Gene Mapping, Cloning, and Sequencing C ytogenet Cell G en et 5 9 :17 -1 9 ( 1992)
Chromosomal localization of the human gene for palmitoyl-CoA ligase (FACL1) H. Stanczak, J.J. Stanczak, and I. Singh D ep artm en t o f P ed iatrics. M edical U n iv ersity o fS o u th C arolina. C harleston SC (USA)
Abstract. We have localized the gene (FACL1) encoding human long chain fatty acid-coenzyme A ligase, E.C.6.2.1.3, also known as palmitoyl-CoA ligase. Using in-situ hybridiza
tion. we have mapped this gene to chromosome region 3ql 3 in the human karyotype.
Activation of fatty acids to their acyl-CoA derivatives by acyl-CoA ligase(s) is the initial and obligatory step in the metab olism of fatty acids. Different acyl-CoA ligases have been recog nized for different chain fatty acids (Groot et al.. 1976). Palmitoyl-CoA ligase (76 kDal), also known as long chain fatty acidCoA ligase. preferentially activates fatty acids of chain length between C |i to C|« (Miyazawa el al.. 1985). Although palmitoylCoA ligase solubilized by detergents from subcellular mem branes (peroxisomes and microsomes) contains also lignoceroyl-CoA ligase activity, mitochondria exhibit only palmitoylCoA ligase activity (Singh et al., 1985, 1988a: Bhushan et al., 1986; Singh and Poulos, 1988; Lazo et al., 1990). Recently, we have isolated the rat cDNA clone for palmitoyl-CoA ligase (Stanczak et al., 1990) and, in this study, we have used this cDNA as a probe to define the chromosomal localization of the gene for palmitoyl-CoA ligase.
In sim hybridzalion. M elaphase chro m o so m e p rep aratio n s w ere prep ared from peripheral blood lym phocytes from random ly selected d o n o rs as described previously (Y unis an d C handler. 1977: D utrillaux and ViegasP cquignot, 1981). C hrom osom e spreads w ere treated w ith ribonuclease A (Sigm a) at a co n cen tratio n o f 100 pg/m l in 2 x S S C at 37 °C for I h. T he slides w ere then rinsed and d ehydrated. The chrom osom al DNA w as d en atu red by im m ersion o f slides in 70% form am ide, 2 x SSC, pH 7.0, a l 70 °C' for 2 m in. follow ed by quick d ehydration in cold ethanol and air-drying. In situ h y b rid i zatio n was perform ed according to the technique o f H arp er and S au n d ers (1981). T he probes were used at 100 ng/m l in hybridization m ixture c o n ta in ing 50% form am ide, 10% dex tran sulfate. 2 x SSC. and 1.000-fold excess o f sonicated salm on sperm D NA d enatured by h eating a t 70 °C fo r 10 m in. F o l low ing hy b rid izatio n , the slides were w ashed several tim es, d eh y d rated , and dried. P rep aratio n s w ere exposed to K odak N T B 2 nuclear track em ulsion lo r 1 0-14 d, developed in K odak Dektol, fixed, an d rinsed w ith w ater. S taining and b anding w ere perform ed after hyb rid izatio n an d au toradio g rap h y . T h e chrom osom es w ere G -banded as described by C h an d ler and Y unis (1978). R -bands were o btained by using the fluoroehrom e-photolysis-G iem sa m ethod o f P erry and W olff (1974). T h e slides were analyzed w ith a Z eiss pholom icroscope using bright field and a planapo objective 63x 1.4.
Material and methods toyl-CoA ligase cD N A (Stanczak et al.. 1990) an d its restriction fragm ents: 0.6-kb £«>R I-«.(iE II. 0.3-kb // / « d i ll , and 0.27-kb /tcoR I-Pvd fragm ents, w hich cover the 5'. m iddle, and 3 ' regions o f the sequence. T he radiolabeling was perfo rm ed aco rd in g to the random p rim e r labeling technique o f Eeinberg and Vogelstein ( 1983) w ith the R andom P rim ed D N A labeling kit from Boehringer-M annheim an d labeled nucleotides (['U jd A T P . | 3H ]d C T P , and (3H ]d T T P ) from New England N uclear. T he specific activity o f probes varied from 1.6 to 4.0 x 10 7 cpm /pg.
