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Association of an ACSL1 gene variant with polyunsaturated fatty acids in bovine skeletal muscle. | |
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MedLine Citation:
PMID: 22078495 Owner: NLM Status: MEDLINE |
BACKGROUND: The intramuscular fat deposition and the fatty acid profiles of beef affect meat quality. High proportions of unsaturated fatty acids are related to beef flavor and are beneficial for the nutritional value of meat. Moreover, a variety of clinical and epidemiologic studies showed that particularly long-chain omega-3 fatty acids from animal sources have a positive impact on human health and disease. RESULTS: To screen for genetic factors affecting fatty acid profiles in beef, we initially performed a microsatellite-based genome scan in a F(2) Charolais × German Holstein resource population and identified a quantitative trait locus (QTL) for fatty acid composition in a region on bovine chromosome 27 where previously QTL affecting marbling score had been detected in beef cattle populations. The long-chain acyl-CoA synthetase 1 (ACSL1) gene was identified as the most plausible functional and positional candidate gene in the QTL interval due to its direct impact on fatty acid metabolism and its position in the QTL interval. ACSL1 is necessary for synthesis of long-chain acyl-CoA esters, fatty acid degradation and phospholipid remodeling. We validated the genomic annotation of the bovine ACSL1 gene by in silico comparative sequence analysis and experimental verification. Re-sequencing of the complete coding, exon-flanking intronic sequences, 3' untranslated region (3'UTR) and partial promoter region of the ACSL1 gene revealed three synonymous mutations in exons 6, 7, and 20, six noncoding intronic gene variants, six polymorphisms in the promoter region, and four variants in the 3' UTR region. The association analysis identified the gene variant in intron 5 of the ACSL1 gene (c.481-233A>G) to be significantly associated with the relative content of distinct fractions and ratios of fatty acids (e.g., n-3 fatty acids, polyunsaturated, n-3 long-chain polyunsaturated fatty acids, trans vaccenic acid) in skeletal muscle. A tentative association of the ACSL1 gene variant with intramuscular fat content indicated that an indirect effect on fatty acid composition via modulation of total fat content of skeletal muscle cannot be excluded. CONCLUSIONS: The initial QTL analysis suggested the ACSL1 gene as a positional and functional candidate gene for fatty acid composition in bovine skeletal muscle. The findings of subsequent association analyses indicate that ACSL1 or a separate gene in close proximity might play a functional role in mediating the lipid composition of beef. |
Philipp Widmann; Karin Nuernberg; Christa Kuehn; Rosemarie Weikard |
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Publication Detail:
Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2011-11-11 |
Journal Detail:
Title: BMC genetics Volume: 12 ISSN: 1471-2156 ISO Abbreviation: BMC Genet. Publication Date: 2011 |
Date Detail:
Created Date: 2012-01-18 Completed Date: 2012-03-12 Revised Date: 2013-06-27 |
Medline Journal Info:
Nlm Unique ID: 100966978 Medline TA: BMC Genet Country: England |
Other Details:
Languages: eng Pagination: 96 Citation Subset: IM |
Research Unit Molecular Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany. |
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MeSH Terms | |
Animals Cattle / genetics*, metabolism Coenzyme A Ligases / genetics* Crosses, Genetic Fatty Acids, Unsaturated / analysis*, biosynthesis Meat / analysis* Muscle, Skeletal / chemistry*, metabolism Polymorphism, Genetic Quantitative Trait Loci |
Chemical | |
Reg. No./Substance:
0/Fatty Acids, Unsaturated; EC 6.2.1.-/Coenzyme A Ligases |
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Full Text | |
Journal Information Journal ID (nlm-ta): BMC Genet ISSN: 1471-2156 Publisher: BioMed Central |
Article Information Download PDF Copyright ©2011 Widmann et al; licensee BioMed Central Ltd. open-access: Received Day: 6 Month: 6 Year: 2011 Accepted Day: 11 Month: 11 Year: 2011 collection publication date: Year: 2011 Electronic publication date: Day: 11 Month: 11 Year: 2011 Volume: 12First Page: 96 Last Page: 96 ID: 3260110 Publisher Id: 1471-2156-12-96 PubMed Id: 22078495 DOI: 10.1186/1471-2156-12-96 |
Association of an ACSL1 gene variant with polyunsaturated fatty acids in bovine skeletal muscle | |
Philipp Widmann1 | Email: firstname.lastname@example.com |
Karin Nuernberg2 | Email: firstname.lastname@example.com |
Christa Kuehn1 | Email: email@example.com |
Rosemarie Weikard1 | Email: firstname.lastname@example.com |
1Research Unit Molecular Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, Dummerstorf, 18196, Germany |
2Research Unit Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, Dummerstorf, 18196, Germany |
In recent decades, the continuing accumulation of knowledge and the increasing number of reports providing evidence regarding the beneficial health effects of polyunsaturated fatty acids (PUFA) have attracted the attention of the medical and public community. Consumers are becoming increasingly aware of the relationships between diet and health and also of the importance of the diet for general physical and mental wellbeing [1,2]. Many clinical and epidemiologic studies have indicated a positive impact of long-chain omega-3 fatty acids (n-3 long-chain polyunsaturated fatty acids, n-3 LC-PUFA) on human health and disease. Beneficial effects of n-3 LC-PUFA are described in infant development, cancer, and cardiovascular diseases (e.g., [3-6]), lipid and glucose metabolism (e.g., [7-10]), inflammation (e.g., [11,12]), and more recently, in various mental illnesses including depression, attention-deficit hyperactivity disorder, and dementia (e.g.,[13]). It has been demonstrated that diets containing higher levels of n-3 LC-PUFA [namely DHA (docosahexaenoic acid; C22:6n-3) and EPA (eicosapentaenoic acid; C20:5n-3)], may reduce cardiovascular risk in diabetes by inhibiting platelet aggregation, improving lipid profiles, and reducing cardiovascular mortality. Thus, n-3 LC-PUFA were particularly recommended to people with diabetes and metabolic disorders associated to obesity [5,14]. Their beneficial health effects may be mediated through multiple distinct mechanisms, including alterations in cell membrane composition and function, gene expression, or eicosanoid biosynthesis [15,16]. It is known that n-3 LC-PUFA can exert important metabolic effects due to their ability to modulate the transcription of regulatory genes with function in lipid metabolism [17-21].
The n-3 LC-PUFA, like DHA and EPA, are particularly abundant in oily cold-water fish and seafood, however, they are also present in other animal products (e.g., ruminant meat and milk) but in lower concentrations. Increases of n-3 LC-PUFA content in the human diet can be achieved by dietary supplementation, but there is also a potential to alter the natural fatty acid (FA) profile in food from animals. FA composition of meat and milk reflects both, FA biosynthesis in the respective animal tissue and FA composition of ingested nutrients. A recent study showed that cattle and lambs fed grass-diet in the period before slaughter had an increased content of beneficial FAs in meat, and that subsequent moderate consumption of the respective meat had resulted in increased plasma and platelet n-3 LC-PUFA concentrations in healthy human individuals [22]. A ruminant diet on grass, which is rich in α-linolenic acid (C18:3n-3, ALA) compared to cereal-based concentrate diet can influence the FA profile of meat in the desired direction and improve its nutritional value [23-25]. However, the link between nutritional intake of FAs and its subsequent concentration in skeletal muscle is stronger in monogastric animals (pigs, poultry) than in ruminants due to hydrogenation of dietary FAs in the rumen (e.g., [26]).
In addition to the environmental conditions, genetic factors may also have a substantial effect on the variability of FA composition in animal products, especially for ruminants [27]. Consequently, genetic selection and breeding of animals with favorably enriched n-3 LC-PUFA content in skeletal muscle can provide a rich source of the desired beneficial FAs for the human diet. Therefore, it is necessary to elucidate the molecular-genetic background of fatty acid composition in bovine skeletal muscle for identifying the genes or gene variants favorable for human nutrition.
Numerous quantitative trait loci (QTL) affecting meat quality traits in cattle like marbling and FA composition have been identified on a variety of bovine chromosomes (http://www.animalgenome.org/cgi-bin/gbrowse/bovine/), which enabled subsequent identification of positional candidate genes, which are located in the vicinity of identified QTL and have putative physiological functions regarding FA synthesis in skeletal muscle. These candidate genes for lipid-associated traits have been studied for their possible role regarding phenotypic variation observed between and within breeds. DNA variants in a variety of genes involved in lipid synthesis and FA metabolism have been found to influence FA composition in bovine muscle tissue and carcass (SCD1, [28-34], SREBP-1 [29], FASN [29,34-37], FABP4 and LXRα [38], GH [29], ACACA [39], myostatin [40,41], leptin [33]).
