). In the current study we utilized interspecific backcross mice to directly examine genetic linkage between Ampd-1 and Ampd-2. Although tissue-specific expression of these genes has not been analyzed in the mouse, we used this approach because of the ease of identifying restriction fragment length variants (RFLV) in an interspecific cross. We reasoned that if the Ampd-1 and Ampd-2 genes were linked in the rat as suggested by the indirect studies discussed above, the homologous mouse loci would be similarly linked. We analyzed the segregation of a RFLV detected with a rat Ampd-2 clone with RFLV previously defined with an Ampd-1 clone (Kingsmore et al., 1990) and with a cDNA clone for the mouse amylase-2 subunit (Amy2) (Moseley and Seldin, 1989). The rat Ampd-2 probe was hybridized under stringent conditions to a seriesof screening blots containing restriction endonuclease-digested DNAs derived from homozygous C3H/HeJ-gldfgld (CC) and heterozygous (CSH/HeJ-gZd/gZd X Mu.s spretus) Fr (SC) mice. As shown in Fig. 1, a 4.3-kb Ampd-2 RFLV unique to M. spretus was generated on TaqI-digested Southern screening blots. Furthermore, only a single band was detected with the rat cDNA, which suggeststhe presence of a single mouse gene that corresponds to the cloned rat cDNA. The Ampd-2 probe was then hybridized to a panel of 338 DNAs from the interspecific backcross [(C3H/HeJ-gZd/gld X M. spretus) F1 X C3H/
AMP deaminase consists of a family of isozymes in higher eukaryotic animals. As a major component of the purine nucleotide cycle, AMP deaminase catalyzes the conversion of AMP to IMP. Within several species, multiple isoforms of AMP deaminase have been identified. In human, four AMP deaminase isozymes have been differentiated on the basis of chromatographic, immunological, and electrophoretic methods (Ogasawara et al., 1982). Hereditary deficiencies in two of these isoforms have been described clinically; deficiency of isozyme M, found only in skeletal muscle, presents clinically with symptoms of exercise-induced muscle cramping and weakness (Fishbein et aZ., 1978). However, deficiency of a second isozyme, isozyme E, has been found in erythrocytes of human subjects exhibiting neither the symptoms described above nor any hematological dysfunction (Ogasawara et al., 1987). The rat has also been shown to express a variety of isoforms with tissue distribution patterns similar to those of man (Ogasawara et al., 1978; Marquetant et al., 1987). Work by Sabina et al. (1987,1989) has identified three stage-specific AMP deaminase transcripts in rat muscle cells at various stages of differentiation: two 2.5-kb transcripts specific to perinatal and adult rat skeletal muscle, and a 3.4-kb transcript found in embryonic muscle, undifferentiated myoblasts, and nonmyogenic tissues. The two smaller transcripts appear to be alternative splicing forms of a primary transcript and thus derived from a single gene (Ampd-1) (Sabina et al., 1989); the larger transcript is transcribed from a different gene (Ampd-2) (Morisaki et al., manuscript submitted). The sequence of a partial cDNA for Ampd-2 isolated from a rat brain library indicates a 69 and a 73% similarity in nucleic acid and amino acid sequence, respectively, when compared with those of rat Ampd-1 gene. Southern blotting demonstrates that distinct restriction fragments in the rat and human
osss-7643/90 $3.00 Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.
3
572
BRIEF
Taq
In human, only the human homolog to Ampd-1, (encoding isozyme M), has been mapped and in is located on lp13-p21 (Sabina et al., manuscript preparation). However, no studies have as yet reported the location of AMPD2 in human. This report identifies Ampd-2 in mouse as being 1.8 CM telomeric to Ampd1, suggesting that AMPDB in human will be situated on the same chromosomal band or on one adjacent to AMPDl since this segment of mouse chromosome 3 appears to be conserved with a segment of human chromosome lp (Moseley and Seldin, 1989). Although sequence homology and genetic linkage data suggest that Ampd-1 and Ampd-2 resulted from a duplication event in a common primordial ancestor, such an event likely occurred relatively early in mammalian evolution, prior to primate-murine divergence, since analogous AMP deaminase isozymes are more similar between species than they are to other isozymes within each species. These suppositions are corroborated by the genetic data described herein. Relatively tight linkage exists between Ampd-1 and Ampd-2, but not to an extent that intergenic crossover events are not observed. In addition, the placement of Amy-2 between Ampd-1 and Ampd-2 suggests that this gene family has been interrupted by insertions during evolution.