Supported by National Institutes of Health grant NS-22576 and March of Dimes Birth Defects Foundation grant 1-1079. Received 20 March 1991: revision accepted 13June 1991. Request reprints from Dr. Inderjit Singh. Department of Pediatrics. Medical Univer sity ofSouth Carolina, 171 Ashley Avenue. Charleston, SC 29425 (USA).
Results and discussion The analysis of silver grains in metaphase spreads of chro mosomes from 80 cells demonstrated that 19% of all the silver grains counted (65/349) were located on the long arm of chro mosome 3 and 69% of these (45/65) were localized on 3q 13. A diagrammatic representation of silver grains on chromosome 3 is shown in Fig. 1. A histogram of grain distribution gives a cleatpeak on one band (3q 13) which we have identified as a locus for palmitoyl-CoA (Fig. 2). Palmitoyl-CoA ligase (76 kDal) is pres ent in mitochondria, microsomes, and peroxisomes and poly clonal antibodies raised against microsomal palmitoyl-CoA ligase cross-react with the 76-kDal protein (Miyazawa et al., 1985) and inhibit the palmitoyl-CoA ligase activity in these three organelles (Singh et al., 1988a; Lazo et al., 1990). The tar geting of the proteins to these subcellular organelles is controlled
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
Preparation o f probes. T he probes used in th is study were the 2.7-kb palm i-
Stanczak/Stanczak/Singh
18
30-
20 io . lD.IJ .
P
1.1.
^
H V rfiV l-t-C th T P
4
1
1
Q
P *
q
P1
3
2
4
q
5
“ 1
1
(Ö 2 0 -
T ïr tf m
fftrp fa lT j
I
P
P 1
Q
6
Q
p '
q 8
7
■Yil-rf1 f r r i T T n r i V i r i T i l i 11 P1 q T V P1 4 P1 9 9
11
10
12
302010 f f t + r in V t V fiH
iw
Q 13
3
q
3
14
11 rV rV f - 1■ f p q P q P q P 9 P q pq P 9 P 16 17 18 19 20 21 22
V itJiritp-r hj
fU lL
P
q 15
T
f-
9 X
1 Y
Chromosomes Fig. 2. A histogram presenting grain distrib u tio n . A significant accu m u la tion o f grains is seen on the long arm o f chrom osom e 3. on the q 13 band.
3 Fig. 1. An idiogram o f G -b an d ed chro m o so m e 3 (at the 500-band level) illu stratin g the d istrib u tio n o f 65 silver grains. T h e m ajority o f the silver grains are localized o n th e 13 band (3 q l 3).
by the presence of specific amino acid sequences called targeting signals. At present no information is available with respect to the targeting of a protein (palmitoyl-CoA ligase) with a given molecular weight and similar immunological properties to three different subcellular organelles: mitochondria, microsomes. and peroxisomes. However, the localization of the gene for palmitoyl-CoA ligase on chromosome 3 supports our previous conclu sion that peroxisomal palmitoyl-CoA ligase is a different en
zyme from lignoceroyl-CoA ligase (Hashmi. 1986; Lazo et al., 1988, 1990; Singh et al.. 1988b; Wanders et al„ 1988); that is, the lignoceroyl-CoA ligase activity is absent in X-adrenoleukodystrophy (X-ALD) whereas palmitoyl-CoA ligase activity is normal. The deficient activity of peroxisomal lignoceroyl-CoA ligase results in abnormal oxidation and. in turn, pathogno monic accumulation of very long chain fatty acids in X-ALD (Singh et al.. 1984a, b). The gene for X-ALD (designated ALD) is localized on the q28 region of the X chromosome (Migeon et al.. 1981). Acknowledgements W e w ould like to thank Ms. Fran S huler for typing th is m anuscript.
Bhushan A. Singh RP, Singh I: Solubilization of rat brain microsomal acyl-CoA ligases: different en zymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA. Archs Biochem Biophys 246:374-380 (1986). Chandler ME, Yunis JJ: A high resolution in situ hybridization technique for the direct visualization of labeled G-banded early metaphase and prophase chromosomes. Cytogenet Cell Genet 22:352-356 (1978). Dulriilaux B. Viegas-Piquignot E: High resolution Rand G-banding on the same preparation. Hum Genet 57:93-95 (1981).
