Progress 12/15/01 to 06/30/04
Outputs
The callipyge gene is a mutation in sheep responsible for pronounced muscle hypertrophy of the fast twitch muscle fibers, primarily in muscles of the pelvic limb. Genetic characterization of the locus has demonstrated a unique mode of inheritance termed polar overdominance, in which only heterozygous offspring inheriting the mutation from their sire express the phenotype. The three other genotypes are normal in appearance. In earlier projects, we localized callipyge to the distal end of ovine chromosome 18, based on linkage to markers TGLA122, CSSM18 and GMBT16. The position of callipyge was then more precisely localized within a 450 Kb chromosome segment after isolation of additional microsatellites, and 250 Kb sequence of this region was obtained. Six genes were predicted to reside within the analyzed segment. Two of these genes, DLK1 and GTL2, have been previously reported in humans, mice and cattle, while the remaining genes, DAT, PEG11, antiPEG11, and MEG8, were
previously unreported. Using primers designed from each of these genes, RT-PCR was performed on several tissues sampled from an 8-week-old lamb, demonstrating that all six transcripts are preferentially expressed in skeletal muscle. Because the corresponding regions in human and mice contain genetically imprinted loci, the imprinting status of these six genes was investigated using single nucleotide polymorphisms (SNPs). These results demonstrated that DLK1, DAT and PEG11 are paternally expressed, while GTL2, antiPEG11, and MEG8 are maternally expressed. In a subsequent study (13), poly A+ RNA was isolated from the longissimus dorsi of 8-week-old animals of the four callipyge genotypes. Northern blotting and/or RT-PCR were used to examine expression of DLK1, GTL2, PEG11, and MEG8 with G3PDH expression serving as a control for similar amounts of mRNA. These experiments demonstrated that the callipyge mutation does not alter the imprinting status of the four genes, but dramatically
enhances their expression level in cis. The paternally expressed genes (DLK1 and PEG11) had increased expression in the C/C and N/C genotypes (where the first and second alleles in the genotype refer to the maternal and paternal chromosomes, respectively), those genotypes that have the paternally-expressed callipyge chromosome. For the maternally expressed genes (GTL2 and MEG8), increased expression occurred in the C/C and C/N genotypes, which have the callipyge mutation on their maternal chromosome. Thus, the callipyge mutation appears to modify the activity of a common regulatory element, which could be either an enhancer (boosted by the callipyge mutation) or a silencer (inhibited by the callipyge mutation). This altered regulatory element may be responsible for the enhanced expression of DLK1, GTL2, PEG11, and MEG8, and possibly other untested genes as well. It is likely that increased expression of one or more of these genes initiates the manifestation of the callipyge
phenotype.
Impacts
Callipyge is a genetic mutation that causes significant muscle hypertrophy in sheep. We have studied several genes that are found in the same chromosomal region as the mutation and found that an increased expression of the genes occurs when they are located on the same parentally inherited chromosome as the mutation (i.e. cis effects of the mutation). There is also evidence that there is increased expression for some of the genes when the callipyge mutation is present in the genotype, but not inherited on the same chromosome (i.e. trans effects).
Publications
- Bidwell, C.A., L.N. Kramer, T.S. Hadfield, D.E. Moody C. Charlier, M. Georges and N.E. Cockett. 2004. Expression of PEG11 and AntiPEG11 transcripts in normal and callipyge sheep. Keystone Symposia: Emerging Mechanisms of Epigenetic Regulation.
- Bidwell, C. A., L. N. Kramer, A. C. Perkins, T. S. Hadfield, D. E. Moody and N. E. Cockett. 2004. Expression of PEG11 and PEG11AS transcripts in normal and callipyge sheep. BMC Biology, 2:17 DOI:10.1186/1741-7007-2-17.
- Cockett, N. E. 2003. Continued characterization of the callipyge mutation in sheep. Proc., Horizons in Livestock Sciences, Gold Coast, Queensland, Australia, 15.
- Davis, E., C. H. Jensen, H. D. Schroder, T. Hadfield, A. Kiem, N. Cockett, M. Georges and C. Charlier. 2004. Ectopic expression of DLK1 protein in skeletal muscle of padumnal heterozygotes causes the callipyge phenotype. Current Biology, 14:1858-1862.
- Perkins, A. C., L. N. Kramer, D. E. Moody, T. S. Hadfiled, S. Eng, N. E. Cockett and C. A. Bidwell. 2004. Developmental expression of paternally expressed genes from the callipyge locus of sheep chromosome 18. Proc., 29th International Conference on Animal Genetics, p. 73.