However, the biochemical processes and the molecular background affecting the genetic variability of the complex polygenic trait of FA composition are not yet completely understood, particularly with regard to European cattle breeds, because the majority of recent studies have been performed on the very specific genetic background of Japanese Black cattle.
Therefore, the aim of this study was to identify genetic factors affecting the variation of FA composition in bovine skeletal muscle. For our study, we took advantage of a unique F2 resource population generated from the major European cattle breeds Charolais and German Holstein by means of embryo transfer and foster mothers [42]. In previous studies, this population had been shown to segregate for two major loci (NCAPG and MSTN) associated with prenatal and pubertal growth, postnatal body composition and general lipid deposition [43,44].
The animals from our resource cross population were kept and fed at standardized uniform conditions and slaughtered at the same age. Therefore, we can exclude exogenous factors due to differences in herd, age, feeding and gender. Consequently, differences in skeletal muscle fatness or FA composition should be due to differences in endogenous factors of the animals like the genetic background. The primary focus of our study was to discover phenotypic differences of FA composition in skeletal muscle between the individual animals of the resource population due to genetic variation.
An initial QTL analysis in the Charolais × German Holstein cross population identified a QTL for FA composition on bovine chromosome 27 (BTA27) as exemplified for n-3 LC-PUFA in Figure 1. In our study, the trait n-3 LC-PUFA represents n-3 PUFA exceeding a carbon chain length of C18. The QTL interval corresponded to a region, where previously QTL affecting marbling had been detected in a Bos indicus × Bos taurus cross and two commercial US Angus populations [45,46]. The QTL explained 20.5% variance in the model calculated as the relative reduction of the residual variance due to including the QTL in the model [47].
Furthermore, QTL for FA composition, myristic acid, (C14:0) and oleic acid (C18:1) content, have been reported in this chromosomal region in a Jersey × Limousin back-cross cattle population [48]. In our study, the QTL interval affecting FA composition in skeletal muscle displayed a peak between 15 and 16 cM on our genetic map of BTA27 corresponding to a genomic position at approximately 16 Mb on the current bovine genome assembly of the chromosome (NCBI mapviewer, build 5.2, http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid = 9913).
Based on its chromosomal position and integration in biochemical pathways of lipid metabolism, we identified the acyl-CoA synthetase long-chain family member 1 (ACSL1) gene as the most plausible positional and functional candidate gene underlying the QTL with effect on FA composition on BTA27. The ACSL1 gene is located exactly under the peak of the QTL interval. Its protein, the ACSL1 enzyme, is known to catalyze the first step of activation of long-chain (LC) FAs by converting them into LC acyl CoA thioesters for channeling towards chain elongation, triacylglyceride synthesis or FA oxidation [49]. ACSL1 has a key function in both the synthesis of cellular lipids and FA degradation, and is also necessary for phospholipid remodeling [50]. Due to its physiological biochemical function, it can be suggested that ACSL1 plays an important role in lipid metabolism, insulin resistance and obesity. Recently, a study in humans reported that a gene variant located in intron 1 of the ACSL1 gene can influence the metabolic syndrome risk (characterized by insulin resistance, dyslipidaemia, abdominal obesity and hypertension associated to type 2 diabetes), and that this ACSL1 genotype-dependent effect can be modulated by dietary PUFA intake suggesting a gene-nutrient interaction [51].
Although sequences for the ACSL1 gene and protein were deposited in the bovine genome databases, we found inconsistencies regarding the structural annotation of the gene in the bovine genome assemblies. A correct and conclusive structural gene annotation is a prerequisite for subsequent screening for gene variants and analysis of their functional relevance. Therefore, the first step of our study focused on the experimental confirmation of the structure of the ACSL1 gene on the genomic and cDNA level. Experimental verification by RT-PCR, re-sequencing and comparative sequence analyses confirmed the genomic annotation of the bovine ACSL1 gene in the alternate UMD_3.1 genome assembly (Figure 2), which is in contrast to the reference genome assembly Btau4.2 (http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid = 9913).
Re-sequencing of DNA from pools and individuals differing in IMF content and Δ9 desaturase activity index included a total of 8.5 Kb of genomic DNA. Comparative sequence analysis revealed a total of 19 single nucleotide polymorphisms (SNPs) in the targeted gene regions (Table 1). Three synonymous exonic (exons 6, 7 and 20), six intronic (introns 5, 6, 9, 13, 16 and 20), six SNPs in the promoter region and four SNPs in the 3'UTR of the bovine ACSL1 gene were detected (Figure 2, Table 1). Eleven out of these SNPs identified in our study were novel and not previously represented in the SNP database (version 133) at NCBI.
The association analysis included all exonic and intronic ACSL1 gene variants (except for the one in intron 16) and one SNP in the promoter region, which were identified by re-sequencing and validated by genotyping in the Charolais × German Holstein resource population. The nine SNPs analyzed in the Holstein × Charolais cross bred population showed a minor allele frequency ≥ 0.2 in the analyzed data set (Table 1). Intragenic linkage disequilibrium (LD) analysis revealed a strong LD between the SNPs in intron 20, exon 20, intron 13 and intron 9 (r2 >0.9), whereas there was only a moderate LD (0.5 < r2 <0.6) between these SNPs and the one in intron 5.
The association analysis with intragenic ACSL1 SNPs revealed that the SNP located in intron 5 of the ACSL1 gene (c.481-233A>G) showed the most significant associations with FA composition in skeletal muscle. The gene variant ACSL1 c.481-233A>G was significantly associated with the relative content of distinct fractions of unsaturated FAs, n-3 FA, PUFA, n-3 LC-PUFA and docosapentaenoic fatty acid (DPA) as well as with the absolute content of total FA, MUFA, and trans vaccenic acid (C18:1trans-11) in M. longissimus dorsi (Table 2). The results revealed that the c.481-233A allele of this gene variant is strongly associated with a higher relative level of n-3 FA, PUFA, DPA, and n-3 LC-PUFA. In contrast, the c.481-233A allele showed a decreasing effect on content of C18:1trans-11, total FA, and MUFA, and tended to be associated with a lower IMF content in skeletal muscle compared to the c.481-233G allele. The c.481-233A allele had a higher freqeuncy (73%) in the analyzed population compared to the alternative allele (27%).
Although the c.481-233A allele tends to be associated with a slightly lower total IMF content, the relative content of the FA fractions, n-3 FA, PUFA, DPA, and n-3 LC-PUFA, known to exert health-beneficial effects in humans is highly increased indicating a higher nutritional value for beef originating from animals with the favorable ACSL1 allele.
The strongest allelic effect of the ACSL1 c.481-233A>G locus was observed for n-3 FA content. This trait also includes the polyunsaturated C18 fatty acids, α-linolenic acid (ALA, C18:3n-3) and stearidonic acid (C18:4n-3). The n-3-FA content is different to the trait n-3 LC-PUFA, which exclusively comprises n-3 FA with a chain length > C18. As an essential FA, ALA cannot be synthesized by mammalian species and must be obtained from the diet. The ALA concentration in skeletal muscle, therefore, could be linked to the dietary absorption. However, the standardized concentrate-based feeding regimen in our study provides uniform feeding conditions for the animals. ALA is the precursor for the n-3 FA pathway [52] by serving as parent FA for the synthesis of stearidonic acid and n-3 LC-PUFA (EPA, DPA, and DHA) via sequential steps of desaturation and/or chain-elongation. The association of ACSL1 c.481-233A>G with DPA and with n-3 LC-PUFA (containing n-3 FA exceeding a chain length of C18) could suggest that a substantial proportion of their precursor ALA might be activated and channeled to chain elongation processes.
The trait PUFA comprises both FA types, the n-6 and n-3 FA. The ACSL1 c.481-233A>G variant showed no significant impact on n-6 FA content and thus, its association with PUFA could be due to its effect on the trait's component n-3 FA.
Interestingly, the ACSL1 gene variant c.481-233A>G that affected FA profiles in bovine skeletal muscle had no significant influence on the ratio n-6/n-3 FA in this tissue. Considering the standardized uniform feeding regimen in our study, this result could support the findings from other studies, which indicate that the n-6/n-3 FA ratio may be affected more by feeding than by genetics [53,54]. In contrast, we found the ACSL1 gene variant c.481-233A>G to be associated with the LA/ALA (C18:2 n-6/C18:3 n-3) ratio. Furthermore, we observed significant associations of this gene variant with the ratios PUFA/SFA and P/S in our study, both representing characteristics of meat quality and widely used to evaluate the nutritional value of meat fat content. Again, the c.481-233A allele revealed an increasing effect on these ratios compared to the c.481-233G allele.