Feinbcrg AP, Vogelstein B: A technique for radiolabel ing DNA restriction endonuclease fragments to high specific activity. Analyt Biochem 132:6-13 (1983). Groot PHE, Scholte HR. Hülsmann WC: Fatty acid activation: specificity, localization and function. Adv Lipid Res 14:72-125 (1976). Harper ME. Saunders GF: Localization of single copy DNA sequences on G-banded human chromo somes by in situ hybridization. Chromosoma 83: 431-439(1981). Hashmi M. Stanley W. Singh I: Lignoceroyl-CoASH ligase: enzyme defect in fatty acid beta-oxidation
system in X-linked childhood adrenoleukodystrophy. FEBS Lett 196:247-250 (1986). Lazo O. Contreras M. Hashmi M, Stanley W. Irazu C. Singh I: Peroxisomal lignoceroyl-CoA ligase defi ciency in childhood adrcnoleukodystrophy and adrenomyeloneuropathy. Proc natl Acad Sei. USA 85:7647-7651 (1988). ’ Lazo O, Contreras M, Yoshida Y. Singh AK. Stanley W, Weise M. Singh I: Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases. J Lipid Res 31:583-595 (1990). Migeon BR. Moser H\V. Moser AB. Axelman J. Sillence
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
References
19
D. Norum RA: Adrenoleukodystrophy: evidence for X-linkage. inactivation and selection favoring the mutant allele in heterozygous cells. Proc natl Acad Sci. USA 78:5066-5070 (1981). Miyazawa S. Hashimoto T. Yokota S: Identity of longchain acyl coenzyme A synthetase of microsomes. mitochondria and peroxisomes in rat liver. J Biochem 98:723-733(1985). Perry P, Wolff S: New Giemsa method for the differ ential staining o f sister chromatids. Nature 251: 156-158(1974). Singh H. Poulos A: Distinct long chain and very long chain fatty acyl-CoA synthetases in rat liver peroxi somes and microsomes. Archs Biochem Biophvs 266:486-495(1988). Singh 1. Bhushan A. Relan NK. HashimotoT: Acyl-CoA ligases from rat brain microsomes: an immuno chemical study. Biochim biophvs Acta 963:509514 (1988a).
Singh I, Lazo O. Stanley W. Hashmi M: Deficiency of peroxisomal lignoceroyl-CoA ligase in X-linked adrenoleukodystrophy. Fed Proc 2:A340 ( 1988b). Singh I. Moser AB. Goldfischer S. Moser HW: l.ignoceric acid is oxidized in the peroxisome: implica tions for the Zellweger cerebro-hepatorenal syn drome and adrenoleukodystrophy. Proc natl Acad Sci, USA 8 1:4203-4207 ( 1984a). Singh I, Moser AB. Moser HW. Kishimoto Y: Adreno leukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts and amniocytes. Pediatr Res 18:286— 290 (1984b). Singh I. Singh RP. Bhushan A, Singh AK: LignoceroylCoA ligase activity in rat brain microsomal frac tion: topographical localization and effect of deter gents and a-cyclodextrin: Archs Biochem Biophys 236:418-426(1985). Slanczak JJ. Stanczak H, Singh I: Cloning and charac
terization of cDNA for palmitoyl-CoA ligase. T rans Am Soc Neurochem 21:A354 (1990). Wanders RJA. Van Roedmund CWT. Van Wijland MJA, Schutgens RUB, Van den Bosch H. Schram AW, Tager JM: Direct demonstration that the defi cient oxidation of very long chain fatty acids in Xlinked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatly acids. Biochem biophys Res Común 153:618— 624(1988). Yunis JJ; Chandler HE: High-resolution chromosome analysis in clinical medicine, in Stefanini M, Hossain AA. Isenberg HD (cds): Progress in Clinical Pathology, Vol VII. pp 267-288 (Grunc and Strat ton, New York 1977).
