Background The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.
Background
The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.Results There was a significant effect of genotype (p < 0.0001) on the transcript abundance of  DLK1, PEG11 , and  MEG8  in the muscles of lambs with the callipyge allele.  DLK1  and  PEG11  transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The  PEG11  locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as  PEG11AS , in skeletal muscle.  PEG11AS  transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the  PEG11  start codon and spanning the entire open reading frame. Analysis of  PEG11  expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals.  PEG11  transcripts were 14-fold more abundant than  PEG11AS  transcripts in the gluteus medius of paternal heterozygous animals.  PEG11AS  transcripts were expressed at higher levels than  PEG11  transcripts in the gluteus medius of animals of the other three genotypes.
Results
There was a significant effect of genotype (p < 0.0001) on the transcript abundance of  DLK1, PEG11 , and  MEG8  in the muscles of lambs with the callipyge allele.  DLK1  and  PEG11  transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The  PEG11  locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as  PEG11AS , in skeletal muscle.  PEG11AS  transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the  PEG11  start codon and spanning the entire open reading frame. Analysis of  PEG11  expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals.  PEG11  transcripts were 14-fold more abundant than  PEG11AS  transcripts in the gluteus medius of paternal heterozygous animals.  PEG11AS  transcripts were expressed at higher levels than  PEG11  transcripts in the gluteus medius of animals of the other three genotypes.DLK1, PEG11MEG8DLK1PEG11PEG11PEG11ASPEG11ASPEG11PEG11PEG11PEG11ASPEG11ASPEG11Conclusions The effect of the callipyge mutation has been to alter the expression of  DLK1 ,  GTL2 ,  PEG11  and  MEG8  in the hypertrophied skeletal muscles. Transcript abundance of  DLK1  and  PEG11  was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of  PEG11  and  PEG11AS  transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in  PEG11  gene regulation and polar overdominance in callipyge sheep.
Conclusions
The effect of the callipyge mutation has been to alter the expression of  DLK1 ,  GTL2 ,  PEG11  and  MEG8  in the hypertrophied skeletal muscles. Transcript abundance of  DLK1  and  PEG11  was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of  PEG11  and  PEG11AS  transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in  PEG11  gene regulation and polar overdominance in callipyge sheep.DLK1GTL2PEG11MEG8DLK1PEG11PEG11PEG11ASPEG11

Background The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 [ 1 - 3 ]. The callipyge phenotype is associated with an altered carcass composition including a 30–40% increase in muscle mass, a 6–7% decrease in carcass fat, decreased organ weights, and a more compact skeleton, all without a net affect on animal growth [ 4 - 7 ]. There is a pronounced hypertrophy of muscles in the loin and pelvic limbs and a lesser degree of hypertrophy in muscles of the thoracic limbs [ 6 - 8 ]. The callipyge phenotype is inherited in a non-Mendelian mode termed polar overdominance [ 9 , 10 ] in which only animals that inherit a normal allele (wild type; +) from the dam and the mutant callipyge allele from the sire ( CLPG Pat ) exhibit the callipyge phenotype. Maternal heterozygotes ( CLPG Mat /+ Pat ) and callipyge allele homozygotes ( CLPG Mat / CLPG Pat ) have muscling and carcass compositions that are similar to normal sheep (wild type homozygotes; +/+). A physical contig spanning the region containing the callipyge mutation was constructed using overlapping ovine bacterial artificial chromosomes [ 11 ] and 215 kb of sequence was obtained from the contig [ 3 ]. Comparisons of the ovine sequence to the human genome sequence and to expressed sequence databases indicated the presence of at least six transcribed genes with allele-specific expression [ 3 ] (Figure  1 ). The gene order along the contig was found to be  Delta-like 1  ( DLK1 ),  DLK associated transcript  ( DAT ), g ene-trap locus 2  ( GTL2 ),  paternal expressed gene 11  ( PEG11  /  PEG11AS ) and  maternal expressed gene 8  ( MEG8 ). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 [ 12 - 15 ]. The  DLK1  locus (also referred to as  PREF-1 ,  Zog-1  and  pG2 ) encodes a transmembrane protein that contains epidermal-growth factor repeats [ 16 - 19 ]. Cleavage of the extracellular domain of  DLK1  produces the circulating protein fetal antigen-1 [ 20 ].  DAT  is a short non-coding RNA that has been proposed to be a cleavage product of extended  DLK1  transcripts [ 21 ]. Both  DLK1 and  DAT  are expressed from the paternal allele [ 3 , 13 - 15 ].  GTL2  (also referred to as  MEG3 ) and  MEG8  genes express non-coding RNA from the maternal allele [ 3 , 13 - 15 ]. The  PEG11  gene contains an intronless open reading frame of 1333 amino acids in sheep [ 3 ]. The human and mouse orthologues known as  retrotransposon-like 1  ( RTL1 / rtl1 ) encode 1358 and 1745 amino acids, respectively. RNA transcripts were detected from the opposite strand of the same gene, referred to as  PEG11AS  (formerly known as  antiPEG11 ) [ 3 ]. In the mouse, two maternally expressed microRNAs have been identified with perfect complementarity to mouse  Rtl1  [ 22 ]. The causative mutation for callipyge is a single base transition of A (wild type; +) to G ( CLPG ) in the intergenic region located between  DLK1  and  GTL2  [ 23 ] (Figure  1 ). This mutation has been shown to be 100% concordant with all animals of the + Mat / CLPG Pat  genotype based on haplotype analysis [ 23 ]. Analysis of sheep from 19 different breeds as well as 13 mammalian species revealed a highly conserved 12 base sequence that includes the single nucleotide polymorphism [ 24 ]. The G polymorphism is unique to direct descendents of the first known callipyge animal, a ram named "Solid Gold". This animal was mosaic for the mutation [ 24 ], providing strong evidence that this single nucleotide polymorphism is the causative mutation. Initial results indicate that the mutation alters the expression of several of the genes within the imprinted cluster [ 25 , 26 ] when they are inherited in  cis  without altering their parent-of-origin-specific expression [ 25 ]. In this study, we analyzed the expression of five genes within the callipyge cluster in the muscles of lambs of all four genotypes. Quantitative analysis of gene expression using a series of orthogonal contrasts showed that  DLK1 ,  PEG11  and  MEG8  exhibited a polar overdominant pattern of gene expression. The expression of  PEG11  and  PEG11AS  transcripts in the muscles of paternal heterozygous callipyge lambs (+ Mat / CLPG Pat ) was different from the other three genotypes. A sense/antisense interaction of  PEG11  and  PEG11AS , such as an RNA interference mechanism, would be consistent with a  trans  interaction between reciprocally imprinted genes that has been previously proposed as a mechanism for polar overdominance [ 27 , 28 ].
