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Assignment of the human glycogen debrancher gene to chromosome 1p21
GENOMICS
13, 931-934 (19%)
Assignment of the Human Glycogen Debrancher Gene to Chromosome 1~21 TERESA L. YANG-FENG, KEQIN ZHENG, JINGWEI Yu,* BING-ZHI YANG,t
YUAN-TSONG CHEN,t AND FA-TEN KAO*
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510; *Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80206; and tDepartment of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710 Received
March
Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but -15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1~21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein. 10 1992 Academic Press, Inc.
INTRODUCTION
Glycogen debranching enzyme is a large monomeric protein (M, = 165,000 + 500) with two independent catalytic activities, 4-Ly-glucantransferase (EC 2.4.1.25) activity and amylo-1,6-glucosidase (EC 3.2.1.33) activity, occurring at separate sites on a single polypeptide chain (Gordon et al., 1972; White and Nelson, 1974; Taylor et al., 1975; Bates et al., 1975; Gillard and Nelson, 1977). The debranching enzyme together with phosphorylase is responsible for complete degradation of glycogen. After phosphorylase has hydrolyzed the outer glucose residues, the glycogen chains contain short outer branches. Debrancher transferase activity transfers three glucose residues from one short branch to the end of another, and then the debrancher glucosidase hydrolyzes the remaining a-1,6 branch-point glucose residue. Full debranching enzyme activity requires both catalytic activities. This enzyme is highly conserved and its presence has been demonstrated in many species, including yeast (Lee et al., 1970), parasite (Werries et al., 1990), fish 931
23, 1992
(Becker et al., 1977), and mammal (Gordon et al., 1972; White and Nelson? 1974; Chen et al., 1987). Genetic deficiency of glycogen debranching enzyme in man (type III glycogen storage disease, GSD-III) causes hepatomegaly, hypoglycemia, short stature, and variable myopathy (Howell and Williams, 1983). Patients with this disease vary remarkably, both clinically and enzymatically. Most patients have disease involving both liver and muscle (type IIIa). However, some patients (-15% of all GSD-III) have only liver involvement without apparent muscle disease (type IIIb). During infancy and childhood the disease may be indistinguishable from type I glycogen storage disease, as hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation are similar predominant features. The liver symptoms improve with age and usually disappear after puberty. Overt liver cirrhosis can occur, but rarely. Muscle weakness, although minimal during childhood, may become predominant in adults with type IIIa disease; these patients may show signs of neuromuscular involvement with slowly progressive weakness and distal muscle wasting. Enzymatically, type IIIa patients have deficient debranching enzyme activity in both liver and muscle, whereas in type IIIb patients the enzyme activity is absent in the liver but retained in the muscle. The molecular mechanism for the control of tissue-specific expression of debranching enzyme is unknown. Regulation is unlikely to be due to the presence of multiple genes that encode isoenzymes because previous studies of the purified protein provide no evidence for subunits or isoenzymes of debranching enzyme and also our data show that the debranching enzyme is 160 kDa in all tissues examined. We hypothesized that glycogen debranching enzyme in liver and muscle is encoded by a single gene. Its expression in liver and muscle, however, is under separate genetic control. We recently reported the isolation and nucleotide sequences of cDNA encoding the complete human muscle debrancher protein (Yang et al., 1992). The debrancher mRNA includes a 4545bp coding region and a 2371-bp 3’-nontranslated region. The calculated molecular mass 0888.7543/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
932
YANG-FENG
of the debrancher protein derived from cDNA sequence is 172,614 Da, consistent with the estimated size of purified protein. Using the cDNA clones, we have localized the debrancher gene to the short arm of chromosome 1 at band p21 by somatic cell hybrid analysis and in situ hybridization.
ET kb
AL.
