374 Nucleic Acids Research, Vol. 20, No. 2
Nucleotide
sequence
of the sheep MyoDi
gene
L.Huynen, J.Bass2, R.C.Gardner and A.R.Bellamy Centre for Gene Technology, Department of Cellular and Molecular Biology, University of Auckland, Auckland and 1MAF Technology, Ruakura Agricultural Centre, Hamilton, New Zealand Submitted September 24, 1991 Central to the development of mammalian skeletal muscle cells is the expression of MyoDi, a single copy gene which encodes a binding factor involved in transcriptional regulation. When transfected into various non-muscle cells the gene triggers musclespecific gene expression (1, 2). MyoDI specifies a nuclear protein that contains a putative basic-helix-loop-helix (BHLH) motif. Deletion mutagenesis has shown the BHLH motif to be essential for binding to sequences in the enhancer region of the musclespecific creatine kinase gene (MCK). The sequences to which MyoDl binds are required for maximal muscle-specific expression of reporter genes under the control of the MCK promoter (3, 4). To isolate the ovine equivalent of MyoDl, a mouse MyoDl cDNA clone (1) was used to screen a cDNA library derived from sheep skeletal muscle and constructed in XgtlO. A cDNA clone of 1554 bp was isolated and sequenced by the dideoxy method (5). Comparison of the inferred amino acid sequence with that of the mouse MyoDl indicated that the clone lacked 125 bp of coding sequence at the 5' terminus. The missing nucleotide data as well as 325 bp of upstream sequence was obtained from a sheep genomic clone isolated from a XEMBL3 library. The sheep genomic clone contains no evidence of any introns and is 51% homologous to the 5' untranslated region of mouse MyoDl. Homology between the mouse and sheep 3' untranslated regions is 62%. The sheep MyoDl protein exhibits 90% amino acid identity to that of the mouse. An additional alanine is present in the sheep MyoDl at amino acid position 174. The area of greatest conservation covers the BHLH motif located between amino acids 101-164 (Figure 1).
EMBL accession no. X62102
ACKNOWLEDGEMENTS We thank H.Weintraub for the full length mouse MyoDl cDNA clone. This work was supported by a grant from the New Zealand Ministry of Agriculture and Fisheries (MAF).
REFERENCES 1. Davis,R.L., Weintraub,H. and Lassar,A.B. (1987) Cell 51, 987-1000. 2. Davis,R.L., Cheng,P.-F., Lassar,A.B. and Weintraub,H. (1990) Cell 60, 773-746. 3. Weintraub,H., Tapscott,S.J., Davis,R.L., Thayer,M.J., Adam,M.K., Lassar,A.B. and Miller,A.D. (1989) Proc. NatL. Acad. Sci. USA 86, 5434-5438. 4. Lassar,A.B., Buskin,J.N., Lockshon,D., Davis,R.L., Apone,S., Hauschka,S.D. and Weintraub,H. (1989) Cell 58, 823-831. 5. Sanger,F., Nicklen,S. and Coulson,A.R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5476. 6. Krause,M., Fire,A., White Harrison,S., Priess,J. and Weintraub,H. (1990) Cell 63, 907-919. 7. Michelson,A.M., Abmayr,S.M., Bate,M., Martinez-Arias,A. and Maniatis,T. (1990) Genes Dev. in press. 8. Hopwood,N.D., Pluck,A. and Gurdon,J.B. (1989) EMBOJ. 8, 3409-3417. 9. Lin,Z., Deschesne,C.A., Eldridge,J. and Patterson,B.M. (1989) Genes Dev. 3, 986-996. Sheep Mouse C .legans
Drosophila Xenopua Chicken
KRKTTNADRRKAATMRERRR KRKTTNADRRKAATMRERRR GPRATKLDRRKMATAZRRR KKKSVTVDRRJAATNRZRRR KRKTTNADRRKAaTMSBRRR KRKTTNADRRXAATMRRRR
bode
LSKVNZAFETLKRCT LSKVNZAFITLKRCT LRXVMRArZVVKQRT LRIVNEAflILKRRT LSKVNWEATLKRYT LSKVNEAFETLKRCT
heizi
SSNPNQRLP SSNPNQRLP CPNPNQRLP SSNPNQRLP SSNPNQRLP STNPKQRLP
h.p
KVEILRNAIRYIEGLQAL
KVSILRUAIRYIEGLOAL KVIILRSAIDYINNLSRM KVEILRMAIEYIESLEDL KVKILRMAIRYISSLQAL KVEILRNAIRYIEELQAL
head
Figure 1. Interspecies comparison of the MyoDl BHLH domains. Protein sequences were obtained from the following sources: mouse (1); C. elegans (6); Drosophila (7); Xenopus (8); chicken (9).