AMPDl
- 23.0 - 9.4 - 6.6
Ampd-2
- 2.3 -2.1 -
1.3
cc SC FIG. 1. Southern blot identification of unique M. spretus 4.3kb RFLV detected with Ampd-2 probe. Molecular size standards in kilobases are shown to the right. Left lane, TaqI-digested DNA from CBH/HeJ-gM/gId (CC) mice. Right lane, TaqI-digested DNA from (CBH/HeJ-gZd/gld X M. spretus)F1 (SC) mice. The Ampd-2 probe was cloned from a 60%bp EcoRI insert from a rat brain cDNA library (Morisaki et al., manuscript submitted).
HeJ-gld/gld], which were then typed as being either CC or SC at Ampd-2 (methods fully discussed in Seldin et al., 1988). The haplotype distribution was then compared with that defined by RFLV detected with Ampd1 and Amy-2 (Table 1). The best gene order (Bishop, 1985) and recombination frequency f standard deviation (Green, 1981) were (centromere)-Ampd-I-1.2 f 0.6 CM-Amy-2-0.6 +- 0.4 CM-Ampd-2-(telomere).
ACKNOWLEDGMENTS Dr. M. F. Seldin is a Charles E. Culpeper Foundation Medical Scholar and the recipient of the Arthritis Foundation Regina S. Loeb Investigator Award; the current work was supported in part by these foundations. Dr. E. W. Holmes is supported by Public Health Service Grant DK12413 from the National Institutes of Health.
TABLE Gene
Mapping
Using
(C3H/HeJ-&d/&d Number
Murine
1
X 1M. spretus) of recombination
F1 X CSH/HeJ-&f/&d
Backcross
Mice
events”
None
gene
573
REPORT
One
Human
homologb
Ampd-1
cc
SC
cc
SC
cc
SC
AMPDl
Amy-2
cc
SC
S”c
ck
cc
SC
AMY2
lp21
Ampd-2
cc
SC
SC
cc
ST!
c”c
AMPD2
N.D.
159
173
3
1
2
0
Number
of mice
332
4
lp13-p21
2
’ Columns indicate the possible genotype combinations of individual backcross mice at three gene loci as contributed by each parent. CC, C3H/HeJ homozygous genotype; SC, Fi heterozygous genotype; x, recombination site. Ampd-2 genotype was determined by RFLV illustrated in Fig. 1 and the Ampd-1 and Amy-2 genotypes were determined as previously described (4, 6). The location of Amy-2 with respect to Ampd2 was previously analyzed (4). b Designated nomenclature of human homologs and their chromosomal assignment based on in situ hybridization to metaphase chromosome spreads. N.D., not determined.
574
BRIEF REPORT REFERENCES
7. OGASAWARA, N., GOTO, H., YAMADA, Y., AND WATANABE, T. (1978). Distribution of AMP-desminase isosymes in rat tissues. Eur.
1. BISHOP, D. T. (1985). The information content of phase-known matings for ordering genetic loci. Genet. Epidemiol. 2: 349-361. 2. FISHBEIN, W. N., ARMJ~RUSTMACHER, V. W., AND GRIFFIN, J. L. (1978). Myoadenylste desminsse deficiency: A new disease of muscle. Science 200: 545-548. 3. GREEN, E. L. (1981). Linkage, recombination and mapping. In “Genetics and Probability in Animal Breeding Experiments” (E. Green, Ed.), pp. 77-113, MacMillian, New York. 4.
KINGSMORE, S. F., MOSELEY, W. S., WATSON, M. L., SABINA, R. L., HOLMES, E. W., AND SELDIN, M. F. (1990). Long-range restriction site mapping of a syntenic segment of human chromosome 1 and mouse chromosome 3. Gerwmics 6: 482-490. 5. MARQUETANT, R., DESAI, N. M., SABINA, R. L., AND HOLMES, E. W. (1987). Evidence for sequential expression of multiple AMP deeminase isoforms during skeletal muscle development. Proc. Natl.
Acad.
Sci. USA
84: 2345-2349.