MCI-I
H
12
3
4
5
6
7
6
9
10
11
12
13
23.0-
9.4-
6.7-
MATERIALS
AND
METHODS 4,4-
Hybridization probes. Two glycogen debrancher cDNA clones were used for somatic cell hybrid analysis: clone 8-l represents the 3’-portion of the cDNA (nucleotides 3430 to 4840, see Yang et al., 1992, for position of the probe) and clone 159-1.3 represents the 5’portion of the cDNA (nucleotides 2328 to 3620). In situ hybridization was carried out using clone 159-1.3. Hybrid cell lines. A mapping panel consisting of 17 mouse-human (NA09925-NA09938, NA09940, NA10324, and NA10567) and 2 Chinese hamster-human (NA10611 and GM07298) hybrids was obtained from the National Institute of General Medicine Sciences Mutant Cell Repository (NIGMS). Characterization and human chromosome content in these hybrids are described in detail in the NIGMS catalog. DNA samples were digested with EcoRI, separated by electrophoresis on agarose gels, transferred to Hybond nylon filters (Amersham), and hybridized to the 32P-labeled probe as described (Yang-Feng et al., 1985). In situ hybridization. The cDNA probe was nick-translated with [3H]dCTP and [3H]dTTP to a specific activity of 4.75 X lo7 cpm/pg. Hybridization of the labeled probe to metaphases was carried out as previously described (Yang-Feng et al., 1985). Chromosomes were treated with RNase, denatured, and hybridized with denatured DNA probe at a concentration of 25 rig/ml for 15 h at 37°C. After a posthybridization wash, the slides were coated with Kodak NTB-2 emulsion, exposed for 10 days, and then developed in Kodak Dektol. Chromosomes were G-banded using Wright’s stain for silver grain analysis.
RESULTS
Southern blot analysis of 19 human and rodent somatic cell hybrids mapped the glycogen debrancher gene to human chromosome 1. The 3’-portion of the cDNA probe (clone 8-1) detected five human EcoRI fragments of 9.4, 4.3, 3.8, 3.6, and 2.7 kb, four mouse hybridizing bands of 13, 7.2, 2.7, and 2.4 kb, and four hamster fragments of 16.5,4.9,3.6, and 2.7 kb (Fig. 1). Except for the 2.7-kb fragment, which was indistinguishable in hybrids, the other four human-specific fragments showed concordant segregation with chromosome 1 (Table 1). To confirm that the glycogen debrancher gene is indeed segregated with a single chromosome, somatic cell analysis with a 5’-portion of the cDNA probe (clone 159-1.3) was also performed. Five human EcoRI fragments of 8.8, 4.1,3.9,3.4,and2.9kb,threemousebandsof8.8,2.9,and 2.4 kb, and two hamster-specific fragments of 16 and 2.9 kb were detected by the 5’ cDNA clone (data not shown). The 2.9-kb human band was not storable in hybrids, and all other human fragments were present only in hybrids containing human chromosome 1. In situ hybridization of the tritium-labeled cDNA probe to human chromosomes resulted in specific labeling at band p21 of chromosome 1 (Fig. 2). Thirty-four of 50 metaphase cells (68%) had silver grains over this spe-
2.3-
FIG. 1. Representative autoradiogram obtained following hybridization of a 3’-portion of the glycogen debrancher cDNA probe with EcoRI-digested DNA from hybrid cells and controls (H, human; M, mouse; CH, Chinese hamster). Lanes 1, 2, 3 and 9: hybrid cell lines containing human chromosome 1. Lanes 4-8 and 10-13: missing human chromosome 1.