Background
The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 [ 1 - 3 ]. The callipyge phenotype is associated with an altered carcass composition including a 30–40% increase in muscle mass, a 6–7% decrease in carcass fat, decreased organ weights, and a more compact skeleton, all without a net affect on animal growth [ 4 - 7 ]. There is a pronounced hypertrophy of muscles in the loin and pelvic limbs and a lesser degree of hypertrophy in muscles of the thoracic limbs [ 6 - 8 ]. The callipyge phenotype is inherited in a non-Mendelian mode termed polar overdominance [ 9 , 10 ] in which only animals that inherit a normal allele (wild type; +) from the dam and the mutant callipyge allele from the sire ( CLPG Pat ) exhibit the callipyge phenotype. Maternal heterozygotes ( CLPG Mat /+ Pat ) and callipyge allele homozygotes ( CLPG Mat / CLPG Pat ) have muscling and carcass compositions that are similar to normal sheep (wild type homozygotes; +/+).CLPGPatCLPGMatPatCLPGMatCLPGPatA physical contig spanning the region containing the callipyge mutation was constructed using overlapping ovine bacterial artificial chromosomes [ 11 ] and 215 kb of sequence was obtained from the contig [ 3 ]. Comparisons of the ovine sequence to the human genome sequence and to expressed sequence databases indicated the presence of at least six transcribed genes with allele-specific expression [ 3 ] (Figure  1 ). The gene order along the contig was found to be  Delta-like 1  ( DLK1 ),  DLK associated transcript  ( DAT ), g ene-trap locus 2  ( GTL2 ),  paternal expressed gene 11  ( PEG11  /  PEG11AS ) and  maternal expressed gene 8  ( MEG8 ). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 [ 12 - 15 ]. The  DLK1  locus (also referred to as  PREF-1 ,  Zog-1  and  pG2 ) encodes a transmembrane protein that contains epidermal-growth factor repeats [ 16 - 19 ]. Cleavage of the extracellular domain of  DLK1  produces the circulating protein fetal antigen-1 [ 20 ].  DAT  is a short non-coding RNA that has been proposed to be a cleavage product of extended  DLK1  transcripts [ 21 ]. Both  DLK1 and  DAT  are expressed from the paternal allele [ 3 , 13 - 15 ].  GTL2  (also referred to as  MEG3 ) and  MEG8  genes express non-coding RNA from the maternal allele [ 3 , 13 - 15 ]. The  PEG11  gene contains an intronless open reading frame of 1333 amino acids in sheep [ 3 ]. The human and mouse orthologues known as  retrotransposon-like 1  ( RTL1 / rtl1 ) encode 1358 and 1745 amino acids, respectively. RNA transcripts were detected from the opposite strand of the same gene, referred to as  PEG11AS  (formerly known as  antiPEG11 ) [ 3 ]. In the mouse, two maternally expressed microRNAs have been identified with perfect complementarity to mouse  Rtl1  [ 22 ].Delta-like 1DLK1DLK associated transcriptDATene-trap locus 2GTL2paternal expressed gene 11PEG11PEG11ASmaternal expressed gene 8MEG8DLK1PREF-1Zog-1pG2DLK1DATDLK1DLK1DATGTL2MEG3MEG8PEG11retrotransposon-like 1RTL1rtl1PEG11ASantiPEG11Rtl1The causative mutation for callipyge is a single base transition of A (wild type; +) to G ( CLPG ) in the intergenic region located between  DLK1  and  GTL2  [ 23 ] (Figure  1 ). This mutation has been shown to be 100% concordant with all animals of the + Mat / CLPG Pat  genotype based on haplotype analysis [ 23 ]. Analysis of sheep from 19 different breeds as well as 13 mammalian species revealed a highly conserved 12 base sequence that includes the single nucleotide polymorphism [ 24 ]. The G polymorphism is unique to direct descendents of the first known callipyge animal, a ram named "Solid Gold". This animal was mosaic for the mutation [ 24 ], providing strong evidence that this single nucleotide polymorphism is the causative mutation. Initial results indicate that the mutation alters the expression of several of the genes within the imprinted cluster [ 25 , 26 ] when they are inherited in  cis  without altering their parent-of-origin-specific expression [ 25 ].CLPGDLK1GTL2MatCLPGPatcisIn this study, we analyzed the expression of five genes within the callipyge cluster in the muscles of lambs of all four genotypes. Quantitative analysis of gene expression using a series of orthogonal contrasts showed that  DLK1 ,  PEG11  and  MEG8  exhibited a polar overdominant pattern of gene expression. The expression of  PEG11  and  PEG11AS  transcripts in the muscles of paternal heterozygous callipyge lambs (+ Mat / CLPG Pat ) was different from the other three genotypes. A sense/antisense interaction of  PEG11  and  PEG11AS , such as an RNA interference mechanism, would be consistent with a  trans  interaction between reciprocally imprinted genes that has been previously proposed as a mechanism for polar overdominance [ 27 , 28 ].DLK1PEG11MEG8PEG11PEG11ASMatCLPGPatPEG11PEG11AStransResults Northern blot analysis Muscle samples were collected from 12- and 8-week-old lambs, when muscle hypertrophy is well established in animals with the callipyge phenotype. Total RNA was extracted from three muscles that undergo hypertrophy including longissimus dorsi (loin), semimembranosus, and gluteus medius (pelvic limb), and one muscle that does not undergo hypertrophy, the supraspinatus, (thoracic limb). Strand specific probes were used to analyze  PEG11  and  PEG11AS  expression. Hybridization of longissimus dorsi, semimembranosus and gluteus medius northern blots with a  PEG11  probe indicated expression of a 6.5 kb  PEG11  transcript in paternal heterozygotes (+ Mat / CLPG Pat ) that was not readily detectable in the other three genotypes (Figure  2 ). Two smaller  PEG11AS  transcripts of 1.7 kb and 0.8 kb were detected in the two genotypes with maternally inherited callipyge alleles ( CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat ) by a probe from the complementary strand.  PEG11  and  PEG11AS  transcripts were not detected in the supraspinatus. The expression of  DLK1  was detectable at various levels in each of the four muscles and all four genotypes, although  DLK1  appeared to be up-regulated in the loin and pelvic limb muscles of + Mat / CLPG Pat  and  CLPG Mat / CLPG Pat  animals (Figure  2 ).  GTL2  transcripts of around 2.4 kb were evident in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes. A distinct 1.9 kb  GTL2  transcript was consistently detected in the + Mat / CLPG Pat  genotype and no  GTL2  transcripts were detected in the +/+ genotype. A 1.8 kb  MEG8  transcript was expressed to a lesser degree than  GTL2  in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes and was also detectable in the + Mat / CLPG Pat  genotype. The expression pattern of  MEG8  was the least consistent in the northern blot analysis of individuals of the four genotypes. PEG11/PEG11AS Ribonuclease protection assays were performed using five riboprobes to map the  PEG11  and  PEG11AS  transcripts to the contig sequence (Figure  3A ). The results for riboprobe P confirm the expression of  PEG11  transcripts in the gluteus medius of + Mat / CLPG Pat  animals and showed a very low level of  PEG11  in the gluteus medius of  CLPG Mat /CLPG Pat  animals.  PEG11AS  transcripts were readily detectable in animals of the +/+,  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes but were slightly above background levels in the + Mat / CLPG Pat  animals (Figure  3B ). Two other  PEG11  probes from the open reading frame (E) and from the 3'UTR (C) showed equivalent results to riboprobe P in the gluteus medius (Figure  3B ). The same expression pattern was seen for  PEG11  and  PEG11AS  transcripts in the supraspinatus, but signal from the protected riboprobes was just above background level. Two probes from upstream of the  PEG11  coding sequence (riboprobes F and G) did not detect any  PEG11  transcripts, indicating that transcription of the 6.5 kb  PEG11  was initiated within 350 bp of the start codon of the open reading frame. Riboprobe F detected  PEG11AS  transcripts in the gluteus medius and supraspinatus of all four genotypes, with the lowest expression in the paternal heterozygous (+ Mat / CLPG Pat ) animals. The  PEG11AS  riboprobe G shows three protected fragments, suggesting variable splice junctions or transcription termination sites for  PEG11AS  transcripts. Quantitative analysis of the effect of genotype on gene expression Gene expression was measured in the gluteus medius and supraspinatus of 8 week-old animals (Figure  4 ) using quantitative PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase was not significantly different across the four genotypes in the gluteus medius and supraspinatus (Table  1 ), indicating that equivalent amounts of RNA were used for cDNA synthesis and quantitative PCR. The effect of the callipyge mutation on genotype-specific expression of  DLK1  and  PEG11  in gluteus medius was the same (Table  1 ) although the magnitude of the response was greater for  PEG11  than  DLK1  (Figure  4A  and  4B ). The paternal heterozygous (+ Mat / CLPG Pat ) animals had the highest transcript abundance (p < 0.05), followed by  CLPG Mat / CLPG Pat  animals, which had significantly greater transcript abundance (p < 0.05) than  CLPG Mat /+ Pat  or +/+ animals. The mRNA abundance of  DLK1  in the gluteus medius was 6-fold and 2.5-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat /CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4A ).  PEG11  mRNA abundance in the gluteus medius was 200-fold and 13-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat / CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4B ). The effect of the callipyge mutation on  DLK1  and  PEG11  expression in the supraspinatus was different (Table  1 ). No differences in  DLK1  transcript abundance in the supraspinatus were found among the four genotypes (Figure  4C ). Although not statistically analyzed, the level of  DLK1  expression was similar between the gluteus medius and supraspinatus (Figure  4A  and  4C ).  PEG11  expression in the supraspinatus had a different genotype-specific pattern than in the gluteus medius. PEG11 expression was elevated 150-fold in + Mat / CLPG Pat  animals (p < 0.05) but was not significantly changed in the other three genotypes (Figure  4D ). The transcript abundance of  PEG11  in supraspinatus was generally much lower than in gluteus medius of the same genotype.  PEG11  expression in the supraspinatus was 12-fold lower than in the gluteus medius of paternal heterozygous animals (Figure  4B  and  4D ). Maternal inheritance of the callipyge allele significantly altered the expression of  MEG8  but not  PEG11AS  in the gluteus medius (Table  1 ). Transcript abundance of  MEG8  in gluteus medius was 6-fold greater (p < 0.05) in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  animals relative to the + Mat / CLPG Pat  and +/+ animals (Figure  4A ). Expression of  MEG8  in the supraspinatus was also affected by the callipyge mutation but to a lesser degree (Figure  4C ). The homozygous callipyge animals had significantly higher  MEG8  transcript abundance than paternal heterozygotes, but those two genotypes were not significantly different from maternal heterozygotes and homozygous wild type lambs. Orthogonal contrasts were used to analyze different models of gene action for the genes that had a significant effect for genotype (Table  1 ). The polar overdominance contrast was significant for transcript abundances of  DLK1 ,  PEG11  and  MEG8  in gluteus medius and for  PEG11  and  MEG8  in the supraspinatus. The additive contrasts were also significant for  DLK1 ,  PEG11  and  MEG8  in gluteus medius but were only significant for  PEG11  in supraspinatus. The maternal dominance contrast was significant for  DLK1  and  PEG11  gluteus medius.
