Photo: Courtesy of Agricultural Research Service
Move over, Dolly. In the famous sheepstakes, Solid Gold (1983-1993) came first. Solid Gold is the first known sheep to have the callipyge condition--Greek for "beautiful buttocks"--and his descendants are shedding light on genomic imprinting, the difference in expression of a gene depending on which parent transmits it. In humans, derailed genomic imprinting causes cancer, autism, bipolar disorder, and other conditions.
In 1983, a lamb was born in Oklahoma that mysteriously developed a huge, firm derriere in the first weeks of life. Instead of shipping Solid Gold to market, the curious producer bred him to normal ewes. Sure enough, about 10% of the offspring began to pack pounds into their posteriors at three weeks of age. (It was not the expected 50% because, researchers later learned, Solid Gold was a mosaic.)
"Solid Gold was a dream come true, a fluke. His mother was old, and she was only able to have one more lamb, and it wasn't callipyge. The producer was astute enough to notice the lamb," recalls Noelle Cockett, dean of the College of Agriculture at Utah State University in Logan.
The unusual ram and his dynasty shone in the show ring. One admirer was Sam P. Jackson, today an associate professor in the Department of Animal Science and Food Technology at Texas Tech University in Lubbock. In 1989, Jackson was just about to start graduate school at Texas Tech, when he met callipyge. "I was seeing these sheep at lamb shows. I asked questions, but no one knew the answers, they just said it was heavily muscled. How did it get that way? How is it inherited? So I went to my adviser and said, 'This looks to me like a PhD project,'" he recalls.
Adviser Ron Green agreed. So he called former graduate student Cockett, whose lab could handle DNA analyses. Michel Georges, then at a Salt Lake City company called Genmark, joined the effort to investigate "dressing percentage"--the proportion of body weight that goes into a saleable carcass. Cockett and Georges named the animal callipyge (pronounced cal-e-peej).
CURIOUS INHERITANCE PATTERN Jackson methodically mated many sheep to follow the inheritance pattern.1 Solid Gold's condition had affected lambs of both sexes, which suggested simple autosomal dominant inheritance. But unlike Mendel's peas, it was not so easy to predict the percentage of offspring that would have giant rears.
"Only sheep that inherit a mutated copy of the callipyge gene from their father and a normal allele from their mother have large bottoms," explains Randy Jirtle, a professor of radiation oncology at Duke University, whose team recently identified the mutation.2 Sheep producers were confused, too; some crosses that were expected to perpetuate the trait were not doing so.
"So we started to listen. Sam crossed callipyge females and callipyge males, and didn't get callipyge offspring," recalls Cockett. The only way to yield all callipyge lambs was to cross callipyge males to females who had two copies of the normal gene variant (allele). That is, a lamb getting the callipyge allele from both parents would, paradoxically, not inherit the rounded rear. So not only is the gene imprinted--it must be inherited from the father--but the mother must provide a normal allele. Cockett, who apparently has a flair for names, coined the term "polar overdominance" to describe this ability of the female genotype to override the paternal imprint.3 "The more it played out, the more bizarre it got," adds Jackson.
Georges, a professor of genetics at the University of Liege in Brussels, Belgium, hypothesizes that the dam's normal allele shuts off synthesis of at least four proteins that form muscle tissue.4 "The mutant allele from the mother blocks expression of certain genes. We call this a posttranslational effect," he says.
Photo: Courtesy of Randy Jirtle, Duke University Medical Center
FINDING THE "GENE" Sheep are not the easiest organisms to dissect genetically, as the markers needed to match gene to chromosome are few and far between. So Cockett borrowed from the bovine, a close relative. On the fourth cow sequence, she hit a bull's-eye: The homologous sequence in sheep tracked only with the endowed individuals. That marker was on cow chromosome 21, the counterpart of sheep chromosome 18. Nine more cow markers pinpointed the part of the sheep chromosome that harbors muscle genes.5
The researchers closed in on the genes, with help from the human genome sequence. "Once we compared human and sheep, suddenly highly conserved regions popped out. Then we identified transcripts for seven genes that are all affected by the callipyge mutation with increased expression," relates Cockett. This molecular evidence supported observations of other effects. For example, the animals have less wool, presumably because they divert more energy into making muscle proteins rather than keratin. "It was very reasonable to suppose the mutation was in a regulatory region, rather than in a single gene, because the mutation alters expression of a lot of genes," Cockett adds. Imprinted genes are expressed in this multiple manner.
The search continued. "It was narrowed down to 3 million bases, then 400,000, and finally 250,000 about two years ago, by Brad Freking at the [US Department of Agriculture] in Clay Center, Nebraska," says Jirtle. For a time interest focused on a sheep gene called DLK1 that controls differentiation of adipose cells. Jirtle's colleagues at Duke, Susan Murphy and Andrew Wylie, entered the picture when they found that the human version of DLK1 is imprinted, expressed only from the paternal allele.6 But none of the muscle-related sheep genes in the imprinted domain, including DLK1, had a mutation. How did callipyge and normal animals differ at the DNA level?