6. MOSELEY, W. S. S., AND SELDIN, M. F. (1989). Definition of mouse chromosome 1 and 3 gene linkage groups that are conserved on human chromosome 1. Genomics 5: 899-905.
J. Biochem.
87:
297-304.
8. OGASAWARA, N., GOTO, H., YAMADA, Y., WATANABE, T., AND ASANO, T. (1982). AMP deaminase isozymes in human tissues. Biochem. Biophys. Acta 714: 298-306. 9. OGASAWARA, N., GOTO, H., YAMADA, Y., NISHIGAKI, I., ITOH, T., HASEGAWA, I., AND PARK, K. S. (1987). Deficiency of AMP desminase in erythrocytes. Hum. Genet. 75: 15-18. 10. SABINA, R. L., MARQUETANT, R., DESAI, N. M., KALETHA, K., AND HOLMES, E. W. (1987). Cloning and sequence of rat myoadenylate deaminase cDNA: Evidence for tissue-specific and developmental regulation. J. Biol. Chem. 262: 12397-12490. 11. SABINA, R. L., OGASAWARA, N., AND HOLMES, E. W. (1989). Expression of three stage-specific transcripts of AMP desminass during myogenesis. Mol. Cell. Btil. 9: 2244-2246. 12. SELDIN, M. F., MORSE, H. C., III, REEVES, J. P., SCRIBNER, C. L., LEBOEUF, R. C., AND STEINBERG, A. D. (1988). Genetic analysis of autoinnnunegZd mice: 1. Identification of a restriction fragment length polymorphism closely linked to the girl mutation within a conserved linkage group. J. Ezp. Med. 167: 688-693.
6,572-574
(1990)
BRIEF REPORT Amp&2 WALTON Departments
of Medicine
Maps to Distal Mouse Chromosome in Linkage with Ampd-I S. MOSELEY, TAKAYUKI MORISAKI, RICHARD L. SABINA, EDWARD W. HOLMES, AND MICHAEL F. SELDIN and Biochemistry, Received
Duke University Medical
July 18, 1989;
revised
November
Center, Durham, North Carolina 277 70 16, 1989
genome hybridize to the Ampd-1 and Ampd-2 cDNAs. Indirect evidence suggeststhat both genes are linked: L6 myoblasts resistant to coformycin coamplify both genes while expressing only Ampd-2 (Morisaki et al., manuscript submitted). Recently, Ampd-1 was localized by linkage analyses to a segment of mouse chromosome 3 that was shown by long-range restriction mapping to be conserved with a segment of human chromosome lp (Kingsmore et al., 1990). In the current study we utilized interspecific backcross mice to directly examine genetic linkage between Ampd-1 and Ampd-2. Although tissue-specific expression of these genes has not been analyzed in the mouse, we used this approach because of the ease of identifying restriction fragment length variants (RFLV) in an interspecific cross. We reasoned that if the Ampd-1 and Ampd-2 genes were linked in the rat as suggested by the indirect studies discussed above, the homologous mouse loci would be similarly linked. We analyzed the segregation of a RFLV detected with a rat Ampd-2 clone with RFLV previously defined with an Ampd-1 clone (Kingsmore et al., 1990) and with a cDNA clone for the mouse amylase-2 subunit (Amy2) (Moseley and Seldin, 1989). The rat Ampd-2 probe was hybridized under stringent conditions to a seriesof screening blots containing restriction endonuclease-digested DNAs derived from homozygous C3H/HeJ-gldfgld (CC) and heterozygous (CSH/HeJ-gZd/gZd X Mu.s spretus) Fr (SC) mice. As shown in Fig. 1, a 4.3-kb Ampd-2 RFLV unique to M. spretus was generated on TaqI-digested Southern screening blots. Furthermore, only a single band was detected with the rat cDNA, which suggeststhe presence of a single mouse gene that corresponds to the cloned rat cDNA. The Ampd-2 probe was then hybridized to a panel of 338 DNAs from the interspecific backcross [(C3H/HeJ-gZd/gld X M. spretus) F1 X C3H/