cific region. Of 89 grains analyzed, 38 (42.7%) were located at 1~21. No other site was labeled above background. DISCUSSION
The human glycogen debrancher gene was mapped to the proximal short arm of chromosome 1, band ~21, by somatic cell hybrid analysis and in situ hybridization. All four human-specific EcoRI fragments detected by the 3’-portion or the 5’-portion of the cDNA clone segregated with chromosome 1 in hybrids, and no secondary sites were detected after in situ hybridization, indicating the presence of a single locus and the absence of related sequences. The assignment of the debrancher gene to a single chromosome site is consistent with our hypothesis that the glycogen debranching enzymes synthesized in liver and muscle are likely to be the products of the same gene. The possibility of two or more tandemly arranged genes located in close proximity at chromosome 1~21, however, cannot be excluded completely. This must await the determination of the chromosomal organization of the debrancher gene(s). The assignment of the glycogen debrancher gene to chromosome 1~21 further illustrates the diversity of chromosomal locations for the genes encoding glycogen metabolism enzymes. Genes for the phosphorylase, phosphorylase kinase, and phosphofructokinase families reside on several different chromosomes (Human Gene Mapping 10). Of the phosphorylase gene family, muscle enzyme maps to llq12-q13.2, liver maps to 14q11.2-q24.3, and brain phosphorylase and its related sequence map to chromosomes 20 and 10, respectively. The loci for muscle, platelet, and liver phosphofructokinase are on chromosomes lcen-q32, lOpter-pll, and
HUMAN
GLYCOGEN
DEBRANCHER
GENE
TABLE Correlation
Presence of sequence/ presence of chromosome
of Human
3
4
5
6
Concordant +/+ -/-
4 15
3 12
3 8
4 7
2 8
4 7
4 7
4 6
Discordant +I-/+
0 0
110 3
6
10 7
8
0 6
0 6
4 12
13
6
discordant hybrids
0
4
7
6
8
8
6
6
5
informative hybrids”
19
19
18
17
18
19
17
16
0
21
39
35
44
42
35
38
% discordant n Human
933
1~21
1 Human
Chromosomes
17
18
19
20
4
4 11
4 10
4 7
12 7
9
0 4
0 2
0 7
2 8
Chromosome 2
Total
CHROMSOME
Glycogen Debrancher Gene with in Human-Rodent Somatic Hybrids
1
Total
IS ON
chromosomes
present
at a frequency
7
8
9
10
11
12
13
14
15
2 9
0 8
2 7
3 11
4 5
4 7
5
10 8
2
0 9
0 5
2 111
3
8
9
2
9
5
3
11
4
2
7
19
14
16
18
16
18
16
18
19
19
16
26
21
50
50
13
50
31
17
58
21
13
from
the analysis.
0 14
of 0.1 or less were
excluded
21q22.3, respectively. The muscle phosphorylase kinase a-subunit maps to Xq12-q13, the P-subunit maps to 16q12-q13, and the y-subunit and its related sequences are on chromosomes 7 and 11, respectively. The glycogen debranching enzyme shares a map position with one of the polysaccharide-degrading enzymes -that is, the amylase gene(s). The human amylase with various allozymes is encoded by multiple genes (Groot et al., 1988,1989), together with a pancreatic enzyme pseu-
16
14 14
0
21
22
X
Y
0 11
1 11
13 5
1
3 2
10
6
4
5
18
18
17
15
17
39
56
35
27
29
dogene, clustered in the region 1~21. The amylases share a functional similarity with the glucosidase activity of the debranching enzyme. Furthermore, a limited short sequence homology can be found between the debranching protein of the debrancher enzyme and the active centers of the amylase. The localization of these two functionally related genes to the same chromosomal region may have some evolutionary significance.
ACKNOWLEDGMENTS This work is supported in part by Grants NIH DOE DE-FG092-91ER 61139 (F.T.K.), NIH DK a grant from the Muscular Dystrophy Association
HD 17449 (F.T.K.), 39078 (Y,T.C.), and (Y.T.C.).
REFERENCES Bates, E. H., Heaton, G. M., Taylor, C., Kernohan, J. C., and Cohen, P. (1975). Debranching enzyme from rabbit skeletal muscle: Evidence for the location of two active centres on a single polypeptide chain. FEBS Lett. 58: 181-185. Becker, J., Long, T. J., and Fischer, properties of debranching enzyme try 16: 291-297.
ti
E. H. (1977). Purification and from dogfish muscle. Biochemis-
Chen, Y-T., He, J-K., Ding, J-H., and Brown, debranching enzyme: Purification, antibody immunoblot analyses of type III glycogen Hum. Genet. 41: 1002-1015.
l
Gillard, B. K., and glucantransferase: study the binding zyme. Biochemistry
l
?
