Functional expression study of igf2 antisense transcript in mouse.

Insulin-like growth factor antisense gene (Igf2as) expression was investigated in different mouse tissues during development, in differentiating C2C12 cells and in a ΔDMR1-U2 knockout mouse model. The expression levels of Igf2as were high in fetal and newborn liver and muscle tissues compared to adults. The Igf2as gene was also expressed in placenta and in brain. The expression data suggests that the Igf2as gene plays a role in early development of the mouse and in placenta. There was no consistent evidence for an interaction between Igf2 and Igf2as transcripts. Furthermore, in knockout placentas lacking Igf2as transcription, Igf2 expression was comparable to that in wild type. These results indicate that Igf2as does not regulate Igf2 sense transcripts. In previous studies, it was suggested that the ΔDMR1-U2 knockout mouse showing intrauterine growth restriction was caused by the absence of placenta-specific Igf2 P0 transcription. We conclude that the ΔDMR1-U2 deletion phenotype should be reconsidered in the light of a functional Igf2as gene.

The Igf2as transcript is exported into cytoplasm and associated with polysomes.

Murine insulin-like growth factor 2 antisense (Igf2as) transcripts originate from the opposite strand of the same Igf2 locus as the Igf2 sense mRNA. The Igf2, insulin 2 (Ins2), and H19 genes form a cluster of imprinted genes on chromosome 7. Loss of imprinting of IGF2 in humans is associated with Beckwith-Wiedemann syndrome and Silver-Russell syndrome, as well as with Wilm's tumor and colorectal cancer. We developed a RNA-FISH protocol to detect Igf2as and Igf2 transcripts. The results from the RNA-FISH were confirmed with quantitative real-time PCR and clearly indicate that the Igf2as transcripts are predominantly located in the cytoplasm of C2C12 cells. In a polysome association study, we showed that the Igf2as sedimented with polysomes in a sucrose gradient. The cellular localization of Igf2as transcripts together with polysome fractionation analysis provides compelling evidence that the Igf2as is protein coding.

Biallelic transcription of the porcine IGF2R gene.

Here we report biallelic IGF2R gene expression in pig. We investigated SNPs in IGF2R exon 37 and in the 3'UTR and found biallelic expression in fetal brain and in livers, muscle and kidney tissues of fetal, newborn and adult pigs. PCR-RFLP and DNA sequencing results show consistently that IGF2R is expressed from both parental alleles although differential allelic expression may occur. The CpG island around IGF2R exon 1 was hypomethylated in all studied tissues and development stages. The CpG island in IGF2R intron 2 was hemimethylated in all studied tissues of fetal, newborn and adult pigs where the maternal allele was hypermethylated. It is therefore a differentially methylated region (DMR) by definition. RT-PCR showed no evidence for AIR transcription. A blast analyses revealed ESTs from intron 1 in sense direction, which are likely internally primed transcript artifacts. We suggest that porcine IGF2R expression widely resembles that of the human ortholog.

Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2.

BACKGROUND: Porcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2). RESULTS: Bisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele. CONCLUSIONS: Our results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle.

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.

Quantification of global DNA methylation with infrared fluorescence in liver and muscle tissues of differentially fed boars.

Methylation of cytosine residues at CpG sites is involved in various biological processes to control gene regulation and gene expression. Global DNA methylation is changed in different tumors and in cloned animals. Global DNA methylation can be accurately quantified by dot blot analysis with infrared (IR) fluorophores. Methylated lambda DNA was used as model DNA to develop and validate an immunochemical assay with IR fluorescence detection. Two different IR fluorophores were used, one to detect 5-methylcytosine and another to account for DNA loading. A sensitive infrared detection method was established which is suitable for accurate and reproducible quantification of global DNA methylation across a wide dynamic range. This method was subsequently employed to quantify global DNA methylation in liver and in muscle tissues of boars which have received either a control diet or a methyl supplemented diet in an ongoing study. A significant difference in global DNA methylation is indicated in muscle but not in liver tissue between the two groups of boars.

The bovine dilated cardiomyopathy locus maps to a 1.0-Mb interval on chromosome 18.

