- Email: [email protected]
Human N-CAM gene: mapping to chromosome 11 by analysis of somatic cell hybrids with mouse and human cDNA probes
Molecular Brain Research, l(l986)
197
197-200
Elsevier BRM 80005
Human N-CAM gene: mapping to chromosome 11 by analysis of somatic cell hybrids with mouse and human cDNA probes F.S. WALSH’, W. PUTT’, J.G. DICKSONl, C.A. QUINN’, R.D. COX’, M. WEBB’,*, N. SPURR’ and P.N. GOODFELLOW’ ‘Instirure of Neurology, London (U.K.) and ‘Imperial Cancer Research Fund, London (V. K.) (Accepted 15 July 1986) Key words: N-CAM cDNA probes-Human
muscle-Gene
mapping
We have used mouse and human cDNA probes to map the chromosomal position of the N-CAM gene in the human genome. Southern analysis of DNA isolated from a panel of mouse- human somatic cell hybrids has assigned the N-CAM gene to chromosome 11. This assignment was found with both mouse and human N-CAM cDNAs.
The neural cell adhesion molecule (N-CAM) is a cell surface glycoprotein expressed by neurones and several other cell types such as skeletal muscle and has been postulated to play a major role in neural tissue morphogenesis and nerve fibre pattern formation3,‘. By immunochemical criteria N-CAM has been identified in a wide variety of species including humans3 suggesting that at least certain epitopes of this molecule are phylogenetically conserved. More recently cDNA clones for mouse5 and chicken8,9 N-CAMS have been isolated and initial studies on the expression of N-CAM-related mRNAs in rodent and avian brain5.9 and skeletal muscle tissue’ have been carried out. In the mouse the N-CAM gene has been mapped to chromosome 9 by segregation analysis using somatic cell hybrids and restriction site polymorphisms in inbred strains of animals4. In addition, a mouse N-CAM cDNA clone pM1.3 (ref. 5) has been used in an in situ hybridisation study of human metaphase chromosomes and locates the human NCAM gene to map position llq23 (ref. 10). Although the short non-homologous pM1.3 probe cross hybridises to human DNA a confirmation of this gene assignment by other methods is desirable. We have isolated N-CAM cDNA clones from a human skeletal muscle library as a first step in the analysis of N-CAM transcripts and their regulation in this tissue. Here we
report that this human muscle cDNA clone maps to human chromosome 11 as does the mouse pM1.3 probe by analysis of a panel of somatic cell hybrids. The mouse N-CAM cDNA clone, pM1.3 (ref. 5) was used to probe DNA obtained from human and mouse cells and human-mouse somatic cell hybrids. A Southern blot of Bgl II digested mouse and human DNA probed with pM1.3 is shown in Fig. la. This digest reveals a clear distinction between the restriction fragment sizes in the mouse and human DNA hybridising to the pM1.3 probe. There are 3 Bgl II fragments (4.6 kb, 6.5 kb, 7.6 kb) in mouse DNA and two fragments in the human (17 kb, 25 kb). The difference in restriction fragment sizes allowed us to determine on which human chromosome the human NCAM gene resides using somatic cell hybrids. Fig. lb shows the pattern of segregation of the human NCAM gene in mouse-human somatic cell hybrids. A list of the cell hybrids tested and whether they contained human DNA fragments hybridising to pM1.3 is shown in Table I. Initial experiments suggested that the human N-CAM gene resided on either human chromosomes 11,13, or 15 and of these chromosome 11 was strongly implicated on the basis of a positive reaction with the cell line HORL9D2Rl. Although HORL9D2Rl cells contain a fragment of the X-chromosome in addition to chromosome 11, this
* Present address: Sandoz Insitute for Medical Research, Gower Place, London WClE 6BN, U.K. Correspondence: F.S. Walsh, Institute of Neurology, Queen Square, London WClN 3BG, U.K. 0169-328X/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
198 a 25 17
TABLE
Segregation of human chromosomes human-mouse hybrids
KbKb-
7*6Kb 6.5Kb56Kb-
I und N-CAM reactivity in
The human cells and human-rodent somatic cell hybrids used in the present study have been described previously’.‘-‘,‘4. The chromosomal content of somatic cell hybrids was determined by combined isoenzyme and karyotype analyses as previously outlined. N.D.. not done
-
Cell line
DUR4.3 CTP34B4 SIR74ii HORL9D2Rl IWILA4
Human chromosomes
3,10,11.12,13,14,15 17,18.20,21,22.X 2,3,6,7,14.16.17 20.x
1,2.4,7,12.11,18 21.22.x ll,Xq II
Control cells 1R (mouse) Human lymphoblasts
can be ruled
out as it is also present
Presence of human specific bands pM1.3
pHFMl
+
+
+
+
N.D.