B. I. (1987). Glycogen characterization, and storage disease. Am. J.
Nelson, T. E. (1977). Amylo-1,6-glucosidase/4-uUse of reversible substrate model inhibitors and active sites of rabbit muscle debranching 16: 3978-3987.
to en-
Gordon, R. B., Brown, D. H., and Brown, B. I. (1972). Studies on the structure and mechanism of action of the glycogen debranching enzymes of muscle and liver. Biochem. Biophys. Acta 289: 97-107. FIG. 2. Silver grain distribution along pairs of G-banded chromosome 1, illustrating
chromosome 1. Three specific labeling at ~21.
Groot, P. C., Bleeker, M. J., Pronk, J. C., Arwert, F., Mager, Planta, R. J., Eriksson, A. W., and Frants, R. R. (1988).
W. H., Human
934 pancreatic Res. 16:
YANG-FENG
amylase 4724.
is encoded
by two different
genes.
Nucleic
Acids
Groot, P. C., Bleeker, M. J., Pronk, J. C., Arwert, F., Mager, W. H., Planta, R. J., Eriksson, A. W., and Frants, R. R. (1989). The human amylase multigene family consists of haplatypes with variable number of enes. Cytogenet. Cell Genet. 51: 1009. Human Gene Mapping 10 (1989). Cytogenet. Cell Genet. 51: (l-4). Howell, R. R., and Williams, J. C. (1983). The glycogen storage diseases. In “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, Eds.), 5th ed., pp. 141-166, McGraw-Hill, New York. Lee, E. Y. C., Carter, J. H., Nielsen, L. D., and Fischer, E. H. (1970). Purification and properties of yeast amylo-1,6-glucosidase-oligo1,4+glucantransferase. Biochemistry 9: 2347-2355. Taylor, C., Cox, A. J., Kernohan, J. C., and Cohen, P. (1975). De-
ET
AL.
branching enzyme from rabbit skeletal muscle, purification, ties and physiological role. Eur. J. Biochem. 51: 105-115. Werries, E., Franz, A., and Geisemeyer, S. (1990). Detection gen-debranching system in trophozoites of E&amoeba J. Protozool. 37(6): 576-580.
properof glycohistolytica.
White, R. C.. and Nelson, T. E. (1974). Reevaluation of the subunit structure and molecular weight of rabbit muscle amylo-1,6-glucosidase/4-cu-glucanotransferase. Biochem. Biophys. Acta 365: 274280. Yang, B-Z., Ding, J-H., Enghild, J. J., Bao, Y., and Chen, Y-T. (1992). Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme. J. Biol. Chem., in press. Yang-Feng, T. L., Floyd-Smith, G., Drouin, J., and Francke, U. (1985). Localization of pronatiodilatin gene to human chromosome 1 and mouse chromosome 4. Am. J. Hum. Genet. 37: 1117-1128.
13, 931-934 (19%)
Assignment of the Human Glycogen Debrancher Gene to Chromosome 1~21 TERESA L. YANG-FENG, KEQIN ZHENG, JINGWEI Yu,* BING-ZHI YANG,t
YUAN-TSONG CHEN,t AND FA-TEN KAO*
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510; *Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80206; and tDepartment of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710 Received
March
Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but -15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1~21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein. 10 1992 Academic Press, Inc.