In contrast to the increasing effect associated with the c.481-233A allele on the relative content of the FA fractions, n-3 FA, PUFA, DPA, and n-3 LC-PUFA, and the PUFA/SFA and P/S ratios, we observed a decreasing effect of this allele on the absolute content of the trans vaccenic acid C18:1trans-11 in skeletal muscle in our study. This effect was in concert with the associated parallel decrease in total FA and MUFA content in the tissue. The effect on C18:1trans-11 is of particular interest, because trans vaccenic acid is a precursor of conjugated linoleic acid (CLAcis-9, trans-11) generation. CLAs are believed to have several important physiological functions, including anti-carcinogenic, anti-atherogenic, immunomodulating, growth and lean body mass promoting effects [55]. Thus, targeted selection of cattle carrying the homozygous c.481-233A/c.481-233A genotype in the ACSL1 gene would possibly be accompanied by detrimental effects on the CLA profile in skeletal muscle.
There is the open question, whether the significant effects of the ACSL1 gene variant c.481-233A>G on FA composition were due to general fatness differences in skeletal muscle, which is supported by several QTL for marbling in the targeted chromosomal region, or whether the effects were associated with the ACSL1 gene variant c.481-233A>G. Alternatively, the effects of this gene variant might modulate the accumulation of specific FAs in skeletal muscle. To address this issue, we extended our association analysis and fitted IMF as a covariate in the model. When adjusting for IMF (Table 2), the association of the ACSL1 gene variant c.481-233A>G with absolute content of trans vaccenic acid in skeletal muscle remained significant, whereas the other associations dropped below a stringent threshold of statistical significance (Bonferroni q < 0.1) and were only tentatively significant (e.g., for relative content of n-3 FA and DPA). Thus, we cannot exclude that variants in the bovine ACSL1 gene may exert a substantial effect on total intramuscular fat content, which indirectly affects intramuscular composition of specific FA fractions. However, as the results for trans vaccenic acid demonstrate, it is suggested that there are also direct effects associated with the ACSL1 gene variant c.481-233A>G on intramuscular content of specific FAs.
Due to our observation that the c.481-233A>G SNP in intron 5 of the ACSL1 gene cannot fully explain the QTL variance (Figure 1), we conclude that this gene variant is presumably not causal, but in LD to another not yet detected polymorphism in its close vicinity affecting FA composition in bovine skeletal muscle. Presumably, these effects are not exclusively the consequence of variation in intramuscular fat content, but due to effects on specific FA. Prior to selective breeding of cattle carrying the desired genotype of the ACSL1 gene variant c.481-233A>G in order to produce meat with specific FA profiles, the association between c.481-233A>G and FA composition has to be confirmed in the particular target cattle population.
Nevertheless, our results indicate that the ACSL1 gene might play a functional role in mediating the FA composition in bovine skeletal muscle and provide a basis to further elucidate the function of the ACSL1 gene and its coordinated network with genes integrated in FA metabolism to dissect the molecular background of lipid composition of beef.
The generation of the Charolais × German Holstein resource cross population (SEGFAM), details regarding feeding and housing of the animals analyzed in our study, have been previously described [43,44]. The animals were kept under standardized environmental and feeding conditions in barn facilities at the Leibniz Institute for Farm Animal Biology (FBN). After birth, the calves were fed a milk/replacer/hay/concentrate diet ad libitum until day 121. Thereafter, the animals received a feed ration of concentrates and chaffed hay with a hay to concentrate ratio of 1:3 and an energy content of 12.7 MJ ME/kg dry matter fed ad libitum until slaughter. The animals were kept in a tight stall barn with individual daily feed recording. At the age of 18 months (547 days of age), the male animals were slaughtered, and a detailed dissection of the carcass was performed. A wide range of phenotypic data related to beef production and beef quality including FA composition were recorded including FA composition of selected skeletal muscles.
Analysis of FA composition of lipids involving 26 different FAs in skeletal muscle (M. longissimus dorsi) was determined for 156 F2 bulls using capillary gas chromatography as described previously [56]. The absolute amount of FAs in skeletal muscle was determined from 2 g of skeletal muscle and calculated as mg/100 g tissue. The relative content of individual fatty acids was calculated as percentage of the total amount of FAs extracted. Based on the data obtained for individual fatty acids, sums of specific fatty acid fractions were calculated: saturated fatty acids (SFA), unsaturated fatty acids (UFA), monounsaturated fatty acids (MUFA), trans fatty acids (TFA), n-3 fatty acids (n-3 FA), n-6 fatty acids (n-6 FA), polyunsaturated fatty acids (PUFA) and n-3 long-chain PUFA (n-3 LC-PUFA). Furthermore, the ratios n-6/n-3 FA, MUFA/SFA, PUFA/SFA, P/S and LA/ALA as well as four different Δ9 desaturase indices [57] were calculated. Intramuscular fat (IMF) content (percentage in 100 g tissue) was ascertained in M. longissimus dorsi by FoodScan Lab (FOSS) as described previously [56]. The phenotypic traits for FA composition of IMF included in our study are summarized in Table 3.
An initial QTL scan comprising 244 microsatellite markers [58] for variation of FA composition in skeletal muscle had pinpointed a region on bovine chromosome 27 (BTA27) with effect on n-3 PUFA content in skeletal muscle. Five microsatellite markers located on BTA27 (BM3507, RM209, BMS689, BM1857, BM203) had been genotyped in all 733 P0, F1, and F2 individuals of the Charolais × German Holstein resource population.
The respective QTL interval pointed to a chromosomal region on BTA27 harboring the acyl-CoA synthetase long-chain family member 1 (ACSL1) gene according to the sequence assembly of the chromosome. Therefore, in a second step of our analysis, nine intragenic ACSL1 SNPs (Figure 1) were added to the initial marker set. All microsatellite markers and all genotyped ACSL1 SNPs were included to calculate a genetic map using CRIMAP Version 2.50 [59], incorporating modifications by Ian Evans and Jill Maddox (University of Melbourne). The resulting genetic map was applied in the QTL analyses with a variance component QTL model as implemented in Qxpak [60] and essentially as described previously [43]:
y=Fb+Zu+Qg+e; |
where y is a vector of phenotypes, b is a vector of the fixed effects (slaughter year, NCAPG I442M genotype), u is the vector of individual infinitesimal polygenic effects, g is a vector of the additive QTL effects not fixed within founder breeds; F, Z and Q represent the incidence matrices for the fixed, polygenic and the QTL effect, respectively, and e is the vector of random residuals. An MCMC algorithm was used to calculate identity-by-descent probabilities as implemented in Qxpak. The NCAPG I442M mutation was included in the model, because previous analyses had shown a major effect of the mutation on carcass lipid deposition and growth in the resource population [44].
Statistical significance of the QTL analyses was tested by a likelihood-ratio test (LRT). Significance thresholds for the LRT were determined according to [61], considering one chromosome with a length of 0.6 M and an average marker density of 0.1 M. The significance thresholds for false positive results with α = 0.05 and α = 0.01 correspond to LRT values > 7.2 and LRT > 10.2, respectively.
The coding sequence of the bovine ACSL1 gene is represented by the reference mRNA sequence NM_001076085.1, which spans 3690 bp and is located on BTA27.
At the beginning of our study, the previous bovine genome assembly Btau4.0 and the current reference assembly Btau4.2 available at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&SHOW_DEFAULTS=on&BLAST_SPEC=OGP__9913__10708, [62]) annotated the bovine ACSL1 gene with a total of 19 protein-coding exons. In silico sequence analysis of the respective mRNA and protein sequences (NM_001076085.1 and NP_001069553) revealed that parts of the sequences could not be aligned to the bovine genome reference assembly Btau4.2. This indicated an incomplete annotation of the bovine ACSL1 gene. However, in the alternative bovine genome assembly Bos_taurus_UMD3.1 (ftp://ftp.cbcb.umd.edu/pub/data/assembly/Bos_taurus/Bos_taurus_UMD_3.1/, [63]) integrated into the recent bovine genome assembly, Build 5.2, at NCBI (http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid=9913), the bovine ACSL1 gene was annotated with a total of 21 protein-coding exons, which is also in agreement with the earlier bovine genome assembly, version Btau3.1. Comparative sequence analysis between gene and protein sequences of the bovine ACSL1 gene and those of the orthologous human counterparts (NM_001995.2 and NP_001986.2) and the current human genome assembly Hsa37.2 (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&SHOW_DEFAULTS=on&SHOW_DEFAULTS=on&BLAST_SPEC=OGP__9606__9558) showed that the mRNA and amino acid sequences of both species display a high similarity (88% and 91% identity, respectively), which supported the annotation of the current Bos_taurus_UMD3.1 and the earlier Btau3.1 assemblies.