+
+
_ +
in cell hybrids
to pM1.3. Further confirmation that the N-CAM gene resides on human chromosome 11 came from the use of a human N-CAM cDNA. We have isolated human muscle N-CAM cDNA clones from a ,Igtll library. The restriction map of one clone designated pHFM1 is shown in Fig. 2. pHFM1 contains a 1.3 kb EcoRl fragment and shares homology with pM1.3 on the large EcoRI-PstI fragment (data not shown). pHFM1 hybridises to the same bands on that do not hybridise
C
D
Fig. 1. Southern analysis of mouse, human and mouse-human somatic cell hybrid DNA. a: shows samples of mouse (track A and human (track B) DNA digested with Bgl II and hybridised with pM1.3. b: samples of DNA digested with Bgl II and hybridised with pM1.3. Track D human DNA, track C mouse DNA. track A DUR4.3 cell hybrids. Track B CTP34B4 cell hybrid. The size of hybridising fragments in given from analysis of the mobility of 1 DNA markers cut with Hind III. The mouse N-CAM cDNA probe designated pM1.3 was generously donated by Dr. C. Goridis. The 0.6 kb insert was excised by EcoRI (Boehringer) purified on a 0.7% agarose gel and labelled with a-[‘*P]d-CTP (3000 Ciimmol) by the replacement synthesis method” to a specific activity > 5 x 10’ cpm//ig. The labelled pM1.3 probe was then used to screen a 1gtll human skeletal muscle cDNA library, and one clone (pHFMl), a I .3 kb cDNA was chosen for the mapping study. The insert from
pHFM1 was purified and labelled with a-[“P]d-CTP
as above.
Full details of the isolation and structure of pM1.3 cross hybridising clones isolated from this library will be published elsewhere. High molecular weight DNA were isolated from human cells and somatic cell hybrids as described previously’. DNA (IO/cg) were digested to completion with Bgl II under manufac-
human and mouse DNA as does pM1.3 although
the
..intensities of reaction are somewhat different. Southern analysis of the homologous N-CAM probe on a variety of somatic cell hybrids (Table I) confirms the
turer’s conditions (Anglian Biotechnology Ltd.) or Hind III (BCL). The digested DNAs were fractioned by electrophoresis in an 0.8% agarose gel and transferred to 0.45 pm nitrocellulose filters (Schleicher and Schull. BA85) according to Southern”. Prehybridisation was carried out for 5 h at 42 “C in the presence of 50% formamide. 5 x SSPE. I x Denhardt’s solution, 10% Dextran sulphate. lOO,uglml calf thymus DNA and 1 ,ug/ml poly (A)h. The pM1.3 and pHFMl probes were then added to the prehybridisation solution for 16 h at 42 “C. The filters were washed twice for 15 min in 5 X SSC. 0.1% SDS at 37 “C before being exposed at -70 “C to Fuji RX film with an intensifying screen.
199 pHFMl
I
I
I
Eco RI
Pst
I
Hind
I Ill
I I
Sac
Eco RI
r
I
O-I Kb Fig. 2. Restriction
location
map of the 1.3 kb pHFM1
of the N-CAM
human
N-CAM
gene to chromosome
cDNA clone.