INTRODUCTION
Glycogen debranching enzyme is a large monomeric protein (M, = 165,000 + 500) with two independent catalytic activities, 4-Ly-glucantransferase (EC 2.4.1.25) activity and amylo-1,6-glucosidase (EC 3.2.1.33) activity, occurring at separate sites on a single polypeptide chain (Gordon et al., 1972; White and Nelson, 1974; Taylor et al., 1975; Bates et al., 1975; Gillard and Nelson, 1977). The debranching enzyme together with phosphorylase is responsible for complete degradation of glycogen. After phosphorylase has hydrolyzed the outer glucose residues, the glycogen chains contain short outer branches. Debrancher transferase activity transfers three glucose residues from one short branch to the end of another, and then the debrancher glucosidase hydrolyzes the remaining a-1,6 branch-point glucose residue. Full debranching enzyme activity requires both catalytic activities. This enzyme is highly conserved and its presence has been demonstrated in many species, including yeast (Lee et al., 1970), parasite (Werries et al., 1990), fish 931
23, 1992
(Becker et al., 1977), and mammal (Gordon et al., 1972; White and Nelson? 1974; Chen et al., 1987). Genetic deficiency of glycogen debranching enzyme in man (type III glycogen storage disease, GSD-III) causes hepatomegaly, hypoglycemia, short stature, and variable myopathy (Howell and Williams, 1983). Patients with this disease vary remarkably, both clinically and enzymatically. Most patients have disease involving both liver and muscle (type IIIa). However, some patients (-15% of all GSD-III) have only liver involvement without apparent muscle disease (type IIIb). During infancy and childhood the disease may be indistinguishable from type I glycogen storage disease, as hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation are similar predominant features. The liver symptoms improve with age and usually disappear after puberty. Overt liver cirrhosis can occur, but rarely. Muscle weakness, although minimal during childhood, may become predominant in adults with type IIIa disease; these patients may show signs of neuromuscular involvement with slowly progressive weakness and distal muscle wasting. Enzymatically, type IIIa patients have deficient debranching enzyme activity in both liver and muscle, whereas in type IIIb patients the enzyme activity is absent in the liver but retained in the muscle. The molecular mechanism for the control of tissue-specific expression of debranching enzyme is unknown. Regulation is unlikely to be due to the presence of multiple genes that encode isoenzymes because previous studies of the purified protein provide no evidence for subunits or isoenzymes of debranching enzyme and also our data show that the debranching enzyme is 160 kDa in all tissues examined. We hypothesized that glycogen debranching enzyme in liver and muscle is encoded by a single gene. Its expression in liver and muscle, however, is under separate genetic control. We recently reported the isolation and nucleotide sequences of cDNA encoding the complete human muscle debrancher protein (Yang et al., 1992). The debrancher mRNA includes a 4545bp coding region and a 2371-bp 3’-nontranslated region. The calculated molecular mass 0888.7543/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
932
YANG-FENG
of the debrancher protein derived from cDNA sequence is 172,614 Da, consistent with the estimated size of purified protein. Using the cDNA clones, we have localized the debrancher gene to the short arm of chromosome 1 at band p21 by somatic cell hybrid analysis and in situ hybridization.
ET kb
AL.
MCI-I
H
12
3
4
5
6
7
6
9
10
11
12
13
23.0-
9.4-
6.7-
MATERIALS
AND
METHODS 4,4-
Hybridization probes. Two glycogen debrancher cDNA clones were used for somatic cell hybrid analysis: clone 8-l represents the 3’-portion of the cDNA (nucleotides 3430 to 4840, see Yang et al., 1992, for position of the probe) and clone 159-1.3 represents the 5’portion of the cDNA (nucleotides 2328 to 3620). In situ hybridization was carried out using clone 159-1.3. Hybrid cell lines. A mapping panel consisting of 17 mouse-human (NA09925-NA09938, NA09940, NA10324, and NA10567) and 2 Chinese hamster-human (NA10611 and GM07298) hybrids was obtained from the National Institute of General Medicine Sciences Mutant Cell Repository (NIGMS). Characterization and human chromosome content in these hybrids are described in detail in the NIGMS catalog. DNA samples were digested with EcoRI, separated by electrophoresis on agarose gels, transferred to Hybond nylon filters (Amersham), and hybridized to the 32P-labeled probe as described (Yang-Feng et al., 1985). In situ hybridization. The cDNA probe was nick-translated with [3H]dCTP and [3H]dTTP to a specific activity of 4.75 X lo7 cpm/pg. Hybridization of the labeled probe to metaphases was carried out as previously described (Yang-Feng et al., 1985). Chromosomes were treated with RNase, denatured, and hybridized with denatured DNA probe at a concentration of 25 rig/ml for 15 h at 37°C. After a posthybridization wash, the slides were coated with Kodak NTB-2 emulsion, exposed for 10 days, and then developed in Kodak Dektol. Chromosomes were G-banded using Wright’s stain for silver grain analysis.