Results
Northern blot analysis Muscle samples were collected from 12- and 8-week-old lambs, when muscle hypertrophy is well established in animals with the callipyge phenotype. Total RNA was extracted from three muscles that undergo hypertrophy including longissimus dorsi (loin), semimembranosus, and gluteus medius (pelvic limb), and one muscle that does not undergo hypertrophy, the supraspinatus, (thoracic limb). Strand specific probes were used to analyze  PEG11  and  PEG11AS  expression. Hybridization of longissimus dorsi, semimembranosus and gluteus medius northern blots with a  PEG11  probe indicated expression of a 6.5 kb  PEG11  transcript in paternal heterozygotes (+ Mat / CLPG Pat ) that was not readily detectable in the other three genotypes (Figure  2 ). Two smaller  PEG11AS  transcripts of 1.7 kb and 0.8 kb were detected in the two genotypes with maternally inherited callipyge alleles ( CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat ) by a probe from the complementary strand.  PEG11  and  PEG11AS  transcripts were not detected in the supraspinatus. The expression of  DLK1  was detectable at various levels in each of the four muscles and all four genotypes, although  DLK1  appeared to be up-regulated in the loin and pelvic limb muscles of + Mat / CLPG Pat  and  CLPG Mat / CLPG Pat  animals (Figure  2 ).  GTL2  transcripts of around 2.4 kb were evident in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes. A distinct 1.9 kb  GTL2  transcript was consistently detected in the + Mat / CLPG Pat  genotype and no  GTL2  transcripts were detected in the +/+ genotype. A 1.8 kb  MEG8  transcript was expressed to a lesser degree than  GTL2  in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes and was also detectable in the + Mat / CLPG Pat  genotype. The expression pattern of  MEG8  was the least consistent in the northern blot analysis of individuals of the four genotypes.
Northern blot analysis
Muscle samples were collected from 12- and 8-week-old lambs, when muscle hypertrophy is well established in animals with the callipyge phenotype. Total RNA was extracted from three muscles that undergo hypertrophy including longissimus dorsi (loin), semimembranosus, and gluteus medius (pelvic limb), and one muscle that does not undergo hypertrophy, the supraspinatus, (thoracic limb). Strand specific probes were used to analyze  PEG11  and  PEG11AS  expression. Hybridization of longissimus dorsi, semimembranosus and gluteus medius northern blots with a  PEG11  probe indicated expression of a 6.5 kb  PEG11  transcript in paternal heterozygotes (+ Mat / CLPG Pat ) that was not readily detectable in the other three genotypes (Figure  2 ). Two smaller  PEG11AS  transcripts of 1.7 kb and 0.8 kb were detected in the two genotypes with maternally inherited callipyge alleles ( CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat ) by a probe from the complementary strand.  PEG11  and  PEG11AS  transcripts were not detected in the supraspinatus. The expression of  DLK1  was detectable at various levels in each of the four muscles and all four genotypes, although  DLK1  appeared to be up-regulated in the loin and pelvic limb muscles of + Mat / CLPG Pat  and  CLPG Mat / CLPG Pat  animals (Figure  2 ).  GTL2  transcripts of around 2.4 kb were evident in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes. A distinct 1.9 kb  GTL2  transcript was consistently detected in the + Mat / CLPG Pat  genotype and no  GTL2  transcripts were detected in the +/+ genotype. A 1.8 kb  MEG8  transcript was expressed to a lesser degree than  GTL2  in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes and was also detectable in the + Mat / CLPG Pat  genotype. The expression pattern of  MEG8  was the least consistent in the northern blot analysis of individuals of the four genotypes.PEG11PEG11ASPEG11PEG11MatCLPGPatPEG11ASCLPGMatPatCLPGMatCLPGPatPEG11PEG11ASDLK1DLK1MatCLPGPatCLPGMatCLPGPatGTL2CLPGMatPatCLPGMatCLPGPatGTL2MatCLPGPatGTL2MEG8GTL2CLPGMatPatCLPGMatCLPGPatMatCLPGPatMEG8PEG11/PEG11AS Ribonuclease protection assays were performed using five riboprobes to map the  PEG11  and  PEG11AS  transcripts to the contig sequence (Figure  3A ). The results for riboprobe P confirm the expression of  PEG11  transcripts in the gluteus medius of + Mat / CLPG Pat  animals and showed a very low level of  PEG11  in the gluteus medius of  CLPG Mat /CLPG Pat  animals.  PEG11AS  transcripts were readily detectable in animals of the +/+,  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes but were slightly above background levels in the + Mat / CLPG Pat  animals (Figure  3B ). Two other  PEG11  probes from the open reading frame (E) and from the 3'UTR (C) showed equivalent results to riboprobe P in the gluteus medius (Figure  3B ). The same expression pattern was seen for  PEG11  and  PEG11AS  transcripts in the supraspinatus, but signal from the protected riboprobes was just above background level. Two probes from upstream of the  PEG11  coding sequence (riboprobes F and G) did not detect any  PEG11  transcripts, indicating that transcription of the 6.5 kb  PEG11  was initiated within 350 bp of the start codon of the open reading frame. Riboprobe F detected  PEG11AS  transcripts in the gluteus medius and supraspinatus of all four genotypes, with the lowest expression in the paternal heterozygous (+ Mat / CLPG Pat ) animals. The  PEG11AS  riboprobe G shows three protected fragments, suggesting variable splice junctions or transcription termination sites for  PEG11AS  transcripts.