11 in
humans. We have shown that the human N-CAM gene maps to chromosome 11. This localisation was determined by two different cDNA clones, one of which was human (pHFM1) in origin and one which was mouse (pM1.3). The data confirms the results found by in situ hybridisation of human metaphase chromosomes using the pM1.3 probe”. These authors point out that their data with pM1.3 is not of the same standard as that generally found with longer homologous probes. This meant that there was a high background on all chromosomes with however significantly increased grain density on the long arm of chromosome 11. As such an alternate mapping procedure was desirable and we have used somatic cell hybrids. In the present studies the mouse pM1.3 probe cross-hybridised with human DNA but as it reacted more strongly with mouse DNA, blots had to be washed at low stringency and exposed for about 14 days in order to detect the human N-CAM sequences. In contrast, the pHFM1 human cDNA probe reacted more strongly with human DNA than with mouse DNA
Collins, M.K.L., Goodfellow, P.N., Dunne, M.J., Spurr, N.. Solomon. E. and Owen, M.J., A human T-cell antigen receptor chain gene maps to chromosome 7. EMBO J., 3 (1984) 2347-2349. Covault, J., Merlie, J.P., Goridis, C. and Sanes, J.R.. Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle, J. Cell Biol., 102 (1986) 731-739. McClain, D.A. and Edelman, G.M., A neural cell adhesion molecule from human brain. Proc. Nufl. Acad. Sci. U.S.A., 79 (1982) 6380-6384. D’Eustachio. P., Owens, G.C., Edelman, G.M. and Cunningham, B.A., Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse, Proc. Narl. Acad. Sci. U.S.A.. 82 (1985) 7631-7635. Goridis, S., Hirn, M., Santoni, M.J., Gennarini, G., Deagostini-Bazin, H.. Jordan, B.R., Kiefer, M. and Steinmetz, M., Isolation of mouse N-CAM related cDNA: detection and cloning using monoclonal antibodies, EMBO J., 4
and high quality autoradiographs were obtained after 48 h exposures. Although the set of somatic cell hybrids used in the study does not have the same fine resolving power as in situ chromosome hybridisation, it was nevertheless possible to show that both probes mapped the N-CAM gene to chromosome 11. It will be of considerable interest to use the human N-CAM probes for in situ chromosome hybridisation studies to confirm and further analyse the N-CAM gene map position on the long arm of chromosome 11. In this respect, both D’Eustachio et al.’ and Nguyen et al.” have noted that the human N-CAM and Thy-l genes map to the same region on chromosome 11. Since both these molecules are expressed by human muscle cells but are not coordinately regulated during differentiation, skeletal muscle may prove to be an appropriate system for assessing any functional significance of their close association in the genome.
We wish to thank Dr. C. Goridis for the generous gift of pM1.3 clone. This work was supported by the Muscular Dystrophy Group of Great Britain.
(1985) 631-635. 6 Maniatis, T., Frisch, E.F. and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1982. 7 Moore, SE. and Walsh, F.S., Specific regulation of N-CAMID2CAM cell adhesion molecule during skeletal muscle development, EMBO J., 4 (1985) 623-630. 8 Murray, B.A., Hemperly, J.J., Gallin, W.J., MacGregor, J.S.. Edelman. G.M. and Cunningham, B.A., Isolation of cDNA clones for the chicken neural cell adhesion molecule (N-CAM). Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 5584-5588. 9 Murray, B.A., Hemperly. J.J., Prediger. E.A., Edelman, G.M. and Cunningham, B.A., Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM), J. Cell Biol., 102 (1986) 189-193. 10 Nguyen, C., Mattei, M.-G., Mattei, J.-F., Santoni, M.-J., Goridis, C. and Jordan, B.R., Location of the human NCAM gene to band q23 of chromosome 11: the third gene
200 coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11. J. Cell Biol., 102 (1986) 711-715. 11 O’Farrell. P.M., Kutter, E. and Nalcanishi. M.. A restriction map of the bacteriophage T4 genome. Mol. Gen. Genet., 179(1980)421-435. 12 Southern. E.M., Gel electrophoresis of restriction fragments. In R. Wu (Ed.). Methods in Enzymology, Vol. 68. Academic Press. New York. 1980, pp. 152-176.
13 Walsh. F.S., Quinn, C.A.. Pym. B. and Goodfellow. P.N.. Cell surface differentiation antigen of human muscle cncoded by a gene (MIC 12) on chromosome 15. C’~~qene~. Cell Gener., 39 (1985) 5 l-56. 14 Woodroofe. M.N.. Tunnacliffe. A., Pym. B.. Goodfellow, P.N. and Walsh. F.S.. Human muscle cell surface antigen 16.3AS is encoded by a gene on chromosome 11. Somat. Cell Mol. Gmer., IO(1984) S3S-540.