AMP deaminase consists of a family of isozymes in higher eukaryotic animals. As a major component of the purine nucleotide cycle, AMP deaminase catalyzes the conversion of AMP to IMP. Within several species, multiple isoforms of AMP deaminase have been identified. In human, four AMP deaminase isozymes have been differentiated on the basis of chromatographic, immunological, and electrophoretic methods (Ogasawara et al., 1982). Hereditary deficiencies in two of these isoforms have been described clinically; deficiency of isozyme M, found only in skeletal muscle, presents clinically with symptoms of exercise-induced muscle cramping and weakness (Fishbein et aZ., 1978). However, deficiency of a second isozyme, isozyme E, has been found in erythrocytes of human subjects exhibiting neither the symptoms described above nor any hematological dysfunction (Ogasawara et al., 1987). The rat has also been shown to express a variety of isoforms with tissue distribution patterns similar to those of man (Ogasawara et al., 1978; Marquetant et al., 1987). Work by Sabina et al. (1987,1989) has identified three stage-specific AMP deaminase transcripts in rat muscle cells at various stages of differentiation: two 2.5-kb transcripts specific to perinatal and adult rat skeletal muscle, and a 3.4-kb transcript found in embryonic muscle, undifferentiated myoblasts, and nonmyogenic tissues. The two smaller transcripts appear to be alternative splicing forms of a primary transcript and thus derived from a single gene (Ampd-1) (Sabina et al., 1989); the larger transcript is transcribed from a different gene (Ampd-2) (Morisaki et al., manuscript submitted). The sequence of a partial cDNA for Ampd-2 isolated from a rat brain library indicates a 69 and a 73% similarity in nucleic acid and amino acid sequence, respectively, when compared with those of rat Ampd-1 gene. Southern blotting demonstrates that distinct restriction fragments in the rat and human
osss-7643/90 $3.00 Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.
3
572
BRIEF
Taq
In human, only the human homolog to Ampd-1, (encoding isozyme M), has been mapped and in is located on lp13-p21 (Sabina et al., manuscript preparation). However, no studies have as yet reported the location of AMPD2 in human. This report identifies Ampd-2 in mouse as being 1.8 CM telomeric to Ampd1, suggesting that AMPDB in human will be situated on the same chromosomal band or on one adjacent to AMPDl since this segment of mouse chromosome 3 appears to be conserved with a segment of human chromosome lp (Moseley and Seldin, 1989). Although sequence homology and genetic linkage data suggest that Ampd-1 and Ampd-2 resulted from a duplication event in a common primordial ancestor, such an event likely occurred relatively early in mammalian evolution, prior to primate-murine divergence, since analogous AMP deaminase isozymes are more similar between species than they are to other isozymes within each species. These suppositions are corroborated by the genetic data described herein. Relatively tight linkage exists between Ampd-1 and Ampd-2, but not to an extent that intergenic crossover events are not observed. In addition, the placement of Amy-2 between Ampd-1 and Ampd-2 suggests that this gene family has been interrupted by insertions during evolution.
AMPDl
- 23.0 - 9.4 - 6.6
Ampd-2
- 2.3 -2.1 -
1.3
cc SC FIG. 1. Southern blot identification of unique M. spretus 4.3kb RFLV detected with Ampd-2 probe. Molecular size standards in kilobases are shown to the right. Left lane, TaqI-digested DNA from CBH/HeJ-gM/gId (CC) mice. Right lane, TaqI-digested DNA from (CBH/HeJ-gZd/gld X M. spretus)F1 (SC) mice. The Ampd-2 probe was cloned from a 60%bp EcoRI insert from a rat brain cDNA library (Morisaki et al., manuscript submitted).
HeJ-gld/gld], which were then typed as being either CC or SC at Ampd-2 (methods fully discussed in Seldin et al., 1988). The haplotype distribution was then compared with that defined by RFLV detected with Ampd1 and Amy-2 (Table 1). The best gene order (Bishop, 1985) and recombination frequency f standard deviation (Green, 1981) were (centromere)-Ampd-I-1.2 f 0.6 CM-Amy-2-0.6 +- 0.4 CM-Ampd-2-(telomere).
ACKNOWLEDGMENTS Dr. M. F. Seldin is a Charles E. Culpeper Foundation Medical Scholar and the recipient of the Arthritis Foundation Regina S. Loeb Investigator Award; the current work was supported in part by these foundations. Dr. E. W. Holmes is supported by Public Health Service Grant DK12413 from the National Institutes of Health.
