Expression of DLK1 splice variants during porcine adipocyte development in vitro and in vivo.

Delta-like 1 (DLK1) belongs to the epidermal growth factor-like transmembrane protein family and is involved in the regulation of adipogenesis. Several splice variants of DLK1 have been identified in various species, of which two have been previously identified in pig. Here, we present two novel porcine DLK1 splice variants DLK1A and DLK1C. The gene expression profile of these variants together with the previously described DLK1B and DLK1C2 variants was studied in adipose tissue depots of pigs and during adipocyte differentiation in vitro. The short DLK1C and DLK1C2 transcripts were most abundantly expressed and their expression was reduced during porcine adipogenesis.

Investigation of a peroxisome proliferator-activated receptor gamma haplotype effect on meat quality and carcass traits in pigs.

Peroxisome proliferator-activated receptor gamma (PPARG) is a key transcription factor that controls adipocyte differentiation and fat deposition in mammals. The primary goal of this study was to investigate PPARG as a candidate gene for meat quality and carcass traits in swine. Part of the PPARG promoter, along with the most 5'-proximal exon of the gene, was amplified by PCR and subsequently screened for polymorphisms by sequencing. A Met59Val substitution was detected in the porcine PPARG gene along with four polymorphisms in the promoter region of the adipose-specific PPARG2. Three of these polymorphisms were chosen for genotyping and tested for association with meat quality, carcass and growth traits, according to the candidate gene approach. More than 1500 animals from different lines and populations were used in the study with records for meat quality and carcass traits. No convincing associations were found between the traits investigated and the PPARG genotypes. It does not appear that variation at the PPARG locus is affecting meat quality, carcass or growth traits in the pig populations studied.

Identification of a novel peroxisome proliferator-activated receptor (PPAR) gamma promoter in man and transactivation by the nuclear receptor RORalpha1.

PPARgamma has been extensively studied for the past decade mainly due to its central role in promoting and maintaining the adipocyte phenotype. To date, three PPARgamma isoforms have been described in man. Here we show the presence of a fourth PPARgamma promoter with its cognate mRNA initiating at exon 1, as evidenced by primer extension analysis. The presence of a putative responsive element (RORE) for RORalpha, a representative of the ROR/RZR orphan receptor superfamily, in the novel promoter was investigated. By gelshift experiments and site-directed mutagenesis we show that this RORE specifically binds the RORalpha1 isoform. We further demonstrate that overexpression of RORalpha1, but not the RORalpha2 and RORalpha3 isoforms, induced a 40-fold increase in promoter activity in transient transfection assays in various cell lines. Considering the strong transcriptional activation it is likely that RORalpha1 forms a part of the multifactorial regulatory mechanisms that control expression of the human PPARgamma gene.

Tissue distribution of porcine peroxisome proliferator-activated receptor alpha: detection of an alternatively spliced mRNA.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) plays a key role in regulating the catabolic pathway of lipids in response to a variety of compounds named peroxisome proliferators (PPs). The cellular responses to PPs differ among mouse/rat and other species and actualize the study in swine, which show close resemblance to human lipid physiology and metabolism. We have isolated the cDNA containing the open reading frame of porcine PPAR alpha whose deduced amino acid sequence revealed an evolutionary distance to mouse/rat that could be implicated in causing the species-dependent response to PPs. Interestingly, an alternatively spliced PPAR alpha mRNA, lacking exon 5, was detected by reverse transcriptase-polymerase chain reaction in several porcine tissues. This deletion alters the reading frame and introduces a premature stop codon of PPAR alpha, presumably giving rise to a C-terminal truncated protein. We have also examined PPAR alpha expression by Northern blot analysis in tissues taken from pigs at three different stages of maturation, including two breeds that differ considerably in body composition and fat deposition. Porcine PPAR alpha was predominantly expressed in kidney and liver in mature individuals. When comparing piglets of a young age, a breed-specific tissue distribution of PPAR alpha mRNA was observed, particularly in liver and heart.

Evolutionary conservation of the apolipoprotein E-C1-C2 gene cluster on bovine chromosome 18q24.

We have constructed a long-range restriction map spanning about 250 kb on bovine chromosome 18q24. Our results show that the apolipoprotein C2 (APOC2) gene is located about 25 kb from the APOE gene. Four putative CpG islands are also indicated in the map. Interestingly, a minisatellite located in the third intron of the human and mouse APOC2 genes was also found at identical position in the bovine gene and revealed high sequence identity comparing with the two corresponding sequences. By means of cosmid mapping, we further demonstrate that the APOE-APOC1-APOC2 gene cluster is evolutionary conserved in cattle.

Characterisation of porcine peroxisome proliferator-activated receptors gamma 1 and gamma 2: detection of breed and age differences in gene expression.