PEG11/PEG11AS
PEG11/PEG11ASRibonuclease protection assays were performed using five riboprobes to map the  PEG11  and  PEG11AS  transcripts to the contig sequence (Figure  3A ). The results for riboprobe P confirm the expression of  PEG11  transcripts in the gluteus medius of + Mat / CLPG Pat  animals and showed a very low level of  PEG11  in the gluteus medius of  CLPG Mat /CLPG Pat  animals.  PEG11AS  transcripts were readily detectable in animals of the +/+,  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  genotypes but were slightly above background levels in the + Mat / CLPG Pat  animals (Figure  3B ). Two other  PEG11  probes from the open reading frame (E) and from the 3'UTR (C) showed equivalent results to riboprobe P in the gluteus medius (Figure  3B ). The same expression pattern was seen for  PEG11  and  PEG11AS  transcripts in the supraspinatus, but signal from the protected riboprobes was just above background level.PEG11PEG11ASPEG11MatCLPGPatPEG11CLPGMat/CLPGPatPEG11ASCLPGMatPatCLPGMatCLPGPatMatCLPGPatPEG11PEG11PEG11ASTwo probes from upstream of the  PEG11  coding sequence (riboprobes F and G) did not detect any  PEG11  transcripts, indicating that transcription of the 6.5 kb  PEG11  was initiated within 350 bp of the start codon of the open reading frame. Riboprobe F detected  PEG11AS  transcripts in the gluteus medius and supraspinatus of all four genotypes, with the lowest expression in the paternal heterozygous (+ Mat / CLPG Pat ) animals. The  PEG11AS  riboprobe G shows three protected fragments, suggesting variable splice junctions or transcription termination sites for  PEG11AS  transcripts.PEG11PEG11PEG11PEG11ASMatCLPGPatPEG11ASPEG11ASQuantitative analysis of the effect of genotype on gene expression Gene expression was measured in the gluteus medius and supraspinatus of 8 week-old animals (Figure  4 ) using quantitative PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase was not significantly different across the four genotypes in the gluteus medius and supraspinatus (Table  1 ), indicating that equivalent amounts of RNA were used for cDNA synthesis and quantitative PCR. The effect of the callipyge mutation on genotype-specific expression of  DLK1  and  PEG11  in gluteus medius was the same (Table  1 ) although the magnitude of the response was greater for  PEG11  than  DLK1  (Figure  4A  and  4B ). The paternal heterozygous (+ Mat / CLPG Pat ) animals had the highest transcript abundance (p < 0.05), followed by  CLPG Mat / CLPG Pat  animals, which had significantly greater transcript abundance (p < 0.05) than  CLPG Mat /+ Pat  or +/+ animals. The mRNA abundance of  DLK1  in the gluteus medius was 6-fold and 2.5-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat /CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4A ).  PEG11  mRNA abundance in the gluteus medius was 200-fold and 13-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat / CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4B ). The effect of the callipyge mutation on  DLK1  and  PEG11  expression in the supraspinatus was different (Table  1 ). No differences in  DLK1  transcript abundance in the supraspinatus were found among the four genotypes (Figure  4C ). Although not statistically analyzed, the level of  DLK1  expression was similar between the gluteus medius and supraspinatus (Figure  4A  and  4C ).  PEG11  expression in the supraspinatus had a different genotype-specific pattern than in the gluteus medius. PEG11 expression was elevated 150-fold in + Mat / CLPG Pat  animals (p < 0.05) but was not significantly changed in the other three genotypes (Figure  4D ). The transcript abundance of  PEG11  in supraspinatus was generally much lower than in gluteus medius of the same genotype.  PEG11  expression in the supraspinatus was 12-fold lower than in the gluteus medius of paternal heterozygous animals (Figure  4B  and  4D ). Maternal inheritance of the callipyge allele significantly altered the expression of  MEG8  but not  PEG11AS  in the gluteus medius (Table  1 ). Transcript abundance of  MEG8  in gluteus medius was 6-fold greater (p < 0.05) in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  animals relative to the + Mat / CLPG Pat  and +/+ animals (Figure  4A ). Expression of  MEG8  in the supraspinatus was also affected by the callipyge mutation but to a lesser degree (Figure  4C ). The homozygous callipyge animals had significantly higher  MEG8  transcript abundance than paternal heterozygotes, but those two genotypes were not significantly different from maternal heterozygotes and homozygous wild type lambs. Orthogonal contrasts were used to analyze different models of gene action for the genes that had a significant effect for genotype (Table  1 ). The polar overdominance contrast was significant for transcript abundances of  DLK1 ,  PEG11  and  MEG8  in gluteus medius and for  PEG11  and  MEG8  in the supraspinatus. The additive contrasts were also significant for  DLK1 ,  PEG11  and  MEG8  in gluteus medius but were only significant for  PEG11  in supraspinatus. The maternal dominance contrast was significant for  DLK1  and  PEG11  gluteus medius.
Quantitative analysis of the effect of genotype on gene expression
Gene expression was measured in the gluteus medius and supraspinatus of 8 week-old animals (Figure  4 ) using quantitative PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase was not significantly different across the four genotypes in the gluteus medius and supraspinatus (Table  1 ), indicating that equivalent amounts of RNA were used for cDNA synthesis and quantitative PCR. The effect of the callipyge mutation on genotype-specific expression of  DLK1  and  PEG11  in gluteus medius was the same (Table  1 ) although the magnitude of the response was greater for  PEG11  than  DLK1  (Figure  4A  and  4B ). The paternal heterozygous (+ Mat / CLPG Pat ) animals had the highest transcript abundance (p < 0.05), followed by  CLPG Mat / CLPG Pat  animals, which had significantly greater transcript abundance (p < 0.05) than  CLPG Mat /+ Pat  or +/+ animals. The mRNA abundance of  DLK1  in the gluteus medius was 6-fold and 2.5-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat /CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4A ).  PEG11  mRNA abundance in the gluteus medius was 200-fold and 13-fold greater in + Mat / CLPG Pat  lambs and  CLPG Mat / CLPG Pat  lambs respectively, relative to normal lambs (+/+; Figure  4B ).DLK1PEG11PEG11DLK1MatCLPGPatCLPGMatCLPGPatCLPGMatPatDLK1MatCLPGPatCLPGMat/CLPGPatPEG11MatCLPGPatCLPGMatCLPGPatThe effect of the callipyge mutation on  DLK1  and  PEG11  expression in the supraspinatus was different (Table  1 ). No differences in  DLK1  transcript abundance in the supraspinatus were found among the four genotypes (Figure  4C ). Although not statistically analyzed, the level of  DLK1  expression was similar between the gluteus medius and supraspinatus (Figure  4A  and  4C ).  PEG11  expression in the supraspinatus had a different genotype-specific pattern than in the gluteus medius. PEG11 expression was elevated 150-fold in + Mat / CLPG Pat  animals (p < 0.05) but was not significantly changed in the other three genotypes (Figure  4D ). The transcript abundance of  PEG11  in supraspinatus was generally much lower than in gluteus medius of the same genotype.  PEG11  expression in the supraspinatus was 12-fold lower than in the gluteus medius of paternal heterozygous animals (Figure  4B  and  4D ).DLK1PEG11DLK1DLK1PEG11MatCLPGPatPEG11PEG11Maternal inheritance of the callipyge allele significantly altered the expression of  MEG8  but not  PEG11AS  in the gluteus medius (Table  1 ). Transcript abundance of  MEG8  in gluteus medius was 6-fold greater (p < 0.05) in the  CLPG Mat /+ Pat  and  CLPG Mat / CLPG Pat  animals relative to the + Mat / CLPG Pat  and +/+ animals (Figure  4A ). Expression of  MEG8  in the supraspinatus was also affected by the callipyge mutation but to a lesser degree (Figure  4C ). The homozygous callipyge animals had significantly higher  MEG8  transcript abundance than paternal heterozygotes, but those two genotypes were not significantly different from maternal heterozygotes and homozygous wild type lambs.MEG8PEG11ASMEG8CLPGMatPatCLPGMatCLPGPatMatCLPGPatMEG8MEG8Orthogonal contrasts were used to analyze different models of gene action for the genes that had a significant effect for genotype (Table  1 ). The polar overdominance contrast was significant for transcript abundances of  DLK1 ,  PEG11  and  MEG8  in gluteus medius and for  PEG11  and  MEG8  in the supraspinatus. The additive contrasts were also significant for  DLK1 ,  PEG11  and  MEG8  in gluteus medius but were only significant for  PEG11  in supraspinatus. The maternal dominance contrast was significant for  DLK1  and  PEG11  gluteus medius.DLK1PEG11MEG8PEG11MEG8DLK1PEG11MEG8PEG11DLK1PEG11Discussion Our results show a clear pattern of increased expression for genes within the imprinted callipyge cluster in muscles that become hypertrophied, whereas expression of these genes was either reduced or absent in a muscle that does not become hypertrophied. The increased gene expression occurred when the mutation was inherited in  cis  and was dependent on each gene's imprinting status, consistent with previous reports [ 25 , 26 ]. These results support the hypothesis that the mutation has disrupted a long range control element [ 27 ]. Two paternally expressed genes,  DLK1  and  PEG11 , had significantly increased transcript abundance when a callipyge allele was inherited from the sire. One