102,046 research outputs found
Complete CSN1S2 characterization, novel alleles identification and association with milk fatty acids composition in river buffalo
No full textThe αs2-casein is one of the phosphoproteins secreted in all ruminants milk and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterised in detail in buffaloes with only two alleles detected (reported as alleles A and B) and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffaloes homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realised at donor splice site of the exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, 6 in the promoter and 3 SNPs in the coding region (g.11072C>T, g.12803A>T and g.14067A>G) with 2 of them responsible of amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at donor splice site of the exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E and F) responsible of seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, B2 are deleted alleles because all characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acids composition was carried out. The SNP g.14067A>G showed a significant association (P<0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker assisted selection programs aiming for the improvement of buffalo milk fatty acid composition
A functional polymorphism influencing the promoter activity of alpaca α-lactalbumin gene (LALBA)
No full textAlpha-lactalbumin (α-La), encoded by LALBA gene, is a Ca2+
binding whey-protein whose key function is to facilitate lactose
synthesis by the galactosyltransferase component, serving as a
regulatory subunit. Other biological functions have been demonstrated
including immune modulation, cell growth regulation,
antimicrobial activity, etc. Gene promoters have transcription
factor (TF) binding sites necessary for gene expression regulation.
Mutations in the promoters may modify the transcription
rates or the mRNA stability, thus affecting the protein yield.
This study aims to sequence the LALBA promoter in alpacas,
identify putative TFs and detect genetic diversity affecting gene
expression.
A DNA fragment (800 bp) spanning the gene promoter until the
exon 1 was amplified and sequenced for 20 alpacas. Multiple
alignments and SNP discovery were accomplished by DNAsis
software, whereas Transfact 7.0 was used for the TF sites search.
Three independent gene reporter assays were achieved by pGL3
specific contructs to test luciferase expression in HEK 293T cells.
Data elaboration was performed using JASP software (p < 0.05,
students’s t-test).
TF binding sites analysis evidenced 16 putative consensus
sequences, including 3 C/EBPα, 3 Sp1, one NF-1, etc. Seven polymorphic
sites were found. Taking as reference the first nucleotide
of the exon 1, one SNP (g.15C > G) was found in the signal peptide,
but it is a silent mutation. The other 6 SNPs were detected
in the promoter (g.-553A > G, g.-428C > T, g.-308C > G, g.-236A > T,
g.-73C > G, g.-51A > G). The SNP g.-553A > G creates a putative
binding site of the TF Sp1. This motif is a well-known enhancer
element for the basal expression of many genes, including milk
proteins.
To assess the SNP effect on the LALBA promoter, we amplified
and cloned a DNA region of 178bp in the pGL3-basic vector from
four homozygous individuals (two g.-553AA and two g.-553GG).
The two different constructs (g.553A and g.-553G), with the pGL3
vector as a control were used to transiently transfect HEK293T
cells. After 48 h, the reporter activity of the variants was measured
using the luciferase assay system. The G variant of this SNP
enhances the promoter activity of the alpaca LALBA (p < 0.01).
Therefore, we suppose an effective role of this binding site in the expression of the α-La in alpaca milk that, consequently, may
affect the functional roles of the protein
A SNP at the exon 1 of the Lipoprotein lipase (LPL) gene is associated with milk traits in the Mediterranean River buffalo
No full textLipoprotein lipase is a key enzyme for the lipid metabolism playing a fundamental role in the composition of fat in adipose
tissue as well as in milk. LPL gene was poorly investigated in dairy ruminants, and it has been never studied in river buffalo.
Aims of this work were to explore the genetic diversity of LPL (from exon 1 to 4) in this species; to characterize the gene
promoter; and to achieve an association study with milk traits. Genomic DNA of 14 individual samples of Mediterranean
Italian breed was amplified and sequenced. Thirteen consensus sequences for transcription factors were found. C/EBPα
and Oct-1 are the main regulatory motifs together with LP-α, although a deeper investigation is necessary to elucidate the
role of the latter element, which in buffalo (and ruminants in general) is lacking the downstream cis-acting motifs (LP-β)
compared to human. A total of 11 SNPs have been detected. The SNP g.107A>G was the only polymorphism found at
exon level, therefore a genotyping was achieved by PCR-RFLP in a population of 523 Mediterranean buffaloes. The allele
frequency was 0.63 and 0.37 respectively for the G and A allele. This SNP was significantly associated with n3-PUFA
(P<0.03; 306 individual milk samples), with the GG genotype showing the highest value (+8.25% and +7.14% compared
with AA and AG, respectively). The AA and GG genotypes showed higher values also for the milk yield compared with AG,
but the estimated difference only approached the level of significance (P=0.07). This study is among the first reports that
show an association between milk fatty acids and LPL genotype in ruminants, and it adds further knowledge in the study
of genes involved in the regulation of milk fatty acids composition in river buffalo
Sequencing and Characterization of αs2-Casein Gene (CSN1S2) in the Old-World Camels Have Proven Genetic Variations Useful for the Understanding of Species Diversification