"So we did brute force sequencing of the whole thing in inbred sheep. Of the 600 polymorphisms identified, only one tracked absolutely with the affected sheep, a single base change of A to G," Jirtle says. The mutation lies within a gene desert, a genomic neighborhood that might once have been dismissed as "junk." The researchers propose that this single-base substitution affects a DNA sequence that either directly or through a corresponding RNA transcript controls the suite of nearby imprinted genes. "This shows that junk DNA can contain genes that regulate others. Knock out one, and you can knock out or alter the expression of others," Jirtle adds.
TRY A LITTLE TENDERNESS While geneticists unravel the controls of callipyge, other researchers are probing anatomy and physiology. A callipyge lamb has small organs, but weighs more, because muscle replaces 30% of the fat. Muscle in the hindquarters grows by increasing cell size, rather than cell number, at the expense of fat under the skin, in and between muscles, and hugging the kidneys. Only certain muscles are affected, and within them, only the fast-twitch fibers. The huge rear that producers at first thought would add value has instead proven a liability. A tradeoff for bulkier callipyge lamb chops is tougher meat.
Roberto Sainz, an associate professor of animal science at the University of California, Davis, explains the nuances of meat quality: "Tenderness depends on both the connective tissue and the myofibrillar component of muscle. Connective tissue is inherently tough. The myofibrillar component consists of the contractile apparatus of the muscle, and it is pretty tough, too."
What callipyge lambs lack is fat. "People want tasty meat, and that means fat. Angus beef is tender because it is marbled with fat. If bulk is increased with muscle, the meat is dry and tough. Slaughterhouses will not presently take callipyge lamb," says Jirtle. But Cockett defends her flock. "In some breeds, callipyge has tough meat, but our lambs aren't tough. Callipyge got a bad rap at the beginning." But the meat can sometimes be manipulated into tastiness. Reports in the meat-science literature detail ways to tenderize callipyge, including marinating, wetting, drying, aging, hanging, and pressing.
Contributing to the toughness is sluggish protein degradation. "After death, proteases break down the myofibrillar structure, tenderizing the meat. This does not work in callipyge sheep," says Sainz. The animals make too much calpastatin, which inhibits the protease calpain. "The increase in calpastatin activity inhibits calpain from degrading myofibrillar proteins, and the meat never becomes tender," explains Steven D. Shackelford, a research food technologist with the US Meat Animal Research Center in Clay Center, Nebraska.
Jirtle hypothesizes a more basic imbalance harkening back to the one-time front-runner candidate gene. "Normally DLK1 expression decreases after birth, but in callipyge it stays up. This could decrease differentiation of adipocytes, transferring energy to muscle, which bulks up."
RAM-IFICATIONS AND EWE-SES The bottom-heavy lambs may turn out to do more for basic biology than for the dinner plate. "Studying callipyge will be incredible for our understanding of imprinting. The mutation allows you to follow imprinting and the animal lives; it just has a big rear," says Cockett, noting that imprinting was first described in doomed fertilized human ova that had two maternal or two paternal genomes.
Jirtle extends observations on callipyge to humans. "Imprinting plays a very important role in cloning and in the etiology of cancer and behavioral disorders. An epigenetic gene regulatory system that evolved over 150 million years ago to create parent-of-origin dependent, monoallelically expressed genes has placed all mammals at an enhanced risk of developing a number of genetic diseases."
The callipyge saga also illustrates how finding a mutation is not an endpoint, but a source of new questions. Says Jackson, "Part of the riddle is that these sheep are born normal, and something triggers those genes. The on/off switch might be the most significant finding about these sheep. Perhaps we can use it for other genes, or in other species."
Green fluorescent lambchops, anyone?
Ricki Lewis (email@example.com) is a contributing editor.
1. S P. Jackson, R.D. Green, "Muscle trait inheritance, growth performance and feed efficiency of sheep exhibiting a muscle hypertrophy phenotype," Journal of Animal Science, 71:241, 1993.
2. B.A. Freking et al., "Identification of the single base change causing the callipyge muscle hypertrophy phenotype, the only known example of polar overdominance in mammals," Genome Research, 12:1-11, October 2002.
3. N.E. Cockett et al., "Polar overdominance at the ovine callipyge locus," Science, 273:236-8, 1996.
4. C. Charlier et al., "The callipyge mutation enhances the expression of coregulated imprinted genes in cis without affecting their imprinting status," Nature Genetics, 27:367-369, 2001.
5. N.E. Cockett et al., "Chromosomal localization of the callipyge gene in sheep (Ovis aries) using bovine DNA markers," Proceedings of the National Academy of Sciences, 91:3019-23, 1994.
6. A. Wylie et al., "Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation," Genome Research, 10:1711-18, 2000.