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
Palmitoyl-CoA ligase gene mapping
Chromosomal localization of the human gene for palmitoyl-CoA ligase (FACL1) H. Stanczak, J.J. Stanczak, and I. Singh D ep artm en t o f P ed iatrics. M edical U n iv ersity o fS o u th C arolina. C harleston SC (USA)
Abstract. We have localized the gene (FACL1) encoding human long chain fatty acid-coenzyme A ligase, E.C.6.2.1.3, also known as palmitoyl-CoA ligase. Using in-situ hybridiza
tion. we have mapped this gene to chromosome region 3ql 3 in the human karyotype.
Activation of fatty acids to their acyl-CoA derivatives by acyl-CoA ligase(s) is the initial and obligatory step in the metab olism of fatty acids. Different acyl-CoA ligases have been recog nized for different chain fatty acids (Groot et al.. 1976). Palmitoyl-CoA ligase (76 kDal), also known as long chain fatty acidCoA ligase. preferentially activates fatty acids of chain length between C |i to C|« (Miyazawa el al.. 1985). Although palmitoylCoA ligase solubilized by detergents from subcellular mem branes (peroxisomes and microsomes) contains also lignoceroyl-CoA ligase activity, mitochondria exhibit only palmitoylCoA ligase activity (Singh et al., 1985, 1988a: Bhushan et al., 1986; Singh and Poulos, 1988; Lazo et al., 1990). Recently, we have isolated the rat cDNA clone for palmitoyl-CoA ligase (Stanczak et al., 1990) and, in this study, we have used this cDNA as a probe to define the chromosomal localization of the gene for palmitoyl-CoA ligase.
In sim hybridzalion. M elaphase chro m o so m e p rep aratio n s w ere prep ared from peripheral blood lym phocytes from random ly selected d o n o rs as described previously (Y unis an d C handler. 1977: D utrillaux and ViegasP cquignot, 1981). C hrom osom e spreads w ere treated w ith ribonuclease A (Sigm a) at a co n cen tratio n o f 100 pg/m l in 2 x S S C at 37 °C for I h. T he slides w ere then rinsed and d ehydrated. The chrom osom al DNA w as d en atu red by im m ersion o f slides in 70% form am ide, 2 x SSC, pH 7.0, a l 70 °C' for 2 m in. follow ed by quick d ehydration in cold ethanol and air-drying. In situ h y b rid i zatio n was perform ed according to the technique o f H arp er and S au n d ers (1981). T he probes were used at 100 ng/m l in hybridization m ixture c o n ta in ing 50% form am ide, 10% dex tran sulfate. 2 x SSC. and 1.000-fold excess o f sonicated salm on sperm D NA d enatured by h eating a t 70 °C fo r 10 m in. F o l low ing hy b rid izatio n , the slides were w ashed several tim es, d eh y d rated , and dried. P rep aratio n s w ere exposed to K odak N T B 2 nuclear track em ulsion lo r 1 0-14 d, developed in K odak Dektol, fixed, an d rinsed w ith w ater. S taining and b anding w ere perform ed after hyb rid izatio n an d au toradio g rap h y . T h e chrom osom es w ere G -banded as described by C h an d ler and Y unis (1978). R -bands were o btained by using the fluoroehrom e-photolysis-G iem sa m ethod o f P erry and W olff (1974). T h e slides were analyzed w ith a Z eiss pholom icroscope using bright field and a planapo objective 63x 1.4.
Material and methods toyl-CoA ligase cD N A (Stanczak et al.. 1990) an d its restriction fragm ents: 0.6-kb £«>R I-«.(iE II. 0.3-kb // / « d i ll , and 0.27-kb /tcoR I-Pvd fragm ents, w hich cover the 5'. m iddle, and 3 ' regions o f the sequence. T he radiolabeling was perfo rm ed aco rd in g to the random p rim e r labeling technique o f Eeinberg and Vogelstein ( 1983) w ith the R andom P rim ed D N A labeling kit from Boehringer-M annheim an d labeled nucleotides (['U jd A T P . | 3H ]d C T P , and (3H ]d T T P ) from New England N uclear. T he specific activity o f probes varied from 1.6 to 4.0 x 10 7 cpm /pg.