197
197-200
Elsevier BRM 80005
Human N-CAM gene: mapping to chromosome 11 by analysis of somatic cell hybrids with mouse and human cDNA probes F.S. WALSH’, W. PUTT’, J.G. DICKSONl, C.A. QUINN’, R.D. COX’, M. WEBB’,*, N. SPURR’ and P.N. GOODFELLOW’ ‘Instirure of Neurology, London (U.K.) and ‘Imperial Cancer Research Fund, London (V. K.) (Accepted 15 July 1986) Key words: N-CAM cDNA probes-Human
muscle-Gene
mapping
We have used mouse and human cDNA probes to map the chromosomal position of the N-CAM gene in the human genome. Southern analysis of DNA isolated from a panel of mouse- human somatic cell hybrids has assigned the N-CAM gene to chromosome 11. This assignment was found with both mouse and human N-CAM cDNAs.
The neural cell adhesion molecule (N-CAM) is a cell surface glycoprotein expressed by neurones and several other cell types such as skeletal muscle and has been postulated to play a major role in neural tissue morphogenesis and nerve fibre pattern formation3,‘. By immunochemical criteria N-CAM has been identified in a wide variety of species including humans3 suggesting that at least certain epitopes of this molecule are phylogenetically conserved. More recently cDNA clones for mouse5 and chicken8,9 N-CAMS have been isolated and initial studies on the expression of N-CAM-related mRNAs in rodent and avian brain5.9 and skeletal muscle tissue’ have been carried out. In the mouse the N-CAM gene has been mapped to chromosome 9 by segregation analysis using somatic cell hybrids and restriction site polymorphisms in inbred strains of animals4. In addition, a mouse N-CAM cDNA clone pM1.3 (ref. 5) has been used in an in situ hybridisation study of human metaphase chromosomes and locates the human NCAM gene to map position llq23 (ref. 10). Although the short non-homologous pM1.3 probe cross hybridises to human DNA a confirmation of this gene assignment by other methods is desirable. We have isolated N-CAM cDNA clones from a human skeletal muscle library as a first step in the analysis of N-CAM transcripts and their regulation in this tissue. Here we
report that this human muscle cDNA clone maps to human chromosome 11 as does the mouse pM1.3 probe by analysis of a panel of somatic cell hybrids. The mouse N-CAM cDNA clone, pM1.3 (ref. 5) was used to probe DNA obtained from human and mouse cells and human-mouse somatic cell hybrids. A Southern blot of Bgl II digested mouse and human DNA probed with pM1.3 is shown in Fig. la. This digest reveals a clear distinction between the restriction fragment sizes in the mouse and human DNA hybridising to the pM1.3 probe. There are 3 Bgl II fragments (4.6 kb, 6.5 kb, 7.6 kb) in mouse DNA and two fragments in the human (17 kb, 25 kb). The difference in restriction fragment sizes allowed us to determine on which human chromosome the human NCAM gene resides using somatic cell hybrids. Fig. lb shows the pattern of segregation of the human NCAM gene in mouse-human somatic cell hybrids. A list of the cell hybrids tested and whether they contained human DNA fragments hybridising to pM1.3 is shown in Table I. Initial experiments suggested that the human N-CAM gene resided on either human chromosomes 11,13, or 15 and of these chromosome 11 was strongly implicated on the basis of a positive reaction with the cell line HORL9D2Rl. Although HORL9D2Rl cells contain a fragment of the X-chromosome in addition to chromosome 11, this
* Present address: Sandoz Insitute for Medical Research, Gower Place, London WClE 6BN, U.K. Correspondence: F.S. Walsh, Institute of Neurology, Queen Square, London WClN 3BG, U.K. 0169-328X/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
198 a 25 17
TABLE
Segregation of human chromosomes human-mouse hybrids
KbKb-
7*6Kb 6.5Kb56Kb-
I und N-CAM reactivity in
The human cells and human-rodent somatic cell hybrids used in the present study have been described previously’.‘-‘,‘4. The chromosomal content of somatic cell hybrids was determined by combined isoenzyme and karyotype analyses as previously outlined. N.D.. not done
-
Cell line
DUR4.3 CTP34B4 SIR74ii HORL9D2Rl IWILA4
Human chromosomes
3,10,11.12,13,14,15 17,18.20,21,22.X 2,3,6,7,14.16.17 20.x
1,2.4,7,12.11,18 21.22.x ll,Xq II
Control cells 1R (mouse) Human lymphoblasts
can be ruled
out as it is also present
Presence of human specific bands pM1.3
pHFMl
+
+
+
+
N.D.