maternally expressed gene,  MEG8 , showed significantly increased transcript abundance when the callipyge allele was inherited from the dam. The changes in gene expression were sustained until 12 weeks of age, when the differences in growth and body composition between callipyge and normal lamb are established and are subsequently maintained [ 7 ]. Northern blot analysis suggests that expression of  GTL2  and  PEG11AS  was increased by maternal inheritance of the callipyge allele. Quantitative analysis was not done for  GTL2  in this study due to the expression of numerous alternatively spliced transcripts that have been reported for mice and sheep [ 26 , 29 ]. The 2.4 kb  GTL2  mRNA seen in this study consisted of a heterogeneous population of alternatively spliced mRNAs. Similarly, two  PEG11AS  transcripts were detected over a 5.6 kb area extending from beyond the 5' end of the  PEG11  transcript to the 3'UTR. The 1.7 and 0.8 kb  PEG11AS  transcripts detected by northern blot analysis using probe P would not be protected by probes C or G unless the  PEG11AS  transcripts undergo intron splicing or there are other transcripts that were not detected in the northern blots. Therefore, the effect of the callipyge mutation on  GTL2  and  PEG11AS  will require a more extensive analysis to fully elucidate their expression patterns in the four genotypes and determine their role in the callipyge model. Due to their paternal allele-specific expression, the  DLK1  and  PEG11  genes are both candidates for an effector gene that is responsible for the skeletal muscle hypertrophy exhibited by paternal heterozygous (+ Mat / CLPG Pat ) animals. In this study, both genes showed a polar overdominant expression pattern in that paternal heterozygotes had significantly higher levels of gene expression than the other three genotypes. The major differences between  DLK1  and  PEG11  were the magnitude and the muscle specificity of the up-regulation.  DLK1  was readily detectable in all muscles and genotypes by northern blot, but the up-regulation in paternal heterozygous animals was restricted to muscles of the loin and pelvic limb. The quantitative results showed a 6-fold increase in  DLK1  transcript abundance in the gluteus medius and no change of  DLK1  abundance in the supraspinatus. This pattern of gene expression is consistent with studies on individual muscle growth that show significant muscle hypertrophy in the gluteus medius but not in the supraspinatus [ 6 , 8 ].  DLK1  transcripts were significantly increased (2.5-fold) in the gluteus medius of  CLPG Mat / CLPG Pat  animals, which do not exhibit muscle hypertrophy. The lack of a phenotype in  CLPG Mat / CLPG Pat  animals could be due to a threshold effect that requires more than a 2.5-fold increase in  DLK1  transcript abundance to change muscle growth. The expression of  PEG11  was very low in the gluteus medius and supraspinatus of normal sheep and was induced in both muscles of callipyge lambs. Northern blot analysis, ribonuclease protection assay and quantitative PCR results all show that the expression of  PEG11  and  PEG11AS  transcripts was much lower in the supraspinatus than the other muscles. The high level of expression of  PEG11  in the gluteus medius relative to the supraspinatus of callipyge lambs indicates that  PEG11  could also be the gene responsible for muscle hypertrophy if there is a threshold level required to change muscle growth. The  PEG11  gene has a long intronless open reading frame but it is not known if a protein is produced or what function it may have. The overdominant nature of the callipyge phenotype through the lack of muscle hypertrophy in animals with the  CLPG Mat / CLPG Pat  genotype is one of the more intriguing aspects of the trait and has led to a hypothesis of  trans  effects by other reciprocally imprinted genes in the callipyge region [ 25 , 27 ]. If the  PEG11  gene has a direct role in muscle hypertrophy, either alone or in concert with  DLK1 , then  PEG11AS  may have a role in a  trans  effect on  PEG11  expression. In + Mat / CLPG Pat  animals,  PEG11  transcripts were 14-fold and 4-fold more abundant than  PEG11AS  transcripts in the gluteus medius and supraspinatus respectively.  PEG11AS  transcripts were more abundant than  PEG11  transcripts in the other three genotypes for both muscles. Therefore, the relative abundance of  PEG11  transcripts to  PEG11AS  transcripts was unique in paternal heterozygous animals. MicroRNAs are a central component of RNA interference mechanisms. In the mouse, two antisense microRNA have been identified for the orthologous  rtl1  locus [ 22 ]. RNA interference mechanisms have been shown both to repress transcription by inducing heterochromatin formation [ 30 - 32 ] and to cause post-transcriptional silencing through nuclear retention or targeted degradation [ 33 - 35 ]. MicroRNAs may be produced from post-transcriptional processing of  PEG11AS  RNA and be involved in normal regulation of the locus and in generating overdominance [ 28 ]. Expression of  PEG11AS  may normally cause repression of the paternal  PEG11  locus since very little  PEG11  mRNA was detectable in the muscles of normal animals. In the animals with a paternally inherited callipyge allele, (+ Mat / CLPG Pat  and  CLPG Mat / CLPG Pat ), the normal repression of the  PEG11  locus has been disrupted by the mutation in a putative long range control element [ 27 ].  PEG11  transcripts only accumulate in significant excess of  PEG11AS  in the paternal heterozygous animals, whereas in the  CLPG Mat / CLPG Pat  animals the  PEG11AS  transcripts remain in excess and may prevent the accumulation of the threshold level of  PEG11  mRNA required to produce a muscle hypertrophy phenotype. Although the expression of  PEG11AS  was not affected by genotype in quantitative PCR, the northern blots and transcript mapping indicate there are multiple transcripts that are likely to undergo different intron splicing. Further analysis of  PEG11AS  expression will be necessary to determine its role in the  PEG11  locus regulation.
Discussion
Our results show a clear pattern of increased expression for genes within the imprinted callipyge cluster in muscles that become hypertrophied, whereas expression of these genes was either reduced or absent in a muscle that does not become hypertrophied. The increased gene expression occurred when the mutation was inherited in  cis  and was dependent on each gene's imprinting status, consistent with previous reports [ 25 , 26 ]. These results support the hypothesis that the mutation has disrupted a long range control element [ 27 ]. Two paternally expressed genes,  DLK1  and  PEG11 , had significantly increased transcript abundance when a callipyge allele was inherited from the sire. One maternally expressed gene,  MEG8 , showed significantly increased transcript abundance when the callipyge allele was inherited from the dam. The changes in gene expression were sustained until 12 weeks of age, when the differences in growth and body composition between callipyge and normal lamb are established and are subsequently maintained [ 7 ].cisDLK1PEG11MEG8Northern blot analysis suggests that expression of  GTL2  and  PEG11AS  was increased by maternal inheritance of the callipyge allele. Quantitative analysis was not done for  GTL2  in this study due to the expression of numerous alternatively spliced transcripts that have been reported for mice and sheep [ 26 , 29 ]. The 2.4 kb  GTL2  mRNA seen in this study consisted of a heterogeneous population of alternatively spliced mRNAs. Similarly, two  PEG11AS  transcripts were detected over a 5.6 kb area extending from beyond the 5' end of the  PEG11  transcript to the 3'UTR. The 1.7 and 0.8 kb  PEG11AS  transcripts detected by northern blot analysis using probe P would not be protected by probes C or G unless the  PEG11AS  transcripts undergo intron splicing or there are other transcripts that were not detected in the northern blots. Therefore, the effect of the callipyge mutation on  GTL2  and  PEG11AS  will require a more extensive analysis to fully elucidate their expression patterns in the four genotypes and determine their role in the callipyge model.GTL2PEG11ASGTL2GTL2PEG11ASPEG11PEG11ASPEG11ASGTL2PEG11ASDue to their paternal allele-specific expression, the  DLK1  and  PEG11  genes are both candidates for an effector gene that is responsible for the skeletal muscle hypertrophy exhibited by paternal heterozygous (+ Mat / CLPG Pat ) animals. In this study, both genes showed a polar overdominant expression pattern in that paternal heterozygotes had significantly higher levels of gene expression than the other three genotypes. The major differences between  DLK1  and  PEG11  were the magnitude and the muscle specificity of the up-regulation.  DLK1  was readily detectable in all muscles and genotypes by northern blot, but the up-regulation in paternal heterozygous animals was restricted to muscles of the loin and pelvic limb. The quantitative results showed a 6-fold increase in  DLK1  transcript abundance in the gluteus medius and no change of  DLK1  abundance in the supraspinatus. This pattern of gene expression is consistent with studies on individual muscle growth that show significant muscle hypertrophy in the gluteus medius but not in the supraspinatus [ 6 , 8 ].  