An experimental confirmation of the bovine ACSL1 gene structure was required because of the inconsistent annotation of the ACSL1 gene. Therefore, fragments completely covering the coding region of the gene and the 5' und 3' UTRs, including the respective critical gene fragments with discordant structure annotation, were validated in our study. Exon-flanking primers (Table 4) were derived from the sequence contigs NW_001494406.2 and NW_930554.1 and used for PCR-amplification with genomic DNA and cDNA. Genomic DNA was isolated from blood leucocytes using standard methods. The cDNA was prepared from liver tissue of a lactating cow. Total RNA extraction and cDNA synthesis by reverse transcription were performed as described recently [43]. To amplify cDNA fragments of the ACSL1 gene, PCR was performed with cDNA using gene-fragment specific primers (Table 4). The PCR-amplicons were isolated from agarose gels using the NucleoSpin® Extract II kit (Macherey & Nagel) and sequenced with PCR primers using BigDye© sequencing chemistry on a capillary sequencer (MEGABACE, GE Healthcare).
Screening for polymorphisms was carried out by re-sequencing and covered the complete coding sequence, exon-flanking intronic regions, the 5' and 3' UTRs and 724 bp of the promoter of the ACSL1 gene. DNA primer pairs for PCR amplification and sequencing were designed based on genomic contig sequences (NW_001494406.2 and NW_930554.1) and the mRNA sequence (NM_001076085.1), respectively (Table 4).
Four genomic DNA pools consisting of selected animals from the Charolais × Holstein resource population differing in their intramuscular fat content and index of delta 9-desaturase were established and subjected to screening for gene variants by comparative re-sequencing. The IMF pools contained DNA from sampling time- and pedigree-matched animals with high (n = 5, 4.93 ± 1.73%) and low (n = 7, 1.78 ± 0.21%) IMF. The Δ9 desaturase index pools consisted of DNA from sampling time- and pedigree-matched animals with a high (n = 7; 50.87 ± 0.89) or low (n = 6, 43.86 ± 0.95) Δ9 desaturase index. Furthermore, two genomic DNA samples from control individuals and two individual DNA samples originating from extreme animals displaying the lowest (1.63%) and highest (6.09%) IMF were included to validate the results received from the pools.
Genomic DNA was isolated from blood leucocytes using standard methods. PCR with exon-flanking primers (Table 4) was performed with a total of 60 ng genomic DNA as described above. The generated PCR products were purified using the peqGOLD Cycle-Pure Kit (PEQLAB) according to the manufacturer's instructions and sequenced. Sequencing was performed on a capillary sequencer (MEGABACE, GE Healthcare) with primers used for targeted PCR amplification. To identify variable DNA positions, the sequences were analyzed meticulously by visual inspection of the sequencing profiles from DNA-pools and individuals' DNA and by sequence alignment to the reference cDNA sequence (NM_001076085.1) as well as to the respective bovine genome sequences. All SNPs identified by sequencing of DNA pools were verified by single sample re-sequencing.
Out of the identified 19 ACSL1 SNPs (see Table 3, Figure 1), nine were genotyped in the Charolais × German Holstein resource population: Two exonic SNPs (c.516C>G, c.1938T>G) and five intronic SNPs (c.481-233A>G, c.580+114C>G, c.845-58T>G, c.1267-100C>T, c.1959+56G>A) were genotyped on an Illumina Beadstation [64] as part of a targeted 384 SNP GoldenGate assay. The SNP in exon 7 (c.584A>G) was analyzed using a PCR-RFLP assay with primers for amplification of the targeted region (Table 4) and the restriction enzyme SacI (Fermentas). The promoter SNP c.-122G>A was genotyped by a Tetra-ARMS PCR assay [65] and validated by direct sequencing. The respective primers are given in Table 4. The NCAPG I442M mutation was genotyped by PCR-RFLP [43].
Prior to association analysis, we tested whether the phenotypic data of the individual traits were normally distributed using the Shapiro Wilk test. For those data displaying distributions significantly different from normality (P < 0.01), we performed natural log (ln) transformation, and the log- transformed data were subjected to association analysis. The respective data are indicated in Tables 2 and 3.
The BTA27 marker haplotypes of the individuals of the resource population were estimated by a Markov chain Monte Carlo (MCMC) algorithm implemented in Qxpak [60]. The corresponding haplotypes were submitted to pairwise LD analysis calculating r2 values using PowerMarker V3.25 [66].
Subsequently to the QTL analyses, association analyses were performed between ACSL1 gene SNPs and the absolute and relative FA composition traits in M. longissimus dorsi. The following model testing for LD as implemented in Qxpak [60] was applied:
yi=ap+ ∑k∑hλikhgk+ ∑m∑nλimngm+ui+eihkmnp |
where yi is the record of individual i, ap is the fixed effect of slaughter year p, λihk is an indicator variable for the NCAPG I442M locus, which is 1 when the allele at the hth haplotype (1 or 2) of the ith individual is 1 and otherwise 0, λimn is a respective indicator variable for the specific ACSL1 SNP, ui is the infinitesimal genetic effect of individual i, gk and gm are the respective allelic effects for NCAPG I442M and the ACSL1 SNP, and eihkmnp is the residual. Analogous to the QTL analyses, the NCAPG I442M mutation was included in the model, because previous analyses had shown a major effect of the mutation on carcass lipid deposition and growth in the resource population [44]. A likelihood-ratio test (likelihood of model with both loci vs. likelihood of model with NCAPG I442M) was applied to test for statistical significance. In order to dissect whether the association of the respective ACSL1 variant with intramuscular FA composition is solely due to indirect effects on IMF or a consequence of direct effects on the specific FA accumulation, we extended the model and fitted IMF as an additional covariate. A Bonferroni correction was calculated (q-value) to account for testing several SNPs in order to avoid false positive associations. The q-values thresholds of 0.05 and 0.1, respectively, indicate an experiment-wise significant or suggestive association, respectively. Finally, an additive fixed effect of the SNP in intron 5 was added in the QTL model described above to test whether this SNP might explain the QTL variance at the identified position on BTA27.
(SCD1): stearoyl-Coenzyme A desaturase 1; (SREBP-1): sterol regulatory element binding protein 1; (FASN): fatty acid synthase; (FABP4): fatty acid binding protein 4; (LXRα): liver X receptor alpha; (GH): growth hormone; (ACACA): acetyl-CoA carboxylase alpha; (NCAPG): non-SMC condensin I complex; subunit G, (MSTN): myostatin.
PW participated in screening for polymorphisms, genotyping and statistical analysis. KN carried out fatty acid analysis. CK conceived the study and performed statistical analysis. RW performed gene structure analysis and screening for polymorphisms. CK and RW wrote the manuscript. All authors read and approved the final manuscript.
Skillful technical assistance of Annett Eberlein, Astrid Kühl, Marlies Fuchs, Birgit Jentz, Maria Dahm, Antje Lehmann and Simone Wöhl is thankfully acknowledged. We thank the German Federal Ministry of Education and Research (BMBF) for the financial support of this work within the scope of the FUGATO QUALIPID project (FKZ 0313391C). Also, we thank our colleagues at the FBN Dummerstorf involved in the generation and care of the SEGFAM F2 resource population for their continuous support of our work and Jill Maddox (University of Melbourne, Australia) for providing the modified CRIMAP version 2.50.