TABLE Gene
Mapping
Using
(C3H/HeJ-&d/&d Number
Murine
1
X 1M. spretus) of recombination
F1 X CSH/HeJ-&f/&d
Backcross
Mice
events”
None
gene
573
REPORT
One
Human
homologb
Ampd-1
cc
SC
cc
SC
cc
SC
AMPDl
Amy-2
cc
SC
S”c
ck
cc
SC
AMY2
lp21
Ampd-2
cc
SC
SC
cc
ST!
c”c
AMPD2
N.D.
159
173
3
1
2
0
Number
of mice
332
4
lp13-p21
2
’ Columns indicate the possible genotype combinations of individual backcross mice at three gene loci as contributed by each parent. CC, C3H/HeJ homozygous genotype; SC, Fi heterozygous genotype; x, recombination site. Ampd-2 genotype was determined by RFLV illustrated in Fig. 1 and the Ampd-1 and Amy-2 genotypes were determined as previously described (4, 6). The location of Amy-2 with respect to Ampd2 was previously analyzed (4). b Designated nomenclature of human homologs and their chromosomal assignment based on in situ hybridization to metaphase chromosome spreads. N.D., not determined.
574
BRIEF REPORT REFERENCES
7. OGASAWARA, N., GOTO, H., YAMADA, Y., AND WATANABE, T. (1978). Distribution of AMP-desminase isosymes in rat tissues. Eur.
1. BISHOP, D. T. (1985). The information content of phase-known matings for ordering genetic loci. Genet. Epidemiol. 2: 349-361. 2. FISHBEIN, W. N., ARMJ~RUSTMACHER, V. W., AND GRIFFIN, J. L. (1978). Myoadenylste desminsse deficiency: A new disease of muscle. Science 200: 545-548. 3. GREEN, E. L. (1981). Linkage, recombination and mapping. In “Genetics and Probability in Animal Breeding Experiments” (E. Green, Ed.), pp. 77-113, MacMillian, New York. 4.
KINGSMORE, S. F., MOSELEY, W. S., WATSON, M. L., SABINA, R. L., HOLMES, E. W., AND SELDIN, M. F. (1990). Long-range restriction site mapping of a syntenic segment of human chromosome 1 and mouse chromosome 3. Gerwmics 6: 482-490. 5. MARQUETANT, R., DESAI, N. M., SABINA, R. L., AND HOLMES, E. W. (1987). Evidence for sequential expression of multiple AMP deeminase isoforms during skeletal muscle development. Proc. Natl.
Acad.
Sci. USA
84: 2345-2349.
6. MOSELEY, W. S. S., AND SELDIN, M. F. (1989). Definition of mouse chromosome 1 and 3 gene linkage groups that are conserved on human chromosome 1. Genomics 5: 899-905.
J. Biochem.
87:
297-304.
8. OGASAWARA, N., GOTO, H., YAMADA, Y., WATANABE, T., AND ASANO, T. (1982). AMP deaminase isozymes in human tissues. Biochem. Biophys. Acta 714: 298-306. 9. OGASAWARA, N., GOTO, H., YAMADA, Y., NISHIGAKI, I., ITOH, T., HASEGAWA, I., AND PARK, K. S. (1987). Deficiency of AMP desminase in erythrocytes. Hum. Genet. 75: 15-18. 10. SABINA, R. L., MARQUETANT, R., DESAI, N. M., KALETHA, K., AND HOLMES, E. W. (1987). Cloning and sequence of rat myoadenylate deaminase cDNA: Evidence for tissue-specific and developmental regulation. J. Biol. Chem. 262: 12397-12490. 11. SABINA, R. L., OGASAWARA, N., AND HOLMES, E. W. (1989). Expression of three stage-specific transcripts of AMP desminass during myogenesis. Mol. Cell. Btil. 9: 2244-2246. 12. SELDIN, M. F., MORSE, H. C., III, REEVES, J. P., SCRIBNER, C. L., LEBOEUF, R. C., AND STEINBERG, A. D. (1988). Genetic analysis of autoinnnunegZd mice: 1. Identification of a restriction fragment length polymorphism closely linked to the girl mutation within a conserved linkage group. J. Ezp. Med. 167: 688-693.