Cardiomyopathies are myocardial diseases that lead to cardiac dysfunction, heart failure, arrhythmia, and sudden death. In human medicine, cardiomyopathies frequently warrant heart transplantation in children and adults. Bovine dilated cardiomyopathy (BDCMP) is a heart muscle disorder that has been observed during the last 30 years in cattle of Holstein-Friesian origin. In Switzerland BDCMP affects Swiss Fleckvieh and Red Holstein breeds. BDCMP is characterized by a cardiac enlargement with ventricular remodeling and chamber dilatation. The common symptoms in affected animals are subacute subcutaneous edema, congestion of the jugular veins, and tachycardia with gallop rhythm. A cardiomegaly with dilatation and hypertrophy of all heart chambers, myocardial degeneration, and fibrosis are typical postmortem findings. It was shown that all BDCMP cases reported worldwide traced back to a red factor-carrying Holstein-Friesian bull, ABC Reflection Sovereign. An autosomal recessive mode of inheritance was proposed for BDCMP. Recently, the disease locus was mapped to a 6.7-Mb interval MSBDCMP06-BMS2785 on bovine Chr 18 (BTA18). In the present study the BDCMP locus was fine mapped by using a combined strategy of homozygosity mapping and association study. A BAC contig of 2.9 Mb encompassing the crucial interval was constructed to establish the correct marker order on BTA18. We show that the disease locus is located in a gene-rich interval of 1.0 Mb and is flanked by the microsatellite markers DIK3006 and MSBDCMP51.

Associations between 2 paternal casein haplotypes and milk yield traits of Swiss Fleckvieh cattle.

Associations between casein haplotypes and milk yield traits of offspring from 5 Swiss Fleckvieh AI test bulls were investigated. The analysis was performed by using a daughter design, where each daughter inherited either paternal haplotype B-A1-A-A or B-A2-A-A for alleles of alpha s1-, beta-, alpha s2- and kappa-casein genes. The substitution effects of paternal CSN2 A1 versus A2 on protein yield deviations (YDs) were significant (P < 0.05), whereas their effects on milk and fat YDs were not. The paternal substitution effects of the CSN2 A1 versus the A2 allele on protein YDs within the 5 sires did not reach the significance level. This is due to the contrary allele substitution effect of a sire compared to the other 4 sires. The effects of maternal haplotypes on milk, protein and fat YDs were not significant. However, it is noteworthy that the effects of haplotypes with a low frequency in the population deviate largely from the most frequent haplotype B-A2-A-A. The effects of beta-lactoglobulin (BLG) genotypes were significant for protein YDs but not for milk and fat YDs. The association between the paternal CSN2 A1 and A2 alleles and milk protein YDs within sires but not milk and fat YDs indicate an interaction, which might be a consequence of CSN2 heterogeneity or a closely linked gene that is contributing to the estimated effects.

Genome-wide identification of quantitative trait loci in a cross between Hampshire and Landrace II: meat quality traits.

BACKGROUND: Meat quality traits are important in pig breeding programs, but they are difficult to include in a traditional selection program. Marker assisted selection (MAS) of meat quality traits is therefore of interest in breeding programs and a Quantitative Trait Locus (QTL) analysis is the key to identifying markers that can be used in MAS. In this study, Landrace and Hampshire intercross and backcross families were used to investigate meat quality traits. Hampshire pigs are commonly used as the sire line in commercial pig breeding. This is the first time a pedigree including Hampshire pigs has been used for a QTL analysis of meat quality traits. RESULTS: In total, we analyzed 39 meat quality traits and identified eight genome-wide significant QTL peaks in four regions: one on chromosome 3, two on chromosome 6 and one on chromosome 16. At least two of the QTLs do not appear to have been detected in previous studies. On chromosome 6 we identified QTLs for water content in M. longissimus dorsi (LD), drip loss in LD and post mortem pH decline in LD. On chromosomes 3 and 16 we identified previously undetected QTLs for protein content in LD and for freezing and cooking loss respectively. CONCLUSION: We identified at least two new meat quality trait QTLs at the genome-wide significance level. We detected two QTLs on chromosome 6 that possibly coincide with QTLs detected in other studies. We were also able to exclude the C1843T mutation in the ryanodine receptor (RYR1) as a causative mutation for one of the chromosome 6 QTLs in this cross.

IGF2 antisense transcript expression in porcine postnatal muscle is affected by a quantitative trait nucleotide in intron 3.

A paternally expressed quantitative trait locus (QTL) for muscle mass was mapped to the insulin-like growth factor II (IGF2) locus at the distal end of pig chromosome 2p. It was recently demonstrated that a G to A transition at position intron 3-nt 3072 is the quantitative trait nucleotide (QTN) underlying this QTL. In this study we report for the first time the existence of imprinted porcine IGF2 antisense transcripts (IGF2-AS) and demonstrate that their expression in postnatal muscle is also affected by the QTN. A coregulated expression of IGF2 and IGF2-AS RNAs in muscle and liver with decreasing transcription from fetal to adult age was demonstrated. Further, the significant difference found in expression of IGF2-AS in postnatal muscle between individuals with different QTL genotypes and the lack of significant differences in fetal muscle and liver reflect completely what has been found for IGF2 sense transcripts.