Supported by National Institutes of Health grant NS-22576 and March of Dimes Birth Defects Foundation grant 1-1079. Received 20 March 1991: revision accepted 13June 1991. Request reprints from Dr. Inderjit Singh. Department of Pediatrics. Medical Univer sity ofSouth Carolina, 171 Ashley Avenue. Charleston, SC 29425 (USA).
Results and discussion The analysis of silver grains in metaphase spreads of chro mosomes from 80 cells demonstrated that 19% of all the silver grains counted (65/349) were located on the long arm of chro mosome 3 and 69% of these (45/65) were localized on 3q 13. A diagrammatic representation of silver grains on chromosome 3 is shown in Fig. 1. A histogram of grain distribution gives a cleatpeak on one band (3q 13) which we have identified as a locus for palmitoyl-CoA (Fig. 2). Palmitoyl-CoA ligase (76 kDal) is pres ent in mitochondria, microsomes, and peroxisomes and poly clonal antibodies raised against microsomal palmitoyl-CoA ligase cross-react with the 76-kDal protein (Miyazawa et al., 1985) and inhibit the palmitoyl-CoA ligase activity in these three organelles (Singh et al., 1988a; Lazo et al., 1990). The tar geting of the proteins to these subcellular organelles is controlled
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
Preparation o f probes. T he probes used in th is study were the 2.7-kb palm i-
Stanczak/Stanczak/Singh
18
30-
20 io . lD.IJ .
P
1.1.
^
H V rfiV l-t-C th T P
4
1
1
Q
P *
q
P1
3
2
4
q
5
“ 1
1
(Ö 2 0 -
T ïr tf m
fftrp fa lT j
I
P
P 1
Q
6
Q
p '
q 8
7
■Yil-rf1 f r r i T T n r i V i r i T i l i 11 P1 q T V P1 4 P1 9 9
11
10
12
302010 f f t + r in V t V fiH
iw
Q 13
3
q
3
14
11 rV rV f - 1■ f p q P q P q P 9 P q pq P 9 P 16 17 18 19 20 21 22
V itJiritp-r hj
fU lL
P
q 15
T
f-
9 X
1 Y
Chromosomes Fig. 2. A histogram presenting grain distrib u tio n . A significant accu m u la tion o f grains is seen on the long arm o f chrom osom e 3. on the q 13 band.
3 Fig. 1. An idiogram o f G -b an d ed chro m o so m e 3 (at the 500-band level) illu stratin g the d istrib u tio n o f 65 silver grains. T h e m ajority o f the silver grains are localized o n th e 13 band (3 q l 3).
by the presence of specific amino acid sequences called targeting signals. At present no information is available with respect to the targeting of a protein (palmitoyl-CoA ligase) with a given molecular weight and similar immunological properties to three different subcellular organelles: mitochondria, microsomes. and peroxisomes. However, the localization of the gene for palmitoyl-CoA ligase on chromosome 3 supports our previous conclu sion that peroxisomal palmitoyl-CoA ligase is a different en
zyme from lignoceroyl-CoA ligase (Hashmi. 1986; Lazo et al., 1988, 1990; Singh et al.. 1988b; Wanders et al„ 1988); that is, the lignoceroyl-CoA ligase activity is absent in X-adrenoleukodystrophy (X-ALD) whereas palmitoyl-CoA ligase activity is normal. The deficient activity of peroxisomal lignoceroyl-CoA ligase results in abnormal oxidation and. in turn, pathogno monic accumulation of very long chain fatty acids in X-ALD (Singh et al.. 1984a, b). The gene for X-ALD (designated ALD) is localized on the q28 region of the X chromosome (Migeon et al.. 1981). Acknowledgements W e w ould like to thank Ms. Fran S huler for typing th is m anuscript.
Bhushan A. Singh RP, Singh I: Solubilization of rat brain microsomal acyl-CoA ligases: different en zymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA. Archs Biochem Biophys 246:374-380 (1986). Chandler ME, Yunis JJ: A high resolution in situ hybridization technique for the direct visualization of labeled G-banded early metaphase and prophase chromosomes. Cytogenet Cell Genet 22:352-356 (1978). Dulriilaux B. Viegas-Piquignot E: High resolution Rand G-banding on the same preparation. Hum Genet 57:93-95 (1981).