Nucleotide
sequence
of the sheep MyoDi
gene
L.Huynen, J.Bass2, R.C.Gardner and A.R.Bellamy Centre for Gene Technology, Department of Cellular and Molecular Biology, University of Auckland, Auckland and 1MAF Technology, Ruakura Agricultural Centre, Hamilton, New Zealand Submitted September 24, 1991 Central to the development of mammalian skeletal muscle cells is the expression of MyoDi, a single copy gene which encodes a binding factor involved in transcriptional regulation. When transfected into various non-muscle cells the gene triggers musclespecific gene expression (1, 2). MyoDI specifies a nuclear protein that contains a putative basic-helix-loop-helix (BHLH) motif. Deletion mutagenesis has shown the BHLH motif to be essential for binding to sequences in the enhancer region of the musclespecific creatine kinase gene (MCK). The sequences to which MyoDl binds are required for maximal muscle-specific expression of reporter genes under the control of the MCK promoter (3, 4). To isolate the ovine equivalent of MyoDl, a mouse MyoDl cDNA clone (1) was used to screen a cDNA library derived from sheep skeletal muscle and constructed in XgtlO. A cDNA clone of 1554 bp was isolated and sequenced by the dideoxy method (5). Comparison of the inferred amino acid sequence with that of the mouse MyoDl indicated that the clone lacked 125 bp of coding sequence at the 5' terminus. The missing nucleotide data as well as 325 bp of upstream sequence was obtained from a sheep genomic clone isolated from a XEMBL3 library. The sheep genomic clone contains no evidence of any introns and is 51% homologous to the 5' untranslated region of mouse MyoDl. Homology between the mouse and sheep 3' untranslated regions is 62%. The sheep MyoDl protein exhibits 90% amino acid identity to that of the mouse. An additional alanine is present in the sheep MyoDl at amino acid position 174. The area of greatest conservation covers the BHLH motif located between amino acids 101-164 (Figure 1).
EMBL accession no. X62102
ACKNOWLEDGEMENTS We thank H.Weintraub for the full length mouse MyoDl cDNA clone. This work was supported by a grant from the New Zealand Ministry of Agriculture and Fisheries (MAF).
REFERENCES 1. Davis,R.L., Weintraub,H. and Lassar,A.B. (1987) Cell 51, 987-1000. 2. Davis,R.L., Cheng,P.-F., Lassar,A.B. and Weintraub,H. (1990) Cell 60, 773-746. 3. Weintraub,H., Tapscott,S.J., Davis,R.L., Thayer,M.J., Adam,M.K., Lassar,A.B. and Miller,A.D. (1989) Proc. NatL. Acad. Sci. USA 86, 5434-5438. 4. Lassar,A.B., Buskin,J.N., Lockshon,D., Davis,R.L., Apone,S., Hauschka,S.D. and Weintraub,H. (1989) Cell 58, 823-831. 5. Sanger,F., Nicklen,S. and Coulson,A.R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5476. 6. Krause,M., Fire,A., White Harrison,S., Priess,J. and Weintraub,H. (1990) Cell 63, 907-919. 7. Michelson,A.M., Abmayr,S.M., Bate,M., Martinez-Arias,A. and Maniatis,T. (1990) Genes Dev. in press. 8. Hopwood,N.D., Pluck,A. and Gurdon,J.B. (1989) EMBOJ. 8, 3409-3417. 9. Lin,Z., Deschesne,C.A., Eldridge,J. and Patterson,B.M. (1989) Genes Dev. 3, 986-996. Sheep Mouse C .legans
Drosophila Xenopua Chicken
KRKTTNADRRKAATMRERRR KRKTTNADRRKAATMRERRR GPRATKLDRRKMATAZRRR KKKSVTVDRRJAATNRZRRR KRKTTNADRRKAaTMSBRRR KRKTTNADRRXAATMRRRR
bode
LSKVNZAFETLKRCT LSKVNZAFITLKRCT LRXVMRArZVVKQRT LRIVNEAflILKRRT LSKVNWEATLKRYT LSKVNEAFETLKRCT
heizi
SSNPNQRLP SSNPNQRLP CPNPNQRLP SSNPNQRLP SSNPNQRLP STNPKQRLP
h.p
KVEILRNAIRYIEGLQAL
KVSILRUAIRYIEGLOAL KVIILRSAIDYINNLSRM KVEILRMAIEYIESLEDL KVKILRMAIRYISSLQAL KVEILRNAIRYIEELQAL
head
Figure 1. Interspecies comparison of the MyoDl BHLH domains. Protein sequences were obtained from the following sources: mouse (1); C. elegans (6); Drosophila (7); Xenopus (8); chicken (9).
