Determination of PRKAG1 coding sequence and mapping of PRKAG1 and PRKAG2 relatively to porcine back fat thickness QTL

PRKAG1, PRKAG2 and PRKAG3 encode three isoforms of AMP-activated protein kinase gamma chain. A major effect on meat quality and a medium effect on back fat thickness of the RN- mutation in the PRKAG3 gene has previously been reported. We have now mapped PRKAG1 and PRKAG2 at expected locations on SSC5 and SSC18 by analysis of radiation hybrids (IMpRH panel). PRKAG2 has been mapped in a region where no quantitative trait loci (QTL) has been reported. PRKAG1 has been mapped close to (but probably outside) a region containing a QTL influencing fatness traits. We have determined the full coding sequence of PRKAG1. No missense mutation was identified when comparing the coding sequence of one Meishan and one Large White boars. Further work is, however, required to determine if a polymorphism in PRKAG1 could be responsible for a part of the variability observed on fatness traits.     

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.     

人PRKAG2全长基因的体外拼接及TA克隆载体的构建

目的:应用体外基因拼接及TA克隆技术构建含PRKAG2基因的载体.方法:通过商业途径购得PRKAG2基因的一个转录变体cDNA质粒(GenBank登记号BC 068598),其在N端缺失了44个氨基酸,根据已知的PRKAG2基因序列(GenBank登记号NM_016203),设计搭桥引物及序列扩增引物,通过PCR搭桥方法,合成完整全序列的PRKAG2基因,并将其克隆到TA载体,将得到的阳性克隆测序鉴定.结果:拼接出全长1759bp的PRKAG2基因,目的基因连接到载体后测序与设计的PRKAG2基因完全一致.结论:成功的构建了全长人PRKAG2基因的TA克隆,为进一步研究PRKAG2基因的功能提供了模板.     

Association between polymorphism in bovine PRKAG3 gene and milk production traits

The aim of the conducted study was to evaluate correlation between genotypes and PRKAG3 compound genotypes and milk production traits (yield of milk, milk fat and milk protein, and protein and fat content in milk). The study covered a herd of 180 Jersey cows. PCR-RFLP method was used for genotyping. The frequencies of alleles that occur mostly and combined genotypes were as follows: T1526G G - 0.57, G 1609A G - 0.92 and for T1526G/G1609A TG/GG - 0.54. The results obtained in the study demonstrated the correlation between analyzed genotypes and selected milk production traits; however they are not statistically significant.     

Polymorphism distribution of RYR1, PRKAG3, HFABP,MYF-5 and MC4R genes in crossbred pigs

This study was designed to screen the crossbred pigs for SNPs in five candidate genes, associated with pork quality traits and to differentiate their genotypes by PCR–RFLP. The results indicated that genotypes of crossbred pigs were NN (90%) and Nn (10%) for RYR1; RR (83%) and QR (17%) for PRKAG3; HH (98%), Hh (1%) and hh (1%) for HFABP; DD (99%) and CD (1%) for MYF-5; and AG (57%), GG (26%) and AA (17%) for MC4R SNPs, respectively. Allelic frequencies for five SNPs {RYR1 (1843C>T), PRKAG3 (c.599G>A), HFABP (c.1322C>T), MYF-5 (c.1205A>C) and MC4R (c.1426A>G)} were 0.95 and 0.05 (N/n), 0.08 and 0.92 (Q/R), 0.99 and 0.01 (H/h), 0.00 and 1.00 (C/D) and 0.45 and 0.55 (A/G), respectively. The effect of RYR1 (1843C>T) SNP was significant on pH₄₅ (P?<?0.05), pH₂₄ (P?<?0.05) and protein % (P?<?0.05). The PRKAG3 (c.599G>A) and MC4R (c.1426A>G) SNP had significant association with dressing percentages. The results revealed that RYR1, PRKAG3 and MC4R SNPs may be used in marker associated selection for pork quality traits in crossbred pigs.     