DLK1  transcripts were significantly increased (2.5-fold) in the gluteus medius of  CLPG Mat / CLPG Pat  animals, which do not exhibit muscle hypertrophy. The lack of a phenotype in  CLPG Mat / CLPG Pat  animals could be due to a threshold effect that requires more than a 2.5-fold increase in  DLK1  transcript abundance to change muscle growth.DLK1PEG11MatCLPGPatDLK1PEG11DLK1DLK1DLK1DLK1CLPGMatCLPGPatCLPGMatCLPGPatDLK1The expression of  PEG11  was very low in the gluteus medius and supraspinatus of normal sheep and was induced in both muscles of callipyge lambs. Northern blot analysis, ribonuclease protection assay and quantitative PCR results all show that the expression of  PEG11  and  PEG11AS  transcripts was much lower in the supraspinatus than the other muscles. The high level of expression of  PEG11  in the gluteus medius relative to the supraspinatus of callipyge lambs indicates that  PEG11  could also be the gene responsible for muscle hypertrophy if there is a threshold level required to change muscle growth. The  PEG11  gene has a long intronless open reading frame but it is not known if a protein is produced or what function it may have.PEG11PEG11PEG11ASPEG11PEG11PEG11The overdominant nature of the callipyge phenotype through the lack of muscle hypertrophy in animals with the  CLPG Mat / CLPG Pat  genotype is one of the more intriguing aspects of the trait and has led to a hypothesis of  trans  effects by other reciprocally imprinted genes in the callipyge region [ 25 , 27 ]. If the  PEG11  gene has a direct role in muscle hypertrophy, either alone or in concert with  DLK1 , then  PEG11AS  may have a role in a  trans  effect on  PEG11  expression. In + Mat / CLPG Pat  animals,  PEG11  transcripts were 14-fold and 4-fold more abundant than  PEG11AS  transcripts in the gluteus medius and supraspinatus respectively.  PEG11AS  transcripts were more abundant than  PEG11  transcripts in the other three genotypes for both muscles. Therefore, the relative abundance of  PEG11  transcripts to  PEG11AS  transcripts was unique in paternal heterozygous animals. MicroRNAs are a central component of RNA interference mechanisms. In the mouse, two antisense microRNA have been identified for the orthologous  rtl1  locus [ 22 ]. RNA interference mechanisms have been shown both to repress transcription by inducing heterochromatin formation [ 30 - 32 ] and to cause post-transcriptional silencing through nuclear retention or targeted degradation [ 33 - 35 ]. MicroRNAs may be produced from post-transcriptional processing of  PEG11AS  RNA and be involved in normal regulation of the locus and in generating overdominance [ 28 ]. Expression of  PEG11AS  may normally cause repression of the paternal  PEG11  locus since very little  PEG11  mRNA was detectable in the muscles of normal animals. In the animals with a paternally inherited callipyge allele, (+ Mat / CLPG Pat  and  CLPG Mat / CLPG Pat ), the normal repression of the  PEG11  locus has been disrupted by the mutation in a putative long range control element [ 27 ].  PEG11  transcripts only accumulate in significant excess of  PEG11AS  in the paternal heterozygous animals, whereas in the  CLPG Mat / CLPG Pat  animals the  PEG11AS  transcripts remain in excess and may prevent the accumulation of the threshold level of  PEG11  mRNA required to produce a muscle hypertrophy phenotype. Although the expression of  PEG11AS  was not affected by genotype in quantitative PCR, the northern blots and transcript mapping indicate there are multiple transcripts that are likely to undergo different intron splicing. Further analysis of  PEG11AS  expression will be necessary to determine its role in the  PEG11  locus regulation.CLPGMatCLPGPattransPEG11DLK1PEG11AStransPEG11MatCLPGPatPEG11PEG11ASPEG11ASPEG11PEG11PEG11ASrtl1PEG11ASPEG11ASPEG11PEG11MatCLPGPatCLPGMatCLPGPatPEG11PEG11PEG11ASCLPGMatCLPGPatPEG11ASPEG11PEG11ASPEG11ASPEG11Conclusions The effect of the callipyge mutation has been to increase transcript abundance of four genes,  DLK1 ,  GTL2 ,  PEG11  and  MEG8 , within the imprinted cluster in skeletal muscles that become hypertrophied. The increase in transcript abundance was consistent with each gene's parental allele-specific expression. The  DLK1  and  PEG11  genes were both expressed at their highest levels in paternal heterozygous animals and exhibited polar overdominant gene expression patterns. Therefore, both genes are candidates for causing muscle hypertrophy.  DLK1  expression was only elevated in muscles that undergo hypertrophy, so its muscle-specific increase was consistent with the callipyge phenotype.  PEG11  was 12-fold more abundant in hypertrophied muscle than non-hypertrophied muscle and only paternal heterozygous animals had  PEG11  transcript levels in excess of  PEG11AS  transcript levels. The unique relationship of  PEG11  and  PEG11AS  in paternal heterozygous animals suggests that an RNA interference mechanism may have a role in regulating the  PEG11  locus and polar overdominance in callipyge sheep.
Conclusions
The effect of the callipyge mutation has been to increase transcript abundance of four genes,  DLK1 ,  GTL2 ,  PEG11  and  MEG8 , within the imprinted cluster in skeletal muscles that become hypertrophied. The increase in transcript abundance was consistent with each gene's parental allele-specific expression. The  DLK1  and  PEG11  genes were both expressed at their highest levels in paternal heterozygous animals and exhibited polar overdominant gene expression patterns. Therefore, both genes are candidates for causing muscle hypertrophy.  DLK1  expression was only elevated in muscles that undergo hypertrophy, so its muscle-specific increase was consistent with the callipyge phenotype.  PEG11  was 12-fold more abundant in hypertrophied muscle than non-hypertrophied muscle and only paternal heterozygous animals had  PEG11  transcript levels in excess of  PEG11AS  transcript levels. The unique relationship of  PEG11  and  PEG11AS  in paternal heterozygous animals suggests that an RNA interference mechanism may have a role in regulating the  PEG11  locus and polar overdominance in callipyge sheep.DLK1GTL2PEG11MEG8DLK1PEG11DLK1PEG11PEG11PEG11ASPEG11PEG11ASPEG11Methods Sample collection A series of planned matings were conducted to produce the four possible callipyge genotypes. The genotypes of all lambs were verified using the single nucleotide polymorphism [ 23 , 24 ] and several markers that flank the callipyge region on chromosome 18. Lambs were slaughtered in accordance with humane practices approved by the Utah State University Institutional Animal Care and Use Committee. Samples were collected from the longissimus dorsi, semimembranosus, gluteus medius and supraspinatus, and preserved in RNAlater (Ambion Inc., Woodlands, TX USA). The tissue samples were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate, 50 mM EDTA, 1% sodium-N-lauroyl-sarcosine and total RNA was sedimented by ultracentrifugation of the homogenate on a cushion of 5.7 M CsCl, 50 mM EDTA [ 36 ]. Purified RNA was quantified by spectrophotometry and the use of a constant mass of RNA for each quantitative assay was based on absorbance at 260 nm. Total RNA was treated with DNase I using DNA  free ™ reagents (Ambion Inc.) to remove trace genomic DNA prior to use in the ribonuclease protection assay and quantitative PCR. Northern blot analysis Northern blots were prepared using denaturing formaldehyde gel electrophoresis (NorthernMax™; Ambion Inc) of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods [ 37 ]. Primer sequences used to amplify probes from the callipyge region are given in Charlier  et al . [ 25 ], and the PCR products were verified by DNA sequencing. Strand specific DNA probes were synthesized by 40 cycles of asymmetric PCR with Strip-EZ™ nucleotides (Ambion Inc.) and 50 μCi of α-[ 32 P]-dATP (Amersham-Pharmacia, Piscataway, NJ USA). Unincorporated nucleotides were removed by spin column chromatography (BioSpin P30; Bio-Rad Inc., Hercules, CA USA). The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate)/0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.). Ribonuclease protection assay Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products (Table  2 ) by ligation of double stranded oligonucleotides containing T7 or SP6 promoter sequences and re-amplification with an adapter and gene specific primer (Lig'n Scribe™, Ambion Inc.). Labeled RNA probes were synthesized using MAXIscript™ reagents (Ambion, Inc.) and 50 μCi of α-[ 32 P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [ 37 ]. Dried gels were exposed to phosphorimaging screens and images were collected using a Cyclone Storage Phosphorimager (Packard Instrument Co., Meriden, CT USA). Quantitative PCR Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents (Invitrogen, Carlsbad, CA USA) with RNase inhibitor supplementation (Superase Inhibitor, Ambion Inc). The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system (Sigma-Aldridge, St. Louis, MO USA). Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 10 8  to 10 2  molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [ 38 ]. The analysis model included genotype as a fixed effect and animal within genotype as a random effect. The number of animals representing each genotype (6 to 8) is given in Table  3 . Orthogonal contrasts were used to evaluate different models of gene action if the effect of genotype on log value of starting abundance was significant. Initially, traditional additive, dominance and reciprocal heterozygote effects were evaluated (see Table  3  for contrasts). If the reciprocal heterozygote effect was significant (p < 0.05), a second set of orthogonal contrasts was used to test additive, maternal dominance and polar overdominance effects as previously described by Freking  et al . [ 10 ] (Table  3 ).