RESULTS
Southern blot analysis of 19 human and rodent somatic cell hybrids mapped the glycogen debrancher gene to human chromosome 1. The 3’-portion of the cDNA probe (clone 8-1) detected five human EcoRI fragments of 9.4, 4.3, 3.8, 3.6, and 2.7 kb, four mouse hybridizing bands of 13, 7.2, 2.7, and 2.4 kb, and four hamster fragments of 16.5,4.9,3.6, and 2.7 kb (Fig. 1). Except for the 2.7-kb fragment, which was indistinguishable in hybrids, the other four human-specific fragments showed concordant segregation with chromosome 1 (Table 1). To confirm that the glycogen debrancher gene is indeed segregated with a single chromosome, somatic cell analysis with a 5’-portion of the cDNA probe (clone 159-1.3) was also performed. Five human EcoRI fragments of 8.8, 4.1,3.9,3.4,and2.9kb,threemousebandsof8.8,2.9,and 2.4 kb, and two hamster-specific fragments of 16 and 2.9 kb were detected by the 5’ cDNA clone (data not shown). The 2.9-kb human band was not storable in hybrids, and all other human fragments were present only in hybrids containing human chromosome 1. In situ hybridization of the tritium-labeled cDNA probe to human chromosomes resulted in specific labeling at band p21 of chromosome 1 (Fig. 2). Thirty-four of 50 metaphase cells (68%) had silver grains over this spe-
2.3-
FIG. 1. Representative autoradiogram obtained following hybridization of a 3’-portion of the glycogen debrancher cDNA probe with EcoRI-digested DNA from hybrid cells and controls (H, human; M, mouse; CH, Chinese hamster). Lanes 1, 2, 3 and 9: hybrid cell lines containing human chromosome 1. Lanes 4-8 and 10-13: missing human chromosome 1.
cific region. Of 89 grains analyzed, 38 (42.7%) were located at 1~21. No other site was labeled above background. DISCUSSION
The human glycogen debrancher gene was mapped to the proximal short arm of chromosome 1, band ~21, by somatic cell hybrid analysis and in situ hybridization. All four human-specific EcoRI fragments detected by the 3’-portion or the 5’-portion of the cDNA clone segregated with chromosome 1 in hybrids, and no secondary sites were detected after in situ hybridization, indicating the presence of a single locus and the absence of related sequences. The assignment of the debrancher gene to a single chromosome site is consistent with our hypothesis that the glycogen debranching enzymes synthesized in liver and muscle are likely to be the products of the same gene. The possibility of two or more tandemly arranged genes located in close proximity at chromosome 1~21, however, cannot be excluded completely. This must await the determination of the chromosomal organization of the debrancher gene(s). The assignment of the glycogen debrancher gene to chromosome 1~21 further illustrates the diversity of chromosomal locations for the genes encoding glycogen metabolism enzymes. Genes for the phosphorylase, phosphorylase kinase, and phosphofructokinase families reside on several different chromosomes (Human Gene Mapping 10). Of the phosphorylase gene family, muscle enzyme maps to llq12-q13.2, liver maps to 14q11.2-q24.3, and brain phosphorylase and its related sequence map to chromosomes 20 and 10, respectively. The loci for muscle, platelet, and liver phosphofructokinase are on chromosomes lcen-q32, lOpter-pll, and
HUMAN
GLYCOGEN
DEBRANCHER
GENE
TABLE Correlation
Presence of sequence/ presence of chromosome
of Human
3
4
5
6
Concordant +/+ -/-
4 15
3 12
3 8
4 7
2 8
4 7
4 7
4 6
Discordant +I-/+
0 0
110 3
6
10 7
8
0 6
0 6
4 12
13
6
discordant hybrids
0
4
7
6
8
8
6
6
5
informative hybrids”
19
19
18
17
18
19
17
16
0
21
39
35
44
42
35
38
% discordant n Human
933
1~21
1 Human
Chromosomes
17
18
19
20
4
4 11
4 10
4 7
12 7
9
0 4
0 2
0 7
2 8
Chromosome 2
Total
CHROMSOME
Glycogen Debrancher Gene with in Human-Rodent Somatic Hybrids
1
Total
IS ON
chromosomes
present
at a frequency
7
8
9
10
11
12
13
14
15
2 9
0 8
2 7
3 11
4 5
4 7
5
10 8
2
0 9
0 5
2 111
3
8
9
2
9
5
3
11
4
2
7
19
14
16
18
16
18
16
18
19
19
16
26
21
50
50
13
50
31
17
58
21
13
from
the analysis.