SNPs rs10224002 in PRKAG2 may disturb gene expression and consequently affect hypertension

Molecular Biology Reports - Genome-wide association studies have identified a large number of genetic loci for blood pressure in European populations. The associations in other populations are...     

Genetic variations of the porcine PRKAG3 gene in Chinese indigenous pig breeds

Four missense substitutions (T30N, G52S, V199I and R200Q) in the porcine PRKAG3 gene were considered as the likely candidate loci affecting meat quality. In this study, the R200Q substitution was investigated in a sample of 62 individuals from Hampshire, Chinese Min and Erhualian pigs, and the genetic variations of T30N, G52S and V199I substitutions were detected in 1505 individuals from 21 Chinese indigenous breeds, 5 Western commercial pig breeds, and the wild pig. Allele 200R was fixed in Chinese Min and Erhualian pigs. Haplotypes II-QQ and IV-QQ were not observed in the Hampshire population, supporting the hypothesis that allele 200Q is tightly linked with allele 199V. Significant differences in allele frequencies of the three substitutions (T30N, G52S and V199I) between Chinese indigenous pigs and Western commercial pigs were observed. Obvious high frequencies of the "favorable" alleles 30T and 52G in terms of meat quality were detected in Chinese indigenous pigs, which are well known for high meat quality. However, the frequency of the "favorable" allele 199I, which was reported to have a greater effect on meat quality in comparison with 30T and 52G, was very low in all of the Chinese indigenous pigs except for the Min pig. The reasons accounting for this discrepancy remain to be addressed. The presence of the three substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations. A novel A/G substitution at position 51 in exon 1 was identified. The results suggest that further studies are required to investigate the associations of these substitutions in the PRKAG3 gene with meat quality of Chinese indigenous pigs, and to uncover other polymorphisms in the PRKAG3 gene with potential effects on meat quality in Chinese indigenous pigs.     

A PRKAG2 mutation causes biphasic changes in myocardial AMPK activity and does not protect against ischemia

Dominant mutations in the gamma2 regulatory subunit of AMP-activated protein kinase (AMPK), encoded by the gene PRKAG2, cause glycogen storage cardiomyopathy. We sought to elucidate the effect of the Thr400Asn (T400N) human mutation in a transgenic mouse (TGT400N) on AMPK activity, and its ability to protect the heart against ischemia-reperfusion injury. TGT400N hearts had markedly vacuolated myocytes, excessive accumulation of glycogen, hypertrophy, and preexcitation. Early activation of myocardial AMPK, followed by depression, and then recovery to wild-type levels was observed. AMPK activity correlated inversely with glycogen content. Partial rescue of the phenotype was observed when TGT400N mice were crossbred with TGalpha2DN mice, which overexpress a dominant negative mutant of the AMPK alpha2 catalytic subunit. TGT400N hearts had greater infarct sizes and apoptosis when subjected to ischemia-reperfusion. Increased AMPK activity is responsible for glycogen storage cardiomyopathy. Despite high glycogen content, the TGT400N heart is not protected against ischemia-reperfusion injury.     

SNP variation in the promoter of the PRKAG3 gene and association with meat quality traits in pig