Methods
Sample collection A series of planned matings were conducted to produce the four possible callipyge genotypes. The genotypes of all lambs were verified using the single nucleotide polymorphism [ 23 , 24 ] and several markers that flank the callipyge region on chromosome 18. Lambs were slaughtered in accordance with humane practices approved by the Utah State University Institutional Animal Care and Use Committee. Samples were collected from the longissimus dorsi, semimembranosus, gluteus medius and supraspinatus, and preserved in RNAlater (Ambion Inc., Woodlands, TX USA). The tissue samples were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate, 50 mM EDTA, 1% sodium-N-lauroyl-sarcosine and total RNA was sedimented by ultracentrifugation of the homogenate on a cushion of 5.7 M CsCl, 50 mM EDTA [ 36 ]. Purified RNA was quantified by spectrophotometry and the use of a constant mass of RNA for each quantitative assay was based on absorbance at 260 nm. Total RNA was treated with DNase I using DNA  free ™ reagents (Ambion Inc.) to remove trace genomic DNA prior to use in the ribonuclease protection assay and quantitative PCR.
Sample collection
A series of planned matings were conducted to produce the four possible callipyge genotypes. The genotypes of all lambs were verified using the single nucleotide polymorphism [ 23 , 24 ] and several markers that flank the callipyge region on chromosome 18. Lambs were slaughtered in accordance with humane practices approved by the Utah State University Institutional Animal Care and Use Committee. Samples were collected from the longissimus dorsi, semimembranosus, gluteus medius and supraspinatus, and preserved in RNAlater (Ambion Inc., Woodlands, TX USA). The tissue samples were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate, 50 mM EDTA, 1% sodium-N-lauroyl-sarcosine and total RNA was sedimented by ultracentrifugation of the homogenate on a cushion of 5.7 M CsCl, 50 mM EDTA [ 36 ]. Purified RNA was quantified by spectrophotometry and the use of a constant mass of RNA for each quantitative assay was based on absorbance at 260 nm. Total RNA was treated with DNase I using DNA  free ™ reagents (Ambion Inc.) to remove trace genomic DNA prior to use in the ribonuclease protection assay and quantitative PCR.freeNorthern blot analysis Northern blots were prepared using denaturing formaldehyde gel electrophoresis (NorthernMax™; Ambion Inc) of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods [ 37 ]. Primer sequences used to amplify probes from the callipyge region are given in Charlier  et al . [ 25 ], and the PCR products were verified by DNA sequencing. Strand specific DNA probes were synthesized by 40 cycles of asymmetric PCR with Strip-EZ™ nucleotides (Ambion Inc.) and 50 μCi of α-[ 32 P]-dATP (Amersham-Pharmacia, Piscataway, NJ USA). Unincorporated nucleotides were removed by spin column chromatography (BioSpin P30; Bio-Rad Inc., Hercules, CA USA). The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate)/0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.).
Northern blot analysis
Northern blots were prepared using denaturing formaldehyde gel electrophoresis (NorthernMax™; Ambion Inc) of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods [ 37 ]. Primer sequences used to amplify probes from the callipyge region are given in Charlier  et al . [ 25 ], and the PCR products were verified by DNA sequencing. Strand specific DNA probes were synthesized by 40 cycles of asymmetric PCR with Strip-EZ™ nucleotides (Ambion Inc.) and 50 μCi of α-[ 32 P]-dATP (Amersham-Pharmacia, Piscataway, NJ USA). Unincorporated nucleotides were removed by spin column chromatography (BioSpin P30; Bio-Rad Inc., Hercules, CA USA). The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate)/0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.).et alRibonuclease protection assay Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products (Table  2 ) by ligation of double stranded oligonucleotides containing T7 or SP6 promoter sequences and re-amplification with an adapter and gene specific primer (Lig'n Scribe™, Ambion Inc.). Labeled RNA probes were synthesized using MAXIscript™ reagents (Ambion, Inc.) and 50 μCi of α-[ 32 P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [ 37 ]. Dried gels were exposed to phosphorimaging screens and images were collected using a Cyclone Storage Phosphorimager (Packard Instrument Co., Meriden, CT USA).
Ribonuclease protection assay
Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products (Table  2 ) by ligation of double stranded oligonucleotides containing T7 or SP6 promoter sequences and re-amplification with an adapter and gene specific primer (Lig'n Scribe™, Ambion Inc.). Labeled RNA probes were synthesized using MAXIscript™ reagents (Ambion, Inc.) and 50 μCi of α-[ 32 P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [ 37 ]. Dried gels were exposed to phosphorimaging screens and images were collected using a Cyclone Storage Phosphorimager (Packard Instrument Co., Meriden, CT USA).Quantitative PCR Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents (Invitrogen, Carlsbad, CA USA) with RNase inhibitor supplementation (Superase Inhibitor, Ambion Inc). The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system (Sigma-Aldridge, St. Louis, MO USA). Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 10 8  to 10 2  molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [ 38 ]. The analysis model included genotype as a fixed effect and animal within genotype as a random effect. The number of animals representing each genotype (6 to 8) is given in Table  3 . Orthogonal contrasts were used to evaluate different models of gene action if the effect of genotype on log value of starting abundance was significant. Initially, traditional additive, dominance and reciprocal heterozygote effects were evaluated (see Table  3  for contrasts). If the reciprocal heterozygote effect was significant (p < 0.05), a second set of orthogonal contrasts was used to test additive, maternal dominance and polar overdominance effects as previously described by Freking  et al . [ 10 ] (Table  3 ).
Quantitative PCR
Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents (Invitrogen, Carlsbad, CA USA) with RNase inhibitor supplementation (Superase Inhibitor, Ambion Inc). The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system (Sigma-Aldridge, St. Louis, MO USA). Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 10 8  to 10 2  molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [ 38 ]. The analysis model included genotype as a fixed effect and animal within genotype as a random effect. The number of animals representing each genotype (6 to 8) is given in Table  3 . Orthogonal contrasts were used to evaluate different models of gene action if the effect of genotype on log value of starting abundance was significant. Initially, traditional additive, dominance and reciprocal heterozygote effects were evaluated (see Table  3  for contrasts). If the reciprocal heterozygote effect was significant (p < 0.05), a second set of orthogonal contrasts was used to test additive, maternal dominance and polar overdominance effects as previously described by Freking  et al . [ 10 ] (Table  3 ).et alAuthors' contributions CB participated in planning the study and developing the experimental design, conducted the northern blot analysis and ribonuclease protection assays, and wrote the drafts of the manuscript. LK and AP isolated RNA and performed the quantitative PCR assays. TH collected the muscle samples and genotyped the animals used in this study. DM participated in experimental design and performed the statistical analysis. NC participated in planning the study, set up a series of matings to generate all four genotypes and provided the experimental animals. All authors read and approved the final manuscript.
Authors' contributions
CB participated in planning the study and developing the experimental design, conducted the northern blot analysis and ribonuclease protection assays, and wrote the drafts of the manuscript. LK and AP isolated RNA and performed the quantitative PCR assays. TH collected the muscle samples and genotyped the animals used in this study. DM participated in experimental design and performed the statistical analysis. NC participated in planning the study, set up a series of matings to generate all four genotypes and provided the experimental animals. All authors read and approved the final manuscript.


Background Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been α7. This subunit can form functional homomeric pentamers (α7) 5 , which can make interpretation of physiological and structural data much simpler. The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner.
Background
Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been α7. This subunit can form functional homomeric pentamers (α7) 5 , which can make interpretation of physiological and structural data much simpler. The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner.Description A7DB   is a new relational database of manually curated experimental physiological data associated with the α7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the α7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1) a simple text query 2) an incremental query builder 3) an interactive query builder and 4) a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations.
Description
A7DB   is a new relational database of manually curated experimental physiological data associated with the α7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the α7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1) a simple text query 2) an incremental query builder 3) an interactive query builder and 4) a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations.Conclusions A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the α7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families.
Conclusions
A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the α7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families.