0 14
of 0.1 or less were
excluded
21q22.3, respectively. The muscle phosphorylase kinase a-subunit maps to Xq12-q13, the P-subunit maps to 16q12-q13, and the y-subunit and its related sequences are on chromosomes 7 and 11, respectively. The glycogen debranching enzyme shares a map position with one of the polysaccharide-degrading enzymes -that is, the amylase gene(s). The human amylase with various allozymes is encoded by multiple genes (Groot et al., 1988,1989), together with a pancreatic enzyme pseu-
16
14 14
0
21
22
X
Y
0 11
1 11
13 5
1
3 2
10
6
4
5
18
18
17
15
17
39
56
35
27
29
dogene, clustered in the region 1~21. The amylases share a functional similarity with the glucosidase activity of the debranching enzyme. Furthermore, a limited short sequence homology can be found between the debranching protein of the debrancher enzyme and the active centers of the amylase. The localization of these two functionally related genes to the same chromosomal region may have some evolutionary significance.
ACKNOWLEDGMENTS This work is supported in part by Grants NIH DOE DE-FG092-91ER 61139 (F.T.K.), NIH DK a grant from the Muscular Dystrophy Association
HD 17449 (F.T.K.), 39078 (Y,T.C.), and (Y.T.C.).
REFERENCES Bates, E. H., Heaton, G. M., Taylor, C., Kernohan, J. C., and Cohen, P. (1975). Debranching enzyme from rabbit skeletal muscle: Evidence for the location of two active centres on a single polypeptide chain. FEBS Lett. 58: 181-185. Becker, J., Long, T. J., and Fischer, properties of debranching enzyme try 16: 291-297.
ti
E. H. (1977). Purification and from dogfish muscle. Biochemis-
Chen, Y-T., He, J-K., Ding, J-H., and Brown, debranching enzyme: Purification, antibody immunoblot analyses of type III glycogen Hum. Genet. 41: 1002-1015.
l
Gillard, B. K., and glucantransferase: study the binding zyme. Biochemistry
l
?
B. I. (1987). Glycogen characterization, and storage disease. Am. J.
Nelson, T. E. (1977). Amylo-1,6-glucosidase/4-uUse of reversible substrate model inhibitors and active sites of rabbit muscle debranching 16: 3978-3987.
to en-
Gordon, R. B., Brown, D. H., and Brown, B. I. (1972). Studies on the structure and mechanism of action of the glycogen debranching enzymes of muscle and liver. Biochem. Biophys. Acta 289: 97-107. FIG. 2. Silver grain distribution along pairs of G-banded chromosome 1, illustrating
chromosome 1. Three specific labeling at ~21.
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W. H., Human
934 pancreatic Res. 16:
YANG-FENG
amylase 4724.
is encoded
by two different
genes.
Nucleic
Acids
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