ABSTRACT: BACKGROUND: The PRKAG3 gene encodes the gamma3 subunit of adenosine monophosphate activated protein kinase (AMPK), a protein that plays a key role in energy metabolism in skeletal muscle. Nonsynonymous single nucleotide polymorphisms (SNPs) in this gene such as I199V are associated with important pork quality traits. The objective of this study was to investigate the relationship between gene expression of the PRKAG3 gene, SNP variation in the PRKAG3 promoter and meat quality phenotypes in pork. RESULTS: PRKAG3 gene expression was found to correlate with a number of traits relating to glycolytic potential (GP) and intramuscular fat (IMF) in three phenotypically diverse F1 crosses comprising of 31 Large White, 23 Duroc and 32 Pietrain sire breeds. The majority of associations were observed in the Large White cross. There was a significant association between genotype at the g.-311A>G locus and PRKAG3 gene expression in the Large White cross. In the same population, ten novel SNPs were identified within a 1.3 kb region spanning the promoter and from this three major haplotypes were inferred. Two tagging SNPs (g.- 995A>G and g.-311A>G) characterised the haplotypes within the promoter region being studied. These two SNPs were subsequently genotyped in larger populations consisting of Large White (n = 98), Duroc (n = 99) and Pietrain (n = 98) purebreds. Four major haplotypes including promoter SNP's g.-995A>G and g.-311A>G and I199V were inferred. In the Large White breed, HAP1 was associated with IMF% in the M. longissmus thoracis et lumborum (LTL) and driploss%. HAP2 was associated with IMFL% GP-influenced traits pH at 24 hr in LTL (pHULT), pH at 45 min in LTL (pH45LT) and pH at 45 min in the M. semimembranosus muscle (pH45SM). HAP3 was associated with driploss%, pHULT pH45LT and b* Minolta. In the Duroc breed, associations were observed between HAP1 and Driploss% and pHUSM. No associations were observed with the remaining haplotypes (HAP2, HAP3 and HAP4) in the Duroc breed. The Pietrain breed was monomorphic in the promoter region. The I199V locus was associated with several GP-influenced traits across all three breeds and IMF% in the Large White and Pietrain breed. No significant difference in promoter function was observed for the three main promoter haplotypes when tested in vitro. CONCLUSION: Gene expression levels of the porcine PRKAG3 are associated with meat quality phenotypes relating to glycolytic potential and IMF% in the Large White breed, while SNP variation in the promoter region of the gene is associated with PRKAG3 gene expression and meat quality phenotypes.     

Comparative sequence analysis of the PRKAG3 region between human and pig: evolution of repetitive sequences and potential new exons

The PRKAG3 gene encodes the gamma3 chain of AMP-activated protein kinase (AMPK). A non-conservative missense mutation in the PRKAG3 gene causes a dominant phenotype involving abnormally high glycogen content in pig skeletal muscle. We have determined >126 kb (in 13 contigs) of porcine genomic sequence surrounding the PRKAG3 gene and the corresponding mouse region covering the gene. A comparison of these PRKAG3 sequences and the human sequence was conducted and used to predict evolutionarily conserved regions, including regulatory regions. A comparison of the human genomic sequence and a porcine BAC sequence containing the PRKAG3 gene, revealed a conserved organization and the presence of three additional genes, CYP27A1 (cytochrome P450, family 27, subfamily A, polypeptide 1), STK36 (Serine Threonine Kinase 36), and the homolog of the unidentified human mRNA KIAA0173. Interspersed repetitive elements constituted 51.4 and 38.6% of this genomic region in human and pig, respectively. We were able to reliably align 12.6 kb of orthologous repeats shared between pig and human and these showed an average sequence identity of 72.4%. Our analysis revealed that the human KIAA0173 gene harbors alternative 5' untranslated exons originating from repetitive elements. This provides an obvious example how transposable elements may affect gene evolution.     

Validation of a paternally imprinted QTL affecting pH24h distinct from PRKAG3 on SSC15

We previously performed a genome scan in a White Duroc × Erhualian F(2) population and identified a QTL on SSC15 with strong effect on pH24h in M. Longissimus dorsi and M. Semimembranous muscle tissue. At that time, the mode of inheritance of this QTL was not clarified, and it was also unclear whether the observed QTL effect was completely or partially caused by mutations in the PRKAG3 gene, which is the only major gene on SSC15 so far known to influence pH24h. In this study, effects of the PRKAG3 gene on meat quality traits were estimated by association analyses. Two substitutions in PRKAG3, p.Ile199Val (p.I199V) and p.Thr30Asn (p.T30N), were found to be segregating in the F(2) population and to significantly affect pH24h and total glycogen in meat, respectively. However, we excluded PRKAG3 as a causative gene for the detected QTL based on the following reasons: (i) the gene was located outside of the QTL confidence interval; (ii) when the PRKAG3 substitution was included as a fixed effect in the QTL model, the F-ratio for the QTL increased rather than decreased; (iii) favourable alleles for pH24h at the QTL and at the PRKAG3 p.I199V locus originated from Erhualian and White Duroc founders, respectively; (iv) more importantly, this QTL showed exclusive maternal expression, differing from the Mendelian expression of PRKAG3. In conclusion, this study is the first to report a maternally-expressed QTL for pH24h on SSC15, which is distinct from PRKAG3.     

