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'Beautiful Buttocks' Pinned Down

15 September 2002: Named from the Greek words for 'beautiful buttocks', the callipyge mutation results in sheep with unusually big and muscular bottoms. Getting to the bottom of the gene which is responsible for the phenotype has proved difficult as the callipyge phenotype is an imprinted trait which is expressed in a unique parent-of-origin dependent manner known as paternal polar overdominance. To date, the callipyge gene is the only known example of this mode of inheritance in mammals.

The callipyge gene, was found in a flock of sheep in Oklahoma in the early 1980s. Some of these sheep had large muscling in their loins and legs. Research revealed that the only sheep with the abnormal muscling were those which had inherited a normal copy of a specific gene from their mother, but a mutated copy of that same gene from their father. The callipyge gene could prove beneficial in breeding because it enables the sheep to convert food into muscle 30% more efficiently than normal sheep. The study of the gene could also provide insights into the processes that give rise to obesity and fat metabolism.

Although it has been known that the mutation was located on a small region near the telomere of sheep chromosome 18, tracking down the exact gene has proved complicated. However, by using a carefully developed DNA panel, researchers from the US Department of Agriculture and Duke University Medical Center have managed to finally pin down the mutation responsible for the callipyge phenotype. The team compared a specific DNA sequence from inbred offspring of the original callipyge sheep against the DNA of normal sheep in order to locate markers. While they found 600 distinct markers in the DNA panel, only one was unique to the callipyge sheep: a single base change from A to G in the DNA sequence. Further testing showed this mutation alone clearly gave rise to the sheep's muscular hypertrophy.

A surprising discovery was that the callipyge mutation appeared to reside in a 'gene desert' on the sheep chromosome where no other known genes have previously been mapped. When comparative mapping in mice and man was conducted, the researchers found that the DNA sequences surrounding the callipyge mutation were highly similar in all three species. As the gene had been conserved in all three species, it was suspected that the gene could have an important biological function. The researchers then tested whether the DNA in this conserved region was used as a template to make RNA within the callipyge sheeps' affected tissue. Surprisingly, they did find an RNA transcript or copy, suggesting that the mutation is located in a previously unidentified gene. According to Randy Jirtle, professor of radiation oncology at Duke and co-author of the study, this is the first time in animals where a mutation has been found that leads to the identification of a new gene, rather than analyzing a known gene to find its mutation. Jirtle commented, "As scientists, we are missing many genes and their mutations by using the traditional approach of linkage analysis to locate and analyze candidate genes."

The next step in the research is to determine how the callipyge gene gives rise to the big-bottomed phenotype. Researchers have long known that a nearby gene, called DLK-1, is over-expressed in the hind quarters of callipyge sheep but no mutations have been found in this gene. The researchers suspect that the mutated callipyge gene is inappropriately regulating the expression of DLK-1 and/or other imprinted genes in this domain. Jirtle said, "We believe the regulation of this imprinted domain is flawed, rather than other genes in this domain being mutated. Mutated callipyge is having a downstream effect on DLK-1, and potentially on other imprinted genes in this region, that in some manner stunts fat cells from maturing while enhancing hind quarter musculature." Previous studies of knock-out mice in which the DLK-1 gene was targeted revealed that mice null for the gene had retarded growth development and accelerated adiposity.

DLK-1 has been studied in other contexts because it is overexpressed in neuroendocrine tumors such as pheochromocytoma and neuroblastoma, and also is involved in the maturation of fat cells and the adrenal gland. DLK-1 is an imprinted gene which lends further credence to the assumption that callipyge is also imprinted. Jirtle commented, "Imprinted genes are like mushrooms, because they are present in groups. Moreover, they display a domino effect, in that one mutation of an imprinted gene could knock out five or 10 genes in one hit, especially when they are all regulated as a group. These are incredibly powerful and subtle genes that bring you into a whole different realm of thinking about gene regulation. In a way, we are at the end of the beginning. We know the specific gene mutation that leads to large bottoms in sheep, but now we have to find how it operates."

According to ARS geneticist Brad Freking of the Roman L. Hruska U.S. Meat Animal Research Center (MARC), USA, one of the research team, this discovery shows the value of obtaining genomic sequences of more agricultural species to align with the human genome in the search for novel genes. Alignment of genomic sequences from several species could help identify important genetic regions not previously recognized in the human genome. This is especially true for regions containing mutations in livestock that have a major impact on the animal, such as the callipyge mutation.

Further work will be conducted to determine the full length of the callipyge, or CLPG1, transcript. The authors of the paper describing the discovery of the mutation speculate that the mutation in the transcript could alter its function as an RNA effector molecule and thus regulate the expression of DLK-1 differently in animals heterozygous for the mutation on the paternal allele.

More information on the discovery of the nature of the callipyge mutation can be found in a paper published in October's issue of Genome Research. The paper, entitled "Identification of the Single Base Change Causing the Callipyge Muscle Hypertrophy Phenotype, the Only Known Example of Polar Overdominance in Mammals" is by Brad A. Freking, Susan K. Murphy, Andrew A. Wylie, Simon J. Rhodes, John W. Keele, Kreg A. Leymaster, Randy L. Jirtle and Timothy P.L. Smith.