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| The Legacy of Solid Gold | |||||||||
| Comparing human and sheep genomic sequences reveals six imprinted genes at the callipyge locus | |||||||||
| By Edward R. Winstead May 7, 2001 
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The mutation appeared to be a miracle. Sheep breeders in Oklahoma had been selecting for increased muscle in their flock, and in 1983 a lamb was born that developed an overly muscular rump. The breeders named the ram Solid Gold and saved him from slaughter. He went on to sire offspring with the pronounced hindquarters, some of which also passed on the mutation. These sheep became known as callipyge, from the Greek for 'beautiful buttocks.'
This week researchers report the shotgun sequencing and annotation of the callipyge region on sheep chromosome 18. The region contains at least six sheep genes, including four novel sequences. Michel Georges, of the Faculty of Veterinary Medicine at the University of Liege, Belgium, and colleagues used comparative genomics to characterize the region: The raw sheep sequence was analyzed next to human chromosome 14, which contains human versions of sheep genes previously mapped to the callipyge locus. "It wasn't until the sequence was determined in sheep and then aligned with the human sequence that the four additional genes were pulled out," says Noelle Cockett, of Utah State University and an author of the study. The sequencing was done at Genoscope, Centre National de Séquençage, in Evry, France, and the results appear in Genome Research. The six sheep genes are expressed in skeletal tissue. "This was important to demonstrate because of the phenotype we are studying," says Georges. His team analyzed tissues from an 8-week-old lamb; signs of callipyge can be detected during the first month of life.
It became clear to breeders in the mid-1980s that callipyge genetics violates just about every known law. Breeders who mated callipyge descendents of Solid Gold got many fewer callipyge animals than they expected. This made no sense to anyone, including scientists. In 1996, a team led by Cockett and Georges explained what was going on: Only lambs that inherit the callipyge mutation from their father but not their mother develop the trait. All other combinations of normal and callipyge chromosomes result in normal sheep. The researchers named the effect polar overdominance. The identity of the callipyge mutation still is not known, but a single mutated gene is no longer a plausible explanation for the dramatic effect. Blaming one gene would be straightforward, and nothing about callipyge is straightforward. "We used to talk about a callipyge gene being responsible for the phenotype, but I've certainly backed away from that," says Cockett. "We now think there's a mutation in a regulatory region that's affecting the expression of these six genes." The callipyge mutation appears to fall in a DNA sequence governing the activity of multiple genes, the researchers found. "Without knowing the identity of the mutation, we were able to show that the callipyge mutation has effects on the expression of a whole series of genes," says Georges. The findings were published last month in Nature Genetics. At the University of Liege, Carole Charlier led an analysis of gene expression for four genes in the callipyge region in normal and callipyge sheep. The expression of genes differed among the four genotypes, and gene expression was influenced by two factors: 1) whether a chromosome was normal or callipyge and 2) whether that chromosome was inherited from the mother or the father.
All six genes in the callipyge region are imprinted, meaning that each is turned on or off in part depending on whether it was inherited from the mother and the father. Of the six, three genes are primarily expressed from the paternal chromosome; the other three are expressed from the maternal chromosome. An overall effect of differential gene expression in the region may be what produces the callipyge phenotype. Genomic imprinting certainly plays a role; the influence of a single regulatory element on groups of genes is associated with genomic imprinting in other species. "Here we have another cluster of imprinted genes that looks similar to well-characterized imprinted domains in humans and mice," says Anne Ferguson-Smith, of the University of Cambridge and an expert on imprinting in mice. She speculates that the callipyge mutation is affecting some sort of 'silencer' element that would normally shut down the expression of certain genes. The mutation allows the genes to stay on inappropriately, according to this theory. "The sheep aren't born with big muscles everywhere so it's not a problem in development," she notes. When imprinted genes in mice stay on too long, the result can be too much muscle throughout the body. Similarly, human growth disorders are associated with the abnormal expression of imprinted genes. Ferguson-Smith's laboratory is screening genomic data for humans, mice and sheep to identify evolutionary features of imprinting. The mechanisms, such as the 'imprint mark,' are likely to be the same across species. "The sheep sequences are facilitating genomic comparisons both between species and within species," she says.
Researchers around the world are investigating imprinting through comparative genomics. The laboratory of Randy L. Jirtle at Duke University Medical Center in Durham, North Carolina, is studying a range of non-human species, including marsupials and hedgehogs, as well as humans. "It will be a high priority for us to line up the human and mouse sequences once mouse comes along," says Andrew Wylie, a member of the Jirtle laboratory. He adds that the work is challenging because it requires the full genomic sequence of an imprinted domain: "An individual gene sequence does not always provide much information about the regulation of the gene." Whatever similarities there are among species, polar overdominance makes callipyge unique. The fact that two copies of the callipyge mutation are harmless strikes some researchers as bizarre. "This is very strange and has always been the most exciting part of the story to me," says Ferguson-Smith, who collaborates with the Belgian group. "It means we will learn new things about how this might operate and about the regulation of imprinted domains." The new study is the latest on the imprinted genes DLK1 and GLT2, which are present in humans, mice and sheep. "Now, we show that there are additional genes in the callipyge region and that they are all subject to imprinting," says Michel Georges. Two of the new genes do not code for proteins, which is true for genes in other imprinted domains. The four novel genes are called DAT, PEG11, antiPEG11, and MEG8. "To understand what is happening in callipyge," says Ferguson-Smith, "we will need to characterize the mutation and understand more about the function of the imprinted genes, particularly DLK1 and GLT2." The French team sequenced DNA from normal rather than callipyge sheep. The plan is to sequence a callipyge chromosome from Solid Gold's descendents in the coming months. "Lining up the normal and callipyge sequences should reveal the callipyge mutation," says Noelle Cockett. "We will confirm the identity of the mutation using Solid Gold's DNA."
"Callipyge is really quite fascinating," says Ferguson-Smith. "In a way, it's a shame this happened in sheep, because there are limits to the experiments one can do." Sheep breed only once per year, and flocks are small. Most studies involve relatively few animals compared to what is possible in mice. The callipyge mutation, whatever it is, may ultimately be more interesting to scientists than the industry. Sheep producers have been cautious about using callipyge genetics because of concerns about the less-than-tender loin chops of the callipyge animal. The loin can be tenderized, but lamb packers have not adopted the practice. In the industry today, callipyge sheep are more of a curiosity than a miracle. . . .
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