- Smit, M., F. Baraldi, E. Davis, X. Tordoir, T. Hadfield, G. Gyapay, N. Cockett, M. Georges and C. Charlier. 2004. Extending the boundaries of the callipyge imprinted gene cluster on ovine chromosome 18. Proc., Plant and Animal Genome XII, p. 233.
- Takeda, H., X. Tordoir, M. Smit, N. Cockett, M. Georges and C. Charlier. 2004. Dnase I hypersensitive sites around the ovine callipyge mutation. Proc., 29th International Conference on Animal Genetics, p. 73.
- M. Smit. 2004. Long range transcriptional regulation of the ovine callipyge imprinted gene cluster. PhD dissertation, Utah State University.
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Progress 01/01/03 to 12/31/03
Outputs
The callipyge phenotype in sheep is an inherited muscular hypertrophy affecting only heterozygous individuals receiving the CLPG mutation from their sire. This unique mode of inheritance is referred to as polar overdominance. In order to identify the callipyge mutation, we have sequenced 184 Kb spanning the DLK1, GTL2, PEG11 and MEG8 imprinted domain and have identified an A to G transition in a highly conserved dodecamer motif between DLK1 and GTL2. This was the only difference found between the callipyge (CLPG) allele and a phylogenetically closely related wild-type allele. This SNP is in perfect association with the callipyge genotype. The demonstration that "Solid Gold" - the alleged founder ram of the callipyge flock - is mosaic for this SNP virtually proves the causality of this SNP in the determinism of the callipyge phenotype. In addition, recent results suggests that polar overdominance of the callipyge locus results from the combined (i) cis-effect of the
CLPG mutation on the expression levels of genes in the DLK1-GTL2 imprinted domain and (ii) a trans-interaction between the products of reciprocally imprinted genes
Impacts
Callipyge is a major gene responsible for a pronounced muscle hypertrophy in sheep. We have initiated a multi-faceted approach for the identification and characterization of the causative gene in callipyge.
Publications
- Cockett, N. E., M. Smit, K. Sergers, T. L. Hadfield, L. Karim, G. D. Snowder, M. Georges and C. Charlier (2003) The callipyge mutation and other genes that affect muscle hypertrophy in sheep. Proc., International Workshop on Major Genes and QTL in Sheep and Goat, Toulouse, France communication 2-23.
- Georges, M., C. Charlier and N. E. Cockett (2003) Polar overdominance at the ovine callipyge locus supports trans interaction between the products of reciprocally imprinted genes. Trends in Genetics 19:248-252.
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Progress 01/01/02 to 12/31/02
Outputs
The callipyge phenotype in sheep is an inherited muscular hypertrophy affecting only heterozygous individuals receiving the CLPG mutation from their sire. This unique mode of inheritance is referred to as polar overdominance. In order to identify the callipyge mutation, we have sequenced 184 Kb spanning the DLK1, GTL2, PEG11 and MEG8 imprinted domain and have identified an A to G transition in a highly conserved dodecamer motif between DLK1 and GTL2. This was the only difference found between the callipyge (CLPG) allele and a phylogenetically closely related wild-type allele. This SNP is in perfect association with the callipyge genotype. The demonstration that "Solid Gold" - the alleged founder ram of the callipyge flock - is mosaic for this SNP virtually proves the causality of this SNP in the determinism of the callipyge phenotype. In addition, recent results suggests that polar overdominance of the callipyge locus results from the combined (i) cis-effect of the
CLPG mutation on the expression levels of genes in the DLK1-GTL2 imprinted domain and (ii) a trans-interaction between the products of reciprocally imprinted genes.
Impacts
Callipyge is a major gene responsible for a pronounced muscle hypertrophy in sheep. We have initiated a multi-faceted approach for the identification and characterization of the causative gene in callipyge.
Publications
- Di Silvestro, F. , C. Charlier, S. Caburet, A. Benesimon, N. Cockett and M. Georges (2002) Monitoring the physical integrity of the callipyge locus by molecular combing. Proc., 27th International Conference on Animal Genetics p. 42.
- Smit, M. A., C. Charlier, N. E. Cockett, B. Rodriguez, T. L. Shay and M. Georges (2002) Expression and imprinting status of genes affected by the callipyge mutation in cultured ovine skeletal muscle satellite cells. Proc., 27th International Conference on Animal Genetics p. 39.
- Smit, M., K. Segers, et al. (2002) Mosaicism of Solid Gold supports the causality of the SNP in determinism of the callipyge phenotype. Genetics (accepted).
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