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Identified SNPs within the ACSL1 locus and positions on the bovine genome assemblies
SNP ID relative to coding sequence* |
Gene region | Variation relative to reference sequence | Position on NW_001494406.2 (Btau4.2) | Position on NW_003104605.1 (UMD_3.1) | Allele frequency |
SNP accession number (dbSNP, NCBI ss#) |
c.-569_570del AC | Promoter | Indel TG | 1918795 | 389858 | Not analyzed | ss469271165 |
c.-224_225del GG | Promoter | Indel (C)5-7 | 1918451 | 389615 | Not analyzed | ss469271166 |
c.-196G>A | Promoter | C>T | 1918422 | 3894586 | Not analyzed | ss469271167 |
c.-167G>C | Promoter | C>G | 1918393 | 3894557 | Not analyzed | ss469271168 |
c.-151G>C | Promoter | C>G | 1918377 | 3894541 | Not analyzed | ss469271169 |
c.-122G>A | Promoter | C>T | 1918348 | 3894512 | 0.67 (G)/0.33 (A) | ss469271170 |
c.481-233A>G | Intron 5 | T>C | 1876389 | 3852106 | 0.73 (A)/0.27 (G) | ss469271171 |
c.516C>G | Exon 6 | G>C | 1876121 | 3852284 | 0.57 (C)/0.43 (G) | ss469271172 |
c.580+114C>G | Intron 6 | G>C | 1875943 | 3852552 | 0.33 (C)/0.67 (G) | ss469271173 |
c.584A>G | Exon 7 | T>C | 1875838 | 3852001 | 0.07 (G)/0.94 (A) | ss469271174 |
c.845-58T>G | Intron 9 | A>C | 1871919 | 3848082 | 0.75 (T)/0.21 (G)5 | ss469271175 |
c.1267-100C>T | Intron 13 | G>A | 1864210 | 3840373 | 0.24 (T)/0.76 (C) | ss469271176 |
c.1525-131C>T | Intron 16 | G>A | 1859370 | 3835533 | Not analyzed | ss469271177 |
c.1938T>G | Exon 20 | A>C | Not annotated | 3831586 | 0.75 (T)/0.25 (G) | ss469271178 |
c.1959+56G>A | Intron 20 | C>T | Not annotated | 3831509 | 0.24 (A)/0.76 (G) | ss469271179 |
c.2099+1023del A | 3'UTR | Indel A | 1853870 | 3829944 | Not analyzed | ss469271180 |
c.2099+1030 C>A | 3'UTR | C>A | 1853863 | 3829937 | Not analyzed | ss469271181 |
c.2099+1032 A>C | 3'UTR | A>C | 1853861 | 3829935 | Not analyzed | ss469271182 |
c.2099+1034 C>A | 3'UTR | C>A | 1853859 | 3829933 | Not analyzed | ss469271183 |
*SNP nomenclature according to the translation start codon ATG, reference sequence NM_001076085.1. SNPs marked in bold were included in the association analysis. Genomic positions of the SNPs were inferred from the current versions of the refererence and alternative bovine genome assemblies Btau4.2 and UMD_3.1 available at NCBI (http://www.ncbi.nlm.nih.gov/genome/guide/cow/index.html).
Association of the SNP in intron 5 of the ACSL1 gene (c.481-233A>G) with variation in intramuscular fatty acid composition and fat content
Model without IMF as covariate | Model with IMF as covariate | ||||||||||||
Trait* | LRT | p-value | Effect allele A | SE | Effect allele G | SE | Var [%]** | LRT | p-value | Effect allele A | SE | Effect allele G | SE |
Total FA [mg]*& | 6.2 | 0.0130 | 3.70 | 0.07 | 3.88 | 0.07 | 3.8 | 0.0507 | |||||
MUFA [mg]*& | 5.9 | 0.0154 | 3.29 | 0.08 | 3.48 | 0.09 | 2.9 | 0.0874 | |||||
n-3 FA [%]*# | 9.7 | 0.0018a | 0.16 | 0.05 | 0.01 | 0.06 | 11.4 | 5.8 | 0.0159 | 0.42 | 0.04 | 0.36 | 0.04 |
PUFA [%]*# | 7.1 | 0.0079b | 1.12 | 0.05 | 0.98 | 0.06 | 3.5 | 3.9 | 0.0477 | ||||
n-3 LC PUFA [%]# | 6.7 | 0.0099b | 0.24 | 0.03 | 0.16 | 0.04 | 3.4 | 3.5 | 0.0628 | ||||
C18:1trans-11 [mg]*& | 10.5 | 0.0012a | 1.31 | 0.10 | 1.63 | 0.12 | 6.5 | 8.1 | 0.0045a | 0.86 | 0.09 | 1.07 | 0.10 |
C22:5n-3 [%]# | 7.2 | 0.0071 b | 0.16 | 0.02 | 0.11 | 0.03 | 3.6 | 5.9 | 0.0150 | 0.27 | 0.02 | 0.23 | 0.02 |
PUFA/SFA* | 7.3 | 0.0069b | -0.79 | 0.06 | -0.94 | 0.07 | 3.6 | 4.2 | 0.0407 | ||||
P/S* | 6.7 | 0.0099b | -1.02 | 0.05 | -1.15 | 0.06 | 3.2 | 3.2 | 0.0758 | ||||
*Trait data log-transformed, &absolute content of fatty acids (mg/100 g of skeletal muscle), #relative content of fatty acids (percentage of the respective fatty acid fraction relative to total fatty acid amount)
LTR: likelihood ratio test, p: significancy of allelic effects, Var: variance explained, SE: standard error, aq-value <0.05, bq-value <0.1, **% variance in the model calculated as the relative reduction of the residual variance due to including the SNP in the model [47].
Allelic effects of the ACSL1 SNP (c.481-233A>G) on different FA fractions: n-3 fatty acids (n-3 FA = C18:3n-3 + C18:4n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3), poly-unsaturated fatty acids (PUFA = n-3 FA + n-6 FA [C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:2n-6 + C22:4n-6]), n-3 long-chain PUFA (n-3 LC-PUFA = C20:3n-3 + C22:6n-3 + C22:5n-3 + C20:5n-3), total FA, mono-unsaturated fatty acids (MUFA), docosapentaenoic fatty acid (DPA, C22:5n-3), trans vaccenic acid (TVA, C18:1trans-11) and the ratios: PUFA/SFA (saturated fatty acids = C12:0 + C14:0 + C16:0 + C17:0 + C18:0 + C20:0 + C24:0), P/S (C18:2 n-6 + C18:3 n-3/C14:0 + C16:0 + C18:0), LA/ALA (C18:2 n-6/C18:3 n-3), and the intramuscular fat content (IMF).
Phenotypic traits characterizing fatty acid composition in skeletal muscle
Trivial name | Abbreviation | Mean of absolute content (mg/100 g) |
SD | Mean of relative content (%) |
Lauric acid | C12:0 | 2.83* | 1.80 | 0.10 | 0.04 |
Myristoleic acid | C14:1cis-9 | 18.53 | 17.85 | 0.62 | 0.31 |
Myristic acid | C14:0 | 92.12* | 65.50 | 3.10* | 0.66 |
Palmitoleic acid | C16:1cis-9 | 109.33* | 76.59 | 3.73 | 0.83 |
Palmitic acid | C16:0 | 823.94* | 490.88 | 28.77 | 1.86 |
Heptadecenoic acid | C17:1cis-10 | 23.48* | 12.51 | 0.85 | 0.18 |
Margaric acid | C17:0 | 34.94* | 18.13 | 1.26* | 0.25 |
Stearic acid | C18:0 | 373.40* | 199.95 | 4.96* | 1.80 |
Oleic acid | C18:1cis-9 | 981.37* | 578.52 | 34.35 | 2.60 |
Vaccenic acid | C18:1cis-11 | 43.60* | 30.60 | 13.41 | 1.65 |
trans Vaccenic acid | C18:1trans-11 | 28.40* | 20.18 | 1.52* | 0.33 |
Linoleic acid (LA) | C18:2n-6 | 121.45* | 29.94 | 0.99* | 0.43 |
Linolelaidic acid | C18:2trans-9, trans-12 | 4.11* | 3.39 | 0.11 | 0.13 |
Conjugated linoleic acid | CLAcis-9, trans-11 | 5.99* | 4.73 | 0.20 | 0.09 |
α-Linolenic acid (ALA) | C18:3n-3 | 12.99 | 4.97 | 0.50* | 0.14 |
Stearidonic acid (SDA) | C18:4n-3 | 2.70 | 3.36 | 0.15 | 0.10 |
Arachidic acid | C20:0 | 2.94 | 1.90 | 0.11 | 0.06 |
Eisosenoic acid | C20:1n-9 | 4.21* | 2.80 | 0.35 | 0.15 |
Eisosatrienoic acid (ETE) | C20:3n-3 | 8.15* | 1.86 | 0.11* | 0.05 |
Arachidonic acid (AA) | C20:4n-6 | 33.89 | 7.60 | 1.48* | 0.74 |
Timnodonic acid, EPA | C20:5n-3 | 2.41* | 0.70 | 0.14* | 0.03 |
Erucic acid | C22:1n-9 | 0.46* | 0.24 | 0.02* | 0.01 |
Adrenic acid | C22:4n-6 | 6.20 | 1.53 | 0.28 | 0.13 |
Clupadonic acid, DPA | C22:5n-3 | 6.51* | 1.34 | 0.26* | 0.11 |
Cervonic acid, DHA | C22:6n-3 | 0.92 | 0.45 | 0.04 | 0.03 |
Lignoceric acid | C24:0 | 0.82 | 0.44 | 0.03* | 0.02 |
∑ Saturated fatty acids | SFA | 1352.06* | 777.93 | 47.55 | 2.38 |
∑ Unsaturated fatty acids | UFA | 1463.62* | 777.61 | 52.45 | 2.38 |
∑ Polyunsaturated fatty acids | PUFA | 208.34* | 44.62 | 8.57* | 3.02 |
∑ Monounsaturated fatty acids | MUFA | 1208.85* | 726.67 | 42.19* | 3.16 |
∑ trans fatty acids | TFA | 43.92* | 24.70 | 1.58 | 0.47 |
∑ n-3 fatty acids | n-3FA | 24.75 | 6.42 | 1.01* | 0.35 |
∑ n-3 long-chain PUFA | n-3 LCPUFA | 9.58 | 2.21 | 0.42* | 0.20 |
∑ n-6 fatty acids | n-6FA | 167.28* | 34.88 | 6.94 | 2.64 |
∑ n-6 long-chain PUFA | n-6 LCPUFA | 40.09 | 8.62 | 1.78 | 0.85 |
∑ Total fatty acids | FA | 2743.81* | 1524.08 | ||
Ratio n-6/n-3 | n-6/n-3 | 6.96 | 1.42 | ||
Ratio MUFA/SFA | MUFA/SFA | 0.89 | 0.09 | ||
Ratio PUFA/SFA | PUFA/SFA | 0.18* | 0.07 | ||
Ratio P/S | P/S | 0.12* | 0.05 | ||
Ratio C18:2n-6/C18:3n-3 | LA/ALA | 9.86* | 2.51 | ||
Δ9-desaturase index MUFA | Δ9MUFA | 46.39 | 2.56 | ||
Δ9-desaturase index C14 | Δ9C14 | 16.31 | 6.65 | ||
Δ9-desaturase index C16 | Δ9C16 | 11.40 | 2.08 | ||
Δ9-desaturase index C18 | Δ9C18 | 72.16 | 3.34 | ||
Intramuscular fat content | IMF | 2.56 | 1.13 |
SD: standard deviation
*Data displaying distributions significantly different from normality (p < 0.01) were log-transformed.