Background Nicotinic acetylcholine receptors (nAChRs) are the most studied members of the cys-loop family of ligand-gated ion channels (LGICs) which also contains γ-aminobutyric acid (GABA) receptors, glycine receptors and 5-HT 3  receptors [ 1 ]. Distinct subtypes of nAChRs mediate, for example, fast synaptic transmission in the brain and at neuromuscular junctions [ 2 ]. All are believed to be pentameric assemblies of various combinations of different subunits (α, β, γ/ε, δ), some of which exist as multiple isoforms [ 3 ]. nAChRs are important targets for novel analgesics as well as new drugs being devised for Alzheimer's disease and schizophrenia [ 4 , 5 ]. Mutations in nAChRs are associated with certain forms of epilepsy [ 6 , 7 ] and several congenital myasthenias [ 8 ]. There is a substantial and growing body of physiological, pharmacological, genomic structural and modeling data on receptors formed from these subunits. The volume and diversity of these data present severe challenges for their efficient storage and interpretation. Here we describe a relational database, initially relating to the α7 subunit, whose aim is to provide an easy to use, extensible web-based interface to access functional data and relate it back, wherever possible, to structural data.
Background
Nicotinic acetylcholine receptors (nAChRs) are the most studied members of the cys-loop family of ligand-gated ion channels (LGICs) which also contains γ-aminobutyric acid (GABA) receptors, glycine receptors and 5-HT 3  receptors [ 1 ]. Distinct subtypes of nAChRs mediate, for example, fast synaptic transmission in the brain and at neuromuscular junctions [ 2 ]. All are believed to be pentameric assemblies of various combinations of different subunits (α, β, γ/ε, δ), some of which exist as multiple isoforms [ 3 ]. nAChRs are important targets for novel analgesics as well as new drugs being devised for Alzheimer's disease and schizophrenia [ 4 , 5 ]. Mutations in nAChRs are associated with certain forms of epilepsy [ 6 , 7 ] and several congenital myasthenias [ 8 ]. There is a substantial and growing body of physiological, pharmacological, genomic structural and modeling data on receptors formed from these subunits. The volume and diversity of these data present severe challenges for their efficient storage and interpretation. Here we describe a relational database, initially relating to the α7 subunit, whose aim is to provide an easy to use, extensible web-based interface to access functional data and relate it back, wherever possible, to structural data.Construction and content The database is currently limited to nAChR α7 subunits. This makes the initial task of populating the database manageable. To allow 3 rd  level normalization, the principal information prototype stored in the database is referred to as an "experiment event". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables (see Figure  1 ) have been designed around the central experiment table [see  Additional file 1 ]. Quite often, several real experiments are performed within any one publication. We wanted to capture all of that data. Thus, all the data for one experiment that was reported have been stored (non-redundantly) as a separate entity. The database is managed by the MySQL relational database management system. PHP scripts query the MySQL system and transform the data into HTML pages for serving to the client. The database currently aims to store molecular information rather than whole-organism genetic information which can be sourced from elsewhere, see for example [ 9 , 10 ]. A typical set of experiments reported in one paper might concern the properties of one mutation at a particular position and its associated changes in physiology and/or pharmacology. All of this data is held in the database. By use of appropriate alignments (either stored within the server or uploaded) the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP [ 11 ]. The model is viewable by either using the Chime plugin, or by setting the browser to use Rasmol as a helper application. User instructions for configuring different browsers are provided on the server, as well as the list of combinations of operating system and browser that we have tested to date. For any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity. The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam [ 12 ], but we do not perform any alignments ourselves. Such tools are already available on numerous web-servers and in any case the user may wish to upload their own alignment. Wherever possible, we link to a well-established resource, as is the practice in for example, SWISS-PROT [ 13 ].
Construction and content
The database is currently limited to nAChR α7 subunits. This makes the initial task of populating the database manageable. To allow 3 rd  level normalization, the principal information prototype stored in the database is referred to as an "experiment event". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables (see Figure  1 ) have been designed around the central experiment table [see  Additional file 1 ]. Quite often, several real experiments are performed within any one publication. We wanted to capture all of that data. Thus, all the data for one experiment that was reported have been stored (non-redundantly) as a separate entity. The database is managed by the MySQL relational database management system. PHP scripts query the MySQL system and transform the data into HTML pages for serving to the client.The database currently aims to store molecular information rather than whole-organism genetic information which can be sourced from elsewhere, see for example [ 9 , 10 ]. A typical set of experiments reported in one paper might concern the properties of one mutation at a particular position and its associated changes in physiology and/or pharmacology. All of this data is held in the database.By use of appropriate alignments (either stored within the server or uploaded) the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP [ 11 ]. The model is viewable by either using the Chime plugin, or by setting the browser to use Rasmol as a helper application. User instructions for configuring different browsers are provided on the server, as well as the list of combinations of operating system and browser that we have tested to date.For any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity.The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam [ 12 ], but we do not perform any alignments ourselves. Such tools are already available on numerous web-servers and in any case the user may wish to upload their own alignment. Wherever possible, we link to a well-established resource, as is the practice in for example, SWISS-PROT [ 13 ].Utility and discussion Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith*). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a 'fast lane' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information. The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database. The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified. The database is very complementary to some existing resources, in particular the LGICdb [ 14 , 15 ]. This database contains sequence, phylogenetic data and sets of coordinates, but does not attempt to store all aspects of individual experiments as we report here. However, this could be used in conjunction with our database to for example explore equivalent positions in related receptors using sequence and phylogenetic information stored therein. Our approach is somewhat similar to that employed by the ProTherm database [ 16 ] which aims to store thermodynamic data for mutant and wildtype proteins [ 17 ]. Although currently there are no entries common to both databases, it is envisaged that thermodynamic data reported for either the α7 nAChR receptor or related channels will appear in the ProTherm database and can thus be used in conjunction with the A7DB to help illustrate for example how loss of function might be related to protein stability. Furthermore, the similarity in design of these databases should make cross-interrogation possible in the future. We should state that the main difference between A7DB and ProTherm is that instead of thermodynamic data we store pharmacological and physiological data and in that sense it is closer to the voltage-gated potassium channel database [ 18 , 19 ]. Finally, the A7DB is also complementary to the Protein Mutant Database [ 20 ] where mutations across many proteins are stored but the searching and data tabulation is primarily text-based. This might be particularly useful if another mutation had been reported in a different protein that bound a similar ligand, for example in acetylcholine also binds to acetylcholinesterase, which we do not store data for.
Utility and discussion
Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith*). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a 'fast lane' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information.The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database.The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified.The database is very complementary to some existing resources, in particular the LGICdb [ 14 , 15 ]. This database contains sequence, phylogenetic data and sets of coordinates, but does not attempt to store all aspects of individual experiments as we report here. However, this could be used in conjunction with our database to for example explore equivalent positions in related receptors using sequence and phylogenetic information stored therein. Our approach is somewhat similar to that employed by the ProTherm database [ 16 ] which aims to store thermodynamic data for mutant and wildtype proteins [ 17 ]. Although currently there are no entries common to both databases, it is envisaged that thermodynamic data reported for either the α7 nAChR receptor or related channels will appear in the ProTherm database and can thus be used in conjunction with the A7DB to help illustrate for example how loss of function might be related to protein stability. Furthermore, the similarity in design of these databases should make cross-interrogation possible in the future. We should state that the main difference between A7DB and ProTherm is that instead of thermodynamic data we store pharmacological and physiological data and in that sense it is closer to the voltage-gated potassium channel database [ 18 , 19 ]. Finally, the A7DB is also complementary to the Protein Mutant Database [ 20 ] where mutations across many proteins are stored but the searching and data tabulation is primarily text-based. This might be particularly useful if another mutation had been reported in a different protein that bound a similar ligand, for example in acetylcholine also binds to acetylcholinesterase, which we do not store data for.Conclusions We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function [ 21 ]. We believe that this resource will grow in usefulness as the amount of data increases. For example, during the construction of this database, atomic coordinates were released for the transmembrane region of the nAChR from  Torpedo marmorata  [ 22 ]. Its development is particularly timely given these recent structural developments which allow three-dimensional information to be correlated with function derived from an ever-increasing body of experimental data.
Conclusions
We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function [ 21 ]. We believe that this resource will grow in usefulness as the amount of data increases. For example, during the construction of this database, atomic coordinates were released for the transmembrane region of the nAChR from  Torpedo marmorata  [ 22 ]. Its development is particularly timely given these recent structural developments which allow three-dimensional information to be correlated with function derived from an ever-increasing body of experimental data.Torpedo marmorataAvailability and requirements The database is freely available at:
Availability and requirements
The database is freely available at:Authors' contributions The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript.
Authors' contributions
The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript.Supplementary Material Additional File 1 Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided. Click here for file
Supplementary Material
Additional File 1
Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided.Click here for file