+
+
_ +
in cell hybrids
to pM1.3. Further confirmation that the N-CAM gene resides on human chromosome 11 came from the use of a human N-CAM cDNA. We have isolated human muscle N-CAM cDNA clones from a ,Igtll library. The restriction map of one clone designated pHFM1 is shown in Fig. 2. pHFM1 contains a 1.3 kb EcoRl fragment and shares homology with pM1.3 on the large EcoRI-PstI fragment (data not shown). pHFM1 hybridises to the same bands on that do not hybridise
C
D
Fig. 1. Southern analysis of mouse, human and mouse-human somatic cell hybrid DNA. a: shows samples of mouse (track A and human (track B) DNA digested with Bgl II and hybridised with pM1.3. b: samples of DNA digested with Bgl II and hybridised with pM1.3. Track D human DNA, track C mouse DNA. track A DUR4.3 cell hybrids. Track B CTP34B4 cell hybrid. The size of hybridising fragments in given from analysis of the mobility of 1 DNA markers cut with Hind III. The mouse N-CAM cDNA probe designated pM1.3 was generously donated by Dr. C. Goridis. The 0.6 kb insert was excised by EcoRI (Boehringer) purified on a 0.7% agarose gel and labelled with a-[‘*P]d-CTP (3000 Ciimmol) by the replacement synthesis method” to a specific activity > 5 x 10’ cpm//ig. The labelled pM1.3 probe was then used to screen a 1gtll human skeletal muscle cDNA library, and one clone (pHFMl), a I .3 kb cDNA was chosen for the mapping study. The insert from
pHFM1 was purified and labelled with a-[“P]d-CTP
as above.
Full details of the isolation and structure of pM1.3 cross hybridising clones isolated from this library will be published elsewhere. High molecular weight DNA were isolated from human cells and somatic cell hybrids as described previously’. DNA (IO/cg) were digested to completion with Bgl II under manufac-
human and mouse DNA as does pM1.3 although
the
..intensities of reaction are somewhat different. Southern analysis of the homologous N-CAM probe on a variety of somatic cell hybrids (Table I) confirms the
turer’s conditions (Anglian Biotechnology Ltd.) or Hind III (BCL). The digested DNAs were fractioned by electrophoresis in an 0.8% agarose gel and transferred to 0.45 pm nitrocellulose filters (Schleicher and Schull. BA85) according to Southern”. Prehybridisation was carried out for 5 h at 42 “C in the presence of 50% formamide. 5 x SSPE. I x Denhardt’s solution, 10% Dextran sulphate. lOO,uglml calf thymus DNA and 1 ,ug/ml poly (A)h. The pM1.3 and pHFMl probes were then added to the prehybridisation solution for 16 h at 42 “C. The filters were washed twice for 15 min in 5 X SSC. 0.1% SDS at 37 “C before being exposed at -70 “C to Fuji RX film with an intensifying screen.
199 pHFMl
I
I
I
Eco RI
Pst
I
Hind
I Ill
I I
Sac
Eco RI
r
I
O-I Kb Fig. 2. Restriction
location
map of the 1.3 kb pHFM1
of the N-CAM
human
N-CAM
gene to chromosome
cDNA clone.