Joint analysis of additive,dominant and first‐order epistatic effects of four genes (IGF2, MC4R,PRKAG3 and LEPR) with known effects on fat content and fat distribution in pigs

LEPR, MC4R, IGF2 and PRKAG3 are genes with known effects on fat content and distribution in pig carcass and pork. In a study performed with Duroc × Landrace/Large White pigs, we have found that IGF2 has strong additive effects on several carcass conformational traits and on fatty acid composition in several anatomical locations. MC4R shows additive effects on saturated fatty acid content in several muscles. On the other side, almost no additive effect has been found for PRKAG3 and very few for LEPR. In this work, no dominant effect has been found for any of the four genes. Using a Bayesian Lasso approach, we have been able now to find first‐order epistatic (mainly dominant–additive) effects between LEPR and PRKAG3 for intramuscular fat content and for saturated fatty acid content in L. dorsii, B. femoralis, Ps. major and whole ham. The presence of interactions between genes in the shaping of traits of such importance as intramuscular fat content and composition highlights the complexity of heritable traits and the difficulty of gene‐assisted selection for such traits.     

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation

Human mutations in PRKAG2, the gene encoding the γ2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TG^T400N), we previously reported initial cardiac hypertrophy (ages 2–8 weeks) followed by dilation and failure (ages 12–20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TG^T400N mice showed significantly increased cardiac mass/body mass ratios up to ～ 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were ～ 2- and ～ 5-fold higher, respectively, in TG^T400N relative to wildtype (WT) mice at age 2 weeks. NF-κB activity and nuclear translocation of the p50 subunit were increased ～ 2- to 3-fold in TG^T400N hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated ～ 2-fold as early as age 2 weeks. To ascertain whether these changes in TG^T400N mice were a consequence of increased AMPK activity, we crossbred TG^T400N with TG^α2DN mice, which express a dominant negative, kinase dead mutant of the AMPK α2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-κB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-κB and Akt signaling pathway, which mediates cardiac hypertrophy.     

Association between polymorphism in bovine <Emphasis Type="Italic">PRKAG3</Emphasis> gene and milk production traits

The aim of the conducted study was to evaluate correlation between genotypes and PRKAG3 compound genotypes and milk production traits (yield of milk, milk fat and milk protein, and protein and fat content in milk). The study covered a herd of 180 Jersey cows. PCR-RFLP method was used for genotyping. The frequencies of alleles that occur mostly and combined genotypes were as follows: T1526G G − 0.57, G1609A G − 0.92 and for T1526G/G1609A TG/GG − 0.54. The results obtained in the study demonstrated the correlation between analyzed genotypes and selected milk production straits; however they are not statistically significant.     

The influence of the <Emphasis Type="Italic">PRKAG3</Emphasis> mutation on glycogen,enzyme activities and fibre types in different skeletal muscles of exercise trained pigs

Background  

AMP-activated protein kinase (AMPK) plays an important role in the regulation of glucose and lipid metabolism in skeletal muscle. Many pigs of Hampshire origin have a naturally occurring dominant mutation in the AMPK γ3 subunit. Pigs carrying this PRKAG3 (R225Q) mutation have, compared to non-carriers, higher muscle glycogen levels and increased oxidative capacity in m. longissimus dorsi, containing mainly type II glycolytic fibres. These metabolic changes resemble those seen when muscles adapt to an increased physical activity level. The aim was to stimulate AMPK by exercise training and study the influence of the PRKAG3 mutation on metabolic and fibre characteristics not only in m. longissimus dorsi, but also in other muscles with different functions.