Two isoforms of peroxisome proliferator-activated receptor gamma (PPAR gamma) cDNAs, gamma 1 and gamma 2, have been isolated and characterised in swine. The relative expression of the two transcripts was studied by northern blot analysis using total RNA isolated from several porcine tissues taken at three different ages (day 1, after 5 weeks and at 100 kg weight). Hybridisation were carried out with two different probes, one binding to both PPAR gamma transcripts and the other being PPAR gamma 2 specific. Strongest hybridisation signals with the PPAR gamma probe binding both variants were detected in adipose tissues and spleen at all three ages, whereas only faint or no signals were detected in other tissues. The tissue distribution pattern of PPAR gamma 1 and gamma 2 suggests a modulation of tissue distribution for the two transcripts and obvious age and breed differences in gene expression in swine.

Characterisation of bovine peroxisome proliferator-activated receptors gamma 1 and gamma 2: genetic mapping and differential expression of the two isoforms.

In this report we describe the isolation and characterisation of the cDNAs encoding two isoforms of peroxisome proliferator-activated receptor gamma (PPAR gamma), gamma 1 and gamma 2, in cattle. The cDNA sequences show strong conservation with the corresponding sequences reported in other species. The distribution of PPAR gamma mRNAs in various bovine tissues was investigated using Northern blot analysis. The highest expression was detected in adipose tissue with about equal amounts of the both transcripts while a differential expression was found in other tissues investigated. PPAR gamma 1 was expressed at relatively high levels in bovine spleen and lung and to a lower extent in ovary, mammary gland, and small intestine. The amount of PPAR gamma 2 was apparently lower than that of PPAR gamma 1 in spleen, lung, and ovary. These results indicate a modulation of tissue distribution for the two transcripts in cattle. Using genetic linkage analysis we have also assigned the PPAR gamma gene to bovine chromosome 22.

Molecular genetics of familial hypercholesterolaemia in Norway.

OBJECTIVES: To characterize mutations in the low density lipoprotein (LDL) receptor gene causing familial hypercholesterolaemia (FH) amongst Norwegian patients. DESIGN: Molecular genetic analyses of the LDL receptor gene have been performed in patients with a clinical diagnosis of FH. SUBJECTS: A total of 742 probands have been studied. Of these, 476 had a diagnosis of definite FH. The rest had a diagnosis of possible FH. RESULTS: Twenty-three different mutations in the LDL receptor gene as well as the apolipoprotein B-3500 mutation have been found. Six of the mutations in the LDL receptor gene are novel mutations. A molecular genetic diagnosis was achieved in 295 of the probands with definite FH (62%) and in 317 probands total. Of the 317 probands, 3% carried the apolipoprotein B-3500 mutation. When family members were included, a total of 624 persons carried a mutation in the LDL receptor gene and 20 carried the apolipoprotein B-3500 mutation. CONCLUSIONS: Approximately 5% of Norwegian FH patients have been provided with a molecular genetic diagnosis. Our data suggest that molecular diagnosis of FH in Norway is feasible and should be implemented in clinical medicine.

[Application of gene technology in the diagnosis of familial hypercholesterolemia]. / Genteknologisk diagnostikk av familiaer hyperkolesterolemi.

Familial hypercholesterolaemia is an autosomal dominant disorder characterized by hypercholesterolaemia, xanthomas and premature coronary heart disease. Treatment of hypercholesterolemia is effective and consists of dietary changes and lipid lowering drugs. Only a minor proportion of familial hypercholesterolaemia patients are adequately treated, however. One explanation for this is assumed to be the relatively vague clinical diagnostic criteria applied. Because familial hypercholesterolaemia is caused by a mutation in the gene encoding the low density lipoprotein (LDL) receptor, mutation analysis of this gene could form the basis for specific diagnosis. 29 different mutations in the LDL receptor gene have been found to cause familial hypercholesterolaemia among Norwegian patients, and a total of 681 patients from 322 unrelated families have been provided with a molecular genetic diagnosis. We conclude that the use of molecular genetic analysis is feasible, and should be used clinically.

Screening for known mutations in the LDL receptor gene causing familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is caused by defective low density lipoprotein (LDL) receptors and is characterized by hypercholesterolemia and premature coronary heart disease. Two strategies can be used to identify the mutation in the LDL receptor gene underlying FH. One strategy is to search for novel mutations by DNA sequencing with or without prior mutation screening. The other strategy is to screen for known mutations. In this study we employed the latter strategy to screen 75 unrelated, Norwegian FH subjects for 38 known mutations. Three of the 38 mutations were detected in our group of FH subjects. Two subjects had FH-Padova, one had FH-Cincinnati-2 and one had FH-Gujerat. When additional unrelated FH heterozygotes were screened for the three mutations, the gene frequencies were 1.3%, 1.0% and 3.0%, respectively. In addition to identifying known mutations we also detected a novel stop codon in codon 541 (S541X). We conclude that screening for known mutations in the LDL receptor gene should be used as a complementary strategy to screening for novel mutations in order to understand the molecular genetics of FH.