Feinbcrg AP, Vogelstein B: A technique for radiolabel ing DNA restriction endonuclease fragments to high specific activity. Analyt Biochem 132:6-13 (1983). Groot PHE, Scholte HR. Hülsmann WC: Fatty acid activation: specificity, localization and function. Adv Lipid Res 14:72-125 (1976). Harper ME. Saunders GF: Localization of single copy DNA sequences on G-banded human chromo somes by in situ hybridization. Chromosoma 83: 431-439(1981). Hashmi M. Stanley W. Singh I: Lignoceroyl-CoASH ligase: enzyme defect in fatty acid beta-oxidation
system in X-linked childhood adrenoleukodystrophy. FEBS Lett 196:247-250 (1986). Lazo O. Contreras M. Hashmi M, Stanley W. Irazu C. Singh I: Peroxisomal lignoceroyl-CoA ligase defi ciency in childhood adrcnoleukodystrophy and adrenomyeloneuropathy. Proc natl Acad Sei. USA 85:7647-7651 (1988). ’ Lazo O, Contreras M, Yoshida Y. Singh AK. Stanley W, Weise M. Singh I: Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases. J Lipid Res 31:583-595 (1990). Migeon BR. Moser H\V. Moser AB. Axelman J. Sillence
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
References
19
D. Norum RA: Adrenoleukodystrophy: evidence for X-linkage. inactivation and selection favoring the mutant allele in heterozygous cells. Proc natl Acad Sci. USA 78:5066-5070 (1981). Miyazawa S. Hashimoto T. Yokota S: Identity of longchain acyl coenzyme A synthetase of microsomes. mitochondria and peroxisomes in rat liver. J Biochem 98:723-733(1985). Perry P, Wolff S: New Giemsa method for the differ ential staining o f sister chromatids. Nature 251: 156-158(1974). Singh H. Poulos A: Distinct long chain and very long chain fatty acyl-CoA synthetases in rat liver peroxi somes and microsomes. Archs Biochem Biophvs 266:486-495(1988). Singh 1. Bhushan A. Relan NK. HashimotoT: Acyl-CoA ligases from rat brain microsomes: an immuno chemical study. Biochim biophvs Acta 963:509514 (1988a).
Singh I, Lazo O. Stanley W. Hashmi M: Deficiency of peroxisomal lignoceroyl-CoA ligase in X-linked adrenoleukodystrophy. Fed Proc 2:A340 ( 1988b). Singh I. Moser AB. Goldfischer S. Moser HW: l.ignoceric acid is oxidized in the peroxisome: implica tions for the Zellweger cerebro-hepatorenal syn drome and adrenoleukodystrophy. Proc natl Acad Sci, USA 8 1:4203-4207 ( 1984a). Singh I, Moser AB. Moser HW. Kishimoto Y: Adreno leukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts and amniocytes. Pediatr Res 18:286— 290 (1984b). Singh I. Singh RP. Bhushan A, Singh AK: LignoceroylCoA ligase activity in rat brain microsomal frac tion: topographical localization and effect of deter gents and a-cyclodextrin: Archs Biochem Biophys 236:418-426(1985). Slanczak JJ. Stanczak H, Singh I: Cloning and charac
terization of cDNA for palmitoyl-CoA ligase. T rans Am Soc Neurochem 21:A354 (1990). Wanders RJA. Van Roedmund CWT. Van Wijland MJA, Schutgens RUB, Van den Bosch H. Schram AW, Tager JM: Direct demonstration that the defi cient oxidation of very long chain fatty acids in Xlinked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatly acids. Biochem biophys Res Común 153:618— 624(1988). Yunis JJ; Chandler HE: High-resolution chromosome analysis in clinical medicine, in Stefanini M, Hossain AA. Isenberg HD (cds): Progress in Clinical Pathology, Vol VII. pp 267-288 (Grunc and Strat ton, New York 1977).
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:44:45 AM
Palmitoyl-CoA ligase gene mapping
