Laura Ordovás

Determination of protein and RNA expression levels of common housekeeping genes in a mouse model of neurodegeneration

The choice of housekeeping proteins or genes for internal standards should be made carefully, taking into account the cell and tissue type, the experimental conditions, and the healthy/disease state(s) under consideration. Furthermore, as the correlation between transcriptional and translational levels of commonly used housekeeping genes is often discussed, this study shed light on the transcriptional levels of β‐actin and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and the translational levels of β‐actin, GAPDH, and β‐tubulin in an amyotrophic lateral sclerosis mouse model.

Structural and functional characterization of the bovine solute carrier family 27 member 1 (SLC27A1) gene

The Solute Carrier Family 27 Member 1 (SLC27A1) is an evolutionarily conserved protein involved in regulating the long chain fatty acid uptake into cells. It has been shown to be expressed in tissues undergoing rapid fatty acid metabolism such as heart, skeletal muscle and adipose tissues, but no expression is detected in liver. Here we report the molecular characterization of the bovine SLC27A1 gene and draw a comparison with orthologous genes of some monogastric species. The bovine SLC27A1 gene is organized in 13 exons and extends over more than 40 kb of genomic DNA. It codes for a protein of 646 amino acids with a predicted molecular weight of 71 kDa which has 92%, 88% and 88% similarity with the human, mouse and rat SLC27A1 proteins respectively. The bovine SLC27A1 RNA expression was high in heart, testis, nervous tissue and muscle and very low in liver. Surprisingly, adipose tissues showed very low RNA expression levels contrary to the results described for both human and mouse genes. On the other hand, discordances observed between the bovine SLC27A1 RNA and protein expression patterns suggest that complex regulation mechanisms may be involved in determining the final SLC27A1 protein levels in each tissue. Finally, we have identified an alternative transcript generated by exon skipping of exon 3 to 7 which could encode a cytosolic SLC27A1 isoform of ∼37 kDa.

Genomic structure and an alternative transcript of bovine mitochondrial glycerol-3-phosphate acyltransferase gene (GPAM)

GPAM maps in BTA26q22, where several QTLs affecting milk production, milk fat and protein content have been mapped. On the basis of the QTL location, the GPAM gene could be considered a good candidate gene for the mentioned traits. Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyses the initial and committed step of glycerolipid synthesis and, therefore, it is a potential site for triacylglycerol synthesis regulation. In this study, the structure of the cDNA and the genomic DNA of the bovine GPAM gene were determined and the expression of its mRNA was studied. The cDNA of the gene was cloned by RT-PCR, 5′ and 3′ rapid amplification of cDNA ends. The GPAM mRNA sequence contains a 2,475-bp coding region and a 3,689-bp 3′ UTR. Its ORF encoded for an 825-amino acid protein and has an 89% homology with the coding regions of previously characterized mouse and human GPAM genes. The predicted amino acid sequence had an 89 and 93% similarity with mouse and human GPAM proteins, respectively. Using a 5′ RACE strategy, two different 5′ UTRs were cloned. Northern blot analysis confirmed the presence of two different transcripts. Adipose tissues and lung had the highest levels of GPAM mRNA expression, whereas it was barely detectable in liver. This expression pattern differs with those of non-ruminant animals where liver is one of the tissues with higher GPAM mRNA expression level.

Identification of 14 new single nucleotide polymorphisms in the bovine SLC27A1 gene and evaluation of their association with milk fat content.

The solute carrier family 27 member 1 (SLC27A1) is an integral membrane protein involved in the transport of long-chain fatty acids across the plasma membrane. This protein has been implicated in diet-induced obesity and is thought to be important in the control of energy homeostasis. In previous reports, our group described the isolation and characterization of the bovine SLC27A1 gene. The bovine gene is organized in 13 exons spanning over more than 40 kb of genomic DNA and maps in BTA 7 where several quantitative trait loci for fat related traits have been described. Because of its key role in lipid metabolism and its genomic localization, in the present work the search for variability in the bovine SLC27A1 gene was carried out with the aim of evaluating its potential association with milk fat content in dairy cattle. By sequencing analysis of all exons and flanking regions 14 new single nucleotide polymorphisms (SNPs) were identified: 1 in the promoter, 7 in introns and 6 in exons. Allele frequencies of all the SNPs were calculated by minisequencing analysis in two groups of Holstein-Friesian animals with highest and lowest milk-fat content estimated breeding values as well as in animals of two Spanish cattle breeds, Asturiana de los Valles and Menorquina. In the conditions assayed, no significant differences between Holstein-Friesian groups were found for any of the SNPs, suggesting that the SLC27A1 gene may have a poor or null effect on milk fat content. In Asturiana and Menorquina breeds all the positions were polymorphic with the exception of SNPs 1 and 8 in which C allele was fixed in both of them.

Prevalence and sequence comparison of Phyllodistomum folium from zebra mussel and from freshwater fish in the Ebro River

We utilised DNA analysis to detect the presence of the digenean Phyllodistomum folium in three cyprinid species, Scardinius erythrophthalmus, Cyprinus carpio and Rutilus rutilus. DNA sequencing of the region containing the genes ITS1-5.8S-ITS2 revealed 100% sequence identity between DNA from the sporocysts found in zebra mussels and DNA from adults located in the urinary system of 29 cyprinid fish. A second genetically different (variation=1.6%) sequence was observed in two samples from R. rutilus. In our opinion, the existence of a complex of species reported as P. folium is supported by recent genetic studies, including our own results. The overall prevalence of P. folium in mussels from the Ebro River was 4.67% in 2006, although during the summer months the rates frequently exceeded 10%.

A false single nucleotide polymorphism generated by gene duplication compromises meat traceability

Controlling meat traceability using SNPs is an effective method of ensuring food safety. We have analyzed several SNPs to create a panel for bovine genetic identification and traceability studies. One of these was the transversion g.329C>T (Genbank accession no. AJ496781) on the cytochrome P450 17A1 gene, which has been included in previously published panels. Using minisequencing reactions, we have tested 701 samples belonging to eight Spanish cattle breeds. Surprisingly, an excess of heterozygotes was detected, implying an extreme departure from Hardy-Weinberg equilibrium (P<0.001). By alignment analysis and sequencing, we detected that the g.329C>T SNP is a false positive polymorphism, which allows us to explain the inflated heterozygotic value. We recommend that this ambiguous SNP, as well as other polymorphisms located in this region, should not be used in identification, traceability or disease association studies. Annotation of these false SNPs should improve association studies and avoid misinterpretations.