MUFA = C14:1 + C16:1 + C17:1 + C18:1 + C20:1 + C22:1 + C18:1cis-9 + C18:1cis-11 + C18:1trans-11
Total FA = sum of all fatty acids determined
n-3FA = C18:3n-3 + C18:4n-3 + C20:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3
n-6FA = C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:2n-6 + C22:4n-6
PUFA = n-3FA + n-6FA
n-3 LC-PUFA = C20:3n-3 + C22:6n-3 + C22:5n-3 + C20:5n-3
TFA = C18:1trans-11 + C18:2trans-9, trans-12
LA/ALA = C18:2n-6/C18:3n-3
P/S = C18:2 n-6 + C18:3 n-3/C14:0 + C16:0 + C18:0
Δ9MUFA = [(C14:1 + C16:1 + C17:1 + C18:1cis-9 + CLAcis-9, trans-11)/(C14:1 + C16:1 + C17:1 + C18:1cis-9 +
C18:1trans-11 + C14:0 + C16:0 + C17:0 + C18:0 + CLAcis-9, trans-11)] × 100
Δ9C18 = [(C18:1cis-9 + CLAcis-9, trans-11/(C18:0 + C18:1cis-9 + C18:1trans-11 + CLAcis-9, trans-11)] × 100
Primer sequences for the bovine ACSL1 gene applied for annotation confirmation, screening for polymorphisms and genotyping
Primer | Sequence (5' → 3') | Gene region | Amplicon (bp) | Position in reference sequence | AccNo. of reference sequence | Application* |
intron 1 -promoter | 844 | 1918181 - 1918198 1919003 - 1919024 |
intron 1 -promoter | 497 | 1918178 - 1918196 1918674 - 1918653 |
promoter | 413 | 1918612 - 1918633 1919003 - 1919024 |
exon 2 | 501 | 1897290 - 1897310 1897770 - 1897790 |
exon 3 | 384 | 1882758 - 1882777 1883120 - 1883141 |
exon 4 | 345 | 1881998 - 1882018 1882320 - 1882342 |
exon 5 | 457 | 1878288 - 1878309 1878724 - 1878744 |
exon 6 | 574 | 1875840 - 1875860 1876392 - 1876413 |
exon 7 | 600 | 1875542 - 1875561 1876122 - 1876141 |
exons 8-9 |
750 | 1872567 - 1872588 1873294 - 1873316 |
exon 10 | 287 | 1871656 - 1871678 1871923 - 1871942 |
exon 11 | 314 | 1869171 - 1869190 1869466 - 1869484 |
exon 12 | 371 | 1866801 - 1866821 1867153 - 1867171 |
exon 13 | 516 | 1864669 - 1864690 1865164 - 1865184 |
exon 14 | 516 | 1863888 - 1863907 1864384 - 1864403 |
exon 15 | 524 | 1862774 - 1862795 1863278 - 1863297 |
exon 16 | 247 | 1860135 - 1860155 1860361 - 1860381 |
exon 17 | 427 | 1859039 - 1859056 1859445 - 1859465 |
exon 18 | 512 | 1857386 - 1857406 1857877 - 1857897 |
exons 19-20 | 517 | 139425 - 139443 139919 - 139941 |
exon 21 | 548 | 138819 - 138836 139345 - 139366 |
exon 21 | 404 | 138633 - 138653 139016 - 139036 |
exon 21 | 650 | 137966 - 137988 138595 - 138615 |
exon 21 | 527 | 137530 - 137552 138032 - 138056 |
intron 1-promoter | 220 | 1918178 - 1918196 1918379 - 1918397 |
exon 7 | 600 | 1875542 - 1875561 1876122 - 1876141 |
promoter | 451 316 175 |
1918052 - 1918070 1918485 - 1918502 1918328 - 1918348 1918348 - 1918368 |
NW_001494406.2 | GT (Tetra-ARMS PCR) |
exons 1-5 |
505 | 38 - 58 521 - 543 |
NM_001076085.1 | cDNA |
exons 5-12 |
649 | 519 - 541 1148 - 1168 |
NM_001076085.1 | cDNA |
exons 11-18 | 735 | 1046-1069 1761 - 1781 |
NM_001076085.1 | cDNA |
exons 17-21 | 399 | 1665 - 1686 2042 - 2064 |
NM_001076085.1 | cDNA |
ACSL1_E21_R3 | GTCAAACTCCCCTCCGCTTC | exons 17-21 | 540 | 2185 - 2205 | NM_001076085.1 | cDNA, RT |
ACSL1_E21_R4 | CAGAAAGAGCAAAGTCCTAACC | 2454 - 2476 | NM_001076085.1 | cDNA, RT |
* cDNA: analysis of cDNA structure, RT: reverse transcription, GT: genotyping, SNP: screening for polymorphisms
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- Valued From
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Go bananas for the ultimate Brazilian churrasco feast at Coco Banana, and your eyes will enjoy a feast of their own with stunning waterfront views of The Entrance
All-you-can-eat Brazilian churrasco feast for two is $39 (value $90)
All-you-can-eat Brazilian churrasco feast for four is $77 (value $180)
Fill up on premium meats and tasty sides including chicken with peri peri sauce, pork with honey and chilli and pineapple with cinnamon and sugar - see the 'Menu' tab for a peek at the whole spread
Delight in the panoramic views from every corner of the restaurant
Embrace the picturesque al fresco setting right on the water's edge
Available on Friday nights - what a way to kick off the weekend!
From the fabulous food and exotic cocktails to the vibrant samba shows and pulsating South-American music, Coco Banana brings you all the passion of carnaval right on the Central Coast.
This protein-filled wonderland is all about the meat - beef, pork, chicken, lamb: you name it, they grill it. Not to mention tasty sides like salad, traditional Brazilian BBQ fried rice, fried cassava, BBQ onion and potato salad. They'll delight you with everything bar the kitchen sink!
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Over 3.2K likes on Facebook
Beautiful beachside location in The Entrance
Waiters will work the room with giant skewers of meat
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We strongly encourage bookings to be made by 24 September 2016. Bookings after this date cannot be guaranteed. If there are no available bookings after 24 September 2016, simply contact our Customer Support Team before 08 October 2016 and you will be entitled to a full credit back to your account
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Shop 4/1 Tuggerah Parade, The Entrance NSW 2261
Churrasco Menu
Chicken Maryland
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If you are looking for something different for a night out, then this is it! Great atmosphere, friendly customer service. Very attentive to all of your needs and the food is AMAZING! The dancers seem to come out every half an hour and they really make the place so lively! Fantastic! This restaurant has quickly become one of my favourites!"TripAdvisor
Amazing food that just keeps coming and coming. A great selection of all sorts of meats cooked Brazilian style. Very authentic and the traditional Brazilian cocktail was a great accompaniment. Brazilian dancers works the crowd with lots of audience participation."TripAdvisor
Thank you Coco Banana, for a great night last night, your food, service and hospitality was amazing. The dancers and host did such a awesome job. Thanks again I can't wait to eat there again."Facebook


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Dr. Ikemefuna Okwuwa is an Assistant Professor in the Department of Family and Community Medicine and also currently serving as the Regional Assistant Dean for Clinical Affairs at Texas Tech University Health Sciences Center of the Permian Basin. He received his medical degree from the University of Benin, Edo State, Nigeria and completed his Family Medicine Residency training at Texas Tech University Health Science Center of the Permian Basin in 2012 where he served as Chief Resident and House Staff President.