11 in
humans. We have shown that the human N-CAM gene maps to chromosome 11. This localisation was determined by two different cDNA clones, one of which was human (pHFM1) in origin and one which was mouse (pM1.3). The data confirms the results found by in situ hybridisation of human metaphase chromosomes using the pM1.3 probe”. These authors point out that their data with pM1.3 is not of the same standard as that generally found with longer homologous probes. This meant that there was a high background on all chromosomes with however significantly increased grain density on the long arm of chromosome 11. As such an alternate mapping procedure was desirable and we have used somatic cell hybrids. In the present studies the mouse pM1.3 probe cross-hybridised with human DNA but as it reacted more strongly with mouse DNA, blots had to be washed at low stringency and exposed for about 14 days in order to detect the human N-CAM sequences. In contrast, the pHFM1 human cDNA probe reacted more strongly with human DNA than with mouse DNA
Collins, M.K.L., Goodfellow, P.N., Dunne, M.J., Spurr, N.. Solomon. E. and Owen, M.J., A human T-cell antigen receptor chain gene maps to chromosome 7. EMBO J., 3 (1984) 2347-2349. Covault, J., Merlie, J.P., Goridis, C. and Sanes, J.R.. Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle, J. Cell Biol., 102 (1986) 731-739. McClain, D.A. and Edelman, G.M., A neural cell adhesion molecule from human brain. Proc. Nufl. Acad. Sci. U.S.A., 79 (1982) 6380-6384. D’Eustachio. P., Owens, G.C., Edelman, G.M. and Cunningham, B.A., Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse, Proc. Narl. Acad. Sci. U.S.A.. 82 (1985) 7631-7635. Goridis, S., Hirn, M., Santoni, M.J., Gennarini, G., Deagostini-Bazin, H.. Jordan, B.R., Kiefer, M. and Steinmetz, M., Isolation of mouse N-CAM related cDNA: detection and cloning using monoclonal antibodies, EMBO J., 4
and high quality autoradiographs were obtained after 48 h exposures. Although the set of somatic cell hybrids used in the study does not have the same fine resolving power as in situ chromosome hybridisation, it was nevertheless possible to show that both probes mapped the N-CAM gene to chromosome 11. It will be of considerable interest to use the human N-CAM probes for in situ chromosome hybridisation studies to confirm and further analyse the N-CAM gene map position on the long arm of chromosome 11. In this respect, both D’Eustachio et al.’ and Nguyen et al.” have noted that the human N-CAM and Thy-l genes map to the same region on chromosome 11. Since both these molecules are expressed by human muscle cells but are not coordinately regulated during differentiation, skeletal muscle may prove to be an appropriate system for assessing any functional significance of their close association in the genome.
We wish to thank Dr. C. Goridis for the generous gift of pM1.3 clone. This work was supported by the Muscular Dystrophy Group of Great Britain.
(1985) 631-635. 6 Maniatis, T., Frisch, E.F. and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1982. 7 Moore, SE. and Walsh, F.S., Specific regulation of N-CAMID2CAM cell adhesion molecule during skeletal muscle development, EMBO J., 4 (1985) 623-630. 8 Murray, B.A., Hemperly, J.J., Gallin, W.J., MacGregor, J.S.. Edelman. G.M. and Cunningham, B.A., Isolation of cDNA clones for the chicken neural cell adhesion molecule (N-CAM). Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 5584-5588. 9 Murray, B.A., Hemperly. J.J., Prediger. E.A., Edelman, G.M. and Cunningham, B.A., Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM), J. Cell Biol., 102 (1986) 189-193. 10 Nguyen, C., Mattei, M.-G., Mattei, J.-F., Santoni, M.-J., Goridis, C. and Jordan, B.R., Location of the human NCAM gene to band q23 of chromosome 11: the third gene
200 coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11. J. Cell Biol., 102 (1986) 711-715. 11 O’Farrell. P.M., Kutter, E. and Nalcanishi. M.. A restriction map of the bacteriophage T4 genome. Mol. Gen. Genet., 179(1980)421-435. 12 Southern. E.M., Gel electrophoresis of restriction fragments. In R. Wu (Ed.). Methods in Enzymology, Vol. 68. Academic Press. New York. 1980, pp. 152-176.
13 Walsh. F.S., Quinn, C.A.. Pym. B. and Goodfellow. P.N.. Cell surface differentiation antigen of human muscle cncoded by a gene (MIC 12) on chromosome 15. C’~~qene~. Cell Gener., 39 (1985) 5 l-56. 14 Woodroofe. M.N.. Tunnacliffe. A., Pym. B.. Goodfellow, P.N. and Walsh. F.S.. Human muscle cell surface antigen 16.3AS is encoded by a gene on chromosome 11. Somat. Cell Mol. Gmer., IO(1984) S3S-540.