Dr. Okwuwa is a member of several state and national organizations including the American Academy of Family Physicians, Texas Medical Association, Texas Academy of Family Physicians (TAFP). He served as Associate Director (Resident), TAFP Board of Directors from 2011-2012, was named TAFP member of the month in April 2012 and received the Dean's Resident Teacher Award for 2011-2012.
Dr. Okwuwa is Board Certified in Family Medicine and his interests include women's healthcare, as well as resident and student education.


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- About me
- Cenantua – What and why?
- Cenantua's pages (bio sketches & so on)
- Citing… this site
November 21, 2008 by Robert Moore
Once again… all this chatter about digital history! What's it all about? Harry has put up an interesting post focusing on a piece written by Robert Frost. He uses it to help explain his interest in the potential for digital history. I get what Harry is saying. Also, consider this… Do we really realize just […]
November 5, 2008 by Robert Moore
Hmmm, I'm intriqued. I wonder how Scribd might be employed within the practice of digital history. I'm probably behind the curve on this one and would bet that it's already in use in some history classes. On another note, in gearing-up to write a literature review of materials relating to "interaction design" in digital history, I took a […]


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If you're involved with cannabis at all, whether recreationally, medically, or from a business standpoint, then you either know about or have probably heard of "dabbing." This method of consumption has been around for at least a decade, but the advent of more advanced extraction methods have led to a flood of cannabis concentrates that have boosted dabbing's popularity.
A dab usually refers to a dose of concentrate that is heated on a hot surface, usually a nail, and then inhaled through a dab rig. It doesn't sound so controversial in those terms, but "blasting dabs" has become a dividing point within the community both because of the intense high that it produces and for the image that it presents to outsiders.
While there are valid concerns to be addressed about the safety of the production and potency of popular concentrates, this new trend isn't all bad. Here's the breakdown on the issues surrounding dabs and how it might actually be a good thing for the legalization movement.
Dabs are concentrated doses of cannabis that are made by extracting THC and other cannabinoids using a solvent like butane or carbon dioxide, resulting in sticky oils also commonly referred to as wax, shatter, budder, and butane hash oil (BHO). While it's possible to extract non-psychoactive compounds like CBD, THC is what's behind the potent effects of dabs, making them the fastest and most efficient way to get really, really medicated. Terpenes, or the aromatic oils that give cannabis flavor, can also be extracted, although it can be difficult to preserve these volatile compounds in the extraction process.
A dab of BHO.
Let's start with the bad news first: dabbing can be dangerous. Actually, it's the extraction that can be dangerous.The process can be tricky, but thanks to online forums and videos, many amateur "scientists" think they have mastered the technique enough to try it on their own. In cases when things go well, the product is probably still pretty poor. When things go bad, houses blow up. A few explosions by ignorant DIYers and suddenly cannabis is being talked about in the same cautionary terms as meth. That's not good.
Additionally, the actual process of dabbing can look quite scary. Glass bongs and oddly-named substances being heated with blow torches have led to the comparison that dabs are the "crack" of pot. It's not that BHO has any similarity to these harsher drugs, but to the uninitiated, unless it's a baked Alaska or a creme brulee, lighting something with a torch never looks tasteful. While this is a matter of choice for most consumers, there are fears that dabbing's ugly looks may hurt the legalization movement.
Another side effect of these home extraction experiments is "dirty" oil that may contain chemical contaminants that could present health hazards to consumers. If the concern is butane, the dangers are most likely minimal since it already occurs in everyday products such as scent and flavor extracts. Whether or not the equipment used in the extraction process is adding additional contaminants is a more viable issue. When done correctly, these extras can be avoided, so as is the case with growing cannabis, it's best to leave it to those who know what they're doing.
One of the most unsettling facts about dabs is that thanks to the super-concentrated power of BHO, for the first time it seems possible to "overdose" on cannabis. While still not lethal, taking more than your personal limit of dabs can lead to uncomfortable highs and, in some cases, passing out. After all of the chanting that "you can't overdose on marijuana," concentrates could be undermining advocates' message of safety. There have also been reports of more intense withdrawal symptoms for dabbers, but again, the information is limited.
All oil is extracted from the tiny crystals on cannabis flowers such as this one.
The biggest positive of concentrates are that they give a powerful dose of medicine to those who truly need it. Patients dealing with severe or chronic pain or extreme nausea report that dabbing can be one of the best ways to get immediate and effective relief. The amount of flowers that would have to be smoked or vaporized to get the same effect is just unfeasible for some patients who need potent medicine quickly.
Yes, the safety issues associated with making extracts are real, but they can be easily controlled in a professional environment. Professional extractors eschew the dangerous "open" extraction method that can be done at home and instead choose closed extraction, which is safer but requires more sophisticated equipment. Also, there are other extraction methods, such as CO2 or ice-water extraction, that are safer and reduce or remove the possibility of explosions.
The relationship between concentrates and technology is symbiotic with the product in need of more research and testing that labs are excitedly advancing technology to meet. Because the industry is expanding and more and more producers are improving and upgrading their methods, it seems most likely that these homemade disasters will remain anomalies. As more places that can legally sell concentrates emerge, there will be less of a need to make your own (though intrepid home chemists will probably still exist). Technology is also probably going to lead to less dabbing in the future, anyway. Improvements in vaporizers mean that more people are using these "no-torch necessary" products to heat their oils. Conveniently, this is the most publicity-friendly path for concentrates to go.
One of the more surprising side effects of the dabbing trend is that it has created an interest in activism in the community's younger members. Industry events such as the Cannabis Cup, which used to focus on flowers, have also become proving grounds for the best concentrates and extraction experts. Most popular among consumers in their twenties, dabs are under the same legislative crackdown as other forms of cannabis, and more activists are starting to get involved.
While dabbing may be going through its awkward phase, overall, concentrates have much to offer patients and cannabis consumers in the future, and dabs are just one option among many.


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Western and Arab media did NOT report that there was another Syrian attending the Munich security conference. The Syrian Muslim Brotherhood dispatched its former leader, Ali Bayanuni, to attend and to keep a close eye on Ma`adh Al-Khatib.
A source on politics, war, the Middle East, Arabic poetry, and art.


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RRassu, Giovanna and Gavini, Elisabetta and Mattana, Antonella and Giunchedi, Paolo (2008) Improvement of antiamoebic activity of Rokitamycin loaded in chitosan microspheres. The Open Drug Delivery Journal, Vol. 2 , p. 38-43. ISSN 1874-1266. Article. |


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A Song; a Psalm of the sons of Korah. Great is Jehovah, and greatly to be praised, In the city of our God, in his holy mountain.
Beautiful in elevation, the joy of the whole earth, Is mount Zion, [on] the sides of the north, The city of the great King.
God hath made himself known in her palaces for a refuge.
For, lo, the kings assembled themselves, They passed by together.
They saw it, then were they amazed; They were dismayed, they hasted away.
Trembling took hold of them there, Pain, as of a woman in travail.
With the east wind Thou breakest the ships of Tarshish.
As we have heard, so have we seen In the city of Jehovah of hosts, in the city of our God: God will establish it for ever. Selah
We have thought on thy lovingkindness, O God, In the midst of thy temple.
As is thy name, O God, So is thy praise unto the ends of the earth: Thy right hand is full of righteousness.
Let mount Zion be glad, Let the daughters of Judah rejoice, Because of thy judgments.
Walk about Zion, and go round about her; Number the towers thereof;
Mark ye well her bulwarks; Consider her palaces: That ye may tell it to the generation following.
For this God is our God for ever and ever: He will be our guide [even] unto death.
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Download How to Train Your Dragon 2 Full Streaming
Watch Movie How to Train Your Dragon 2 High Quality with duration 102 Min and released on 2014-06-13 and MPAA rating is 284.- Original Title : How to Train Your Dragon 2
- Movie title in your country : How to Train Your Dragon 2
- Year of movie : 2014
- Genres of movie : Action, Adventure, Animation, Comedy, Fantasy, Family,
- Status of movie : Released
- Release date of movie : 2014-06-13
- Companies of movie : DreamWorks Animation,
- Countries of movie : United States of America,
- Language of movie : English,
- Durationof movie : 102 Min
- Average vote of movie : 8.4
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- Jay Baruchel ( Hiccup (voice) )
- Gerard Butler ( Stoick the Vast (voice) )
- Kristen Wiig ( Ruffnut (voice) )
- Jonah Hill ( Snotlout (voice) )
- Cate Blanchett ( Valka (voice) )
- Djimon Hounsou ( Drago Bludvist (voice) )
- Kit Harington ( Eret, Son of Eret (voice) )
- Christopher Mintz-Plasse ( Fishlegs (voice) )
- T. J. Miller ( Tuffnut (voice) )
- America Ferrera ( Astrid (voice) )
- Craig Ferguson ( Gobber (voice) )
How to Train Your Dragon 2 Synopsis:
Watch Streaming How to Train Your Dragon 2 The thrilling second chapter of the epic How To Train Your Dragon trilogy brings back the fantastical world of Hiccup and Toothless five years later. While Astrid, Snotlout and the rest of the gang are challenging each other to dragon races (the island's new favorite contact sport), the now inseparable pair journey through the skies, charting unmapped territories and exploring new worlds. When one of their adventures leads to the discovery of a secret ice cave that is home to hundreds of new wild dragons and the mysterious Dragon Rider, the two friends find themselves at the center of a battle to protect the peace. Download How to Train Your Dragon 2 For Free
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Story : Cressida Cowell, Producer : Bonnie Arnold, Producer : Chris Sanders, Music : John Powell, Production Controller : Brent Hutchinson, Production Manager : John Swanson, Visual Effects Design Consultant : Nicolas Weis, Director : Dean DeBlois, Screenplay : Dean DeBlois,
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You are here: Home>Exhibitions>'Mrs Best at the piano'
Graphite on paperBequeathed, Dr R.I. Best, the sitter's husband, 1959NGI 3310
'Gabriel could not listen while Mary Jane was playing her Academy piece, full of runs and difficult passages, to the hushed drawing room.'The Dead, p.209*
Edith Best née Oldham (1868-1950) was a talented pianist who, with her husband Dr Richard Best, organised the Dublin Feis Ceoil musical event for many years. Yeats, who was a friend of the couple, depicts Mrs Best at a concert piano, engrossed in her playing. In 1959 Dr Best bequeathed their collection of Irish paintings to the National Gallery of Ireland. The collection included Jack Yeats's painting 'In the tram', on display in this show.
*Please note: page numbers cited in the labels refer to The O'Brien Press 2012 edition of Dubliners by James Joyce, published to coincide with the 'One City, One Book' festival 2012. This edition is for sale in the NGI Shop.
Find out more about the Dublin: One City, One Book festival, an initiative of Dublin City Public Libraries


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10-17-2012, 10:52 AM | #1 (permalink) |
Hey There from Northern California!
Hi All,
My name is Jess and I'm located in Northern California. Originally from Baltimore though, so I am an Orioles fan. I collected baseball cards as a kid for many years, but then took a break for a long time. This year though my 4yr old daughter fell in love with baseball (and Manny Machado), so we ended up at our local sports card shop buying her first pack of Topps and a Machado rookie card. I've been having a lot of fun this year getting back in to collecting alongside my daughter. Glad to have found this forum and looking forward to chatting with you guys! Thanks! Jess |
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Skin infections (see above) are the most common type of disease produced by Staphylococcus. Staph infections of the skin can progress to impetigo (a crusting of the skin) or cellulitis (inflammation of the deeper layers of skin and connective tissue under the skin, leading to swelling and redness of the area). In rare situations, a serious complication known as scalded skin syndrome (see below) can develop. In breastfeeding women, staph can result in mastitis (inflammation of the breast) or in abscess of the breast. Staphylococcal breast abscesses can release bacteria into the mother's milk.
Melissa Conrad Stöppler, MD, is a U.S. board-certified Anatomic Pathologist with subspecialty training in the fields of Experimental and Molecular Pathology. Dr. Stöppler's educational background includes a BA with Highest Distinction from the University of Virginia and an MD from the University of North Carolina. She completed residency training in Anatomic Pathology at Georgetown University followed by subspecialty fellowship training in molecular diagnostics and experimental pathology.
Dr. Shiel received a Bachelor of Science degree with honors from the University of Notre Dame. There he was involved in research in radiation biology and received the Huisking Scholarship. After graduating from St. Louis University School of Medicine, he completed his Internal Medicine residency and Rheumatology fellowship at the University of California, Irvine. He is board-certified in Internal Medicine and Rheumatology.
Find out what women really need.


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This is Caleb...
Last summer, Colin had a very exciting and fun week at TriRidgefield's Kids Camp. For about a year before, Colin had great interest in running and that extended to triathlon training the more and more he heard about it.
He had no hesitation about signing up again this year. When I asked Caleb if he wanted to go, he said. "No, that's Colin's thing. My thing is baseball." I could not influence him to change his mind.
Then his school held a Rod Dickson Kids Marathon training event and Caleb got the bug. His interest did a 180 degree turn. He was in for Kids Camp 120%.
I was thrilled to see Caleb excited about this discipline, pleased to see him and his brother share an interest and tickled to know that he would be under the tutelage of Coach Cliff Scherb, Ironman.
Cliff and Caleb both have type 1 diabetes, use the OmniPod and DexCom Seven Plus systems. They both have rocking buzz cuts which enhance their speediness. :)
Caleb had a fantastic week. He had so much fun. I was worried it would be a little too intense for him. He said the workouts were exhausting, but only when I asked. His consistent reference to his days at camp were "That was fun. Camp is so much fun. I can't wait to go back because it's so fun. Fun, fun, fun!!"
Since last year's camp experience, Colin got a new bike. It is just about the only thing he got for Christmas this year. He saved up every penny that came his way and asked anyone interested in getting him a gift to contribute to his bike. After the first day of camp he felt validated in this investment. I wouldn't describe camp as uber competitive, but there are some serious kid athletes in attendance. Colin made a great improvement this year over last and finished second in his age group.
Caleb didn't place, but he did fabulously. I was so proud of both of them.
When I interviewed Cliff in 2010, I asked for advice on Colin's behalf. He emphasized swimming for the younger crowd. I saw firsthand how important this part of the race is. Colin was not the fastest swimmer, but he came out pretty strong and made up some time in the bike and run. I could see how not having any prior swim experience other than goofing around in the pool impacted Caleb's performance. He and Lila are now in weekly swim lessons! But I have to say that the improvement in his technique in just the one week of tricamp was astounding.
I cannot say enough about what nice, professional and encouraging people Evan (the camp director) and Coach Cliff are. The kids were great, the parents were great - it was a fabulous week all around.
It's been said over and over what a terrific guy Cliff is, but you'll have to tolerate me saying it yet again. He really is one of the nicest people I've ever met. This was not a diabetes camp, but Caleb and Cliff had that connection. Just knowing that there was someone around him that understood was a great influence to Caleb, and the fact that it was Cliff, a humble, kind and skilled athlete, made the experience even sweeter.
I was lucky enough to contribute to an article in Diabetes Forecast that featured Cliff that I invite you to check out. He talks about being an Ironman and living with diabetes and balancing the two. It's really no joke - this guy is an expert when is comes to endurance training and managing blood sugars. Every time I talk to him I get a new tip. I am so blessed to have had so much direct exposure to that brain trust. If training of this nature is at all an interest of yours, please read Cliff's story. He is the founder and principal coach of TriStar Labs in Norwalk, CT. We're looking forward to cheering him on this August when he races in the Ironman US Championship in New York City.
That's great that he's that excited about it at his age! Those are all very much lifetime sports that will get you in great shape and, even if you aren't at a "world championship" level, you can have a great time doing it! Then again, if you start when you're young, the sky's the limit!
What a wonderful story!
Regardless of the outcome, I think he was a winner and champion from the start! You're a great mom. I am also encouraging my son, Jacob to go forward in sports and all he chooses! What a great story!


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Highland is a hamlet (and census-designated place) in Ulster County, New York, United States. The population was 5,647 at the 2010 census. Highland is a community in the Town of Lloyd, on U.S. Route 9W. Routes 44 and 55 run through it as well. It is the closest town to the western end of the Mid-Hudson Bridge. Natives stress both syllables when pronouncing the name; it does not rhyme with "island." [edit] Research Tips


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