No full textThe CSN1S2 gene encodes αs2-casein, the third most abundant protein in camel milk. Despite its importance in foals, human nutrition, and dairy processing, the CSN1S2 gene in camels has received little attention. This study presents the first complete characterization of the CSN1S2 gene sequence in Old-World camels (Camelus bactrianus and Camelus dromedarius). Additionally, the gene promoter, consisting of 752 bp upstream of exon 1, was analyzed. The entire gene comprises 17 exons, ranging in length from 24 bp (exons 4, 8, 11, and 13) to 280 bp (exon 17). Interesting was the identification of the exon 12 in both species. The promoter analysis revealed 24 putative binding sites in the Bactrian camel and 22 in dromedary camel. Most of these sites were typical elements associated with milk protein, such as C/EBP-α, C/EBP-β, Oct-1, and AP1. The SNP discovery showed relatively high genetic diversity compared to other camel casein genes (CSN1S1, CSN2, and CSN3), with a total of 34 polymorphic sites across the two species. Particularly noteworthy is the transition g.311G>A in the CSN1S2 promoter, creating a new putative consensus binding site for a C/EBP-β in the Bactrian camel. At the exon level, two novel variants were found. One was detected in exon 6 of the Bactrian camel (g.3639C>G), resulting in an amino acid replacement, p.36Ile>Met. The second variant was found in noncoding exon 17 of dromedary CSN1S2 (g.1511G>T). Although this mutation occurs in the 3'-UnTranslated Region, it represents the first example of exonic polymorphism in the CSN1S2 for this species. This SNP also affects the binding sites of different microRNAs, including the seed sequence of the miRNA 4662a-3p, highlighting its role as a regulatory factor for CSN1S2 gene. A PCR-RFLP was set up for genotyping a dromedary Tunisian population (n = 157), and the minor allele frequency was found to be 0.27 for the G allele, indicating a potential yield improvement margin. The interspersed elements (INEs) analysis revealed 10 INEs covering 7.34% and 8.14% of the CSN1S2 sequence in the Bactrian and dromedary camels, respectively. Furthermore, six elements (A, B, F, H, I, and L) are shared among cattle and camels and are partially found in other ruminants, suggesting a common ancestral origin of these retrotransposons. Conversely, elements C, D, E, and G are specific to camels
Sequencing of lipoprotein lipase gene in the Mediterranean river buffalo identified novel variants affecting gene expression
No full textLipoprotein lipase (LPL) is a key enzyme for lipid metabolism, playing a fundamental role in the composition of fat in adipose tissue and milk. The LPL gene has been seldom investigated in dairy ruminants and barely studied in river buffalo (Bubalus bubalis). The aim of this work was to explore the genetic diversity of LPL and its promoter and to identify functional mutations, using a combined approach based on sequencing, dual-color electrophoretic mobility shift assay, and quantitative PCR. Thirteen consensus sequences for transcription factors were found in the promoter. Eleven SNP were detected, and the attention was focused on the SNP with potential functional effects: g.-446A>G, because the presence of G created a consensus motif for the transcription factor Sp1, and g.107A>G, which was the only exonic SNP. We developed PCR-RFLP methods for genotyping the 2 SNP and calculated the allele frequencies. A strong linkage disequilibrium (D′ = 1; r2 = 0.903) was found between the 2 SNP. The dual-color electrophoretic mobility shift assay demonstrated that only genotype g.-446GG allowed the binding of the Sp1 transcription factor, resulting in overexpression of the gene (~2.5 fold), as confirmed by the quantitative PCR results. Haploinsufficiency is proposed as a regulation mechanism. This study adds further knowledge on the structure of the LPL gene and its expression in river buffalo, with potential effects on milk qualitative and quantitative productio
Multiple-breed genomic evaluation by principal component analysis in small size populations
No full textIn this study, the effects of breed composition and predictor dimensionality on the accuracy of direct genomic values (DGV) in a multiple breed (MB) cattle population were investigated. A total of 3559 bulls of three breeds were genotyped at 54 001 single nucleotide polymorphisms: 2093 Holstein (H), 749 Brown Swiss (B) and 717 Simmental (S). DGV were calculated using a principal component (PC) approach for either single (SB) or MB scenarios. Moreover, DGV were computed using all SNP genotypes simultaneously with SNPBLUP model as comparison. A total of seven data sets were used: three with a SB each, three with different pairs of breeds (HB, HS and BS), and one with all the three breeds together (HBS), respectively. Editing was performed separately for each scenario. Reference populations differed in breed composition, whereas the validation bulls were the same for all scenarios. The number of SNPs retained after data editing ranged from 36 521 to 41 360. PCs were extracted from actual genotypes. The total number of retained PCs ranged from 4029 to 7284 in Brown Swiss and HBS respectively, reducing the number of predictors by about 85% (from 82% to 89%). In all, three traits were considered: milk, fat and protein yield. Correlations between deregressed proofs and DGV were used to assess prediction accuracy in validation animals. In the SB scenarios, average DGV accuracy did not substantially change when either SNPBLUP or PC were used. Improvement of DGV accuracy were observed for some traits in Brown Swiss, only when MB reference populations and PC approach were used instead of SB-SNPBLUP (+10% HBS, +16%HB for milk yield and +3% HBS and +7% HB for protein yield, respectively). With the exclusion of the abovementioned cases, similar accuracies were observed using MB reference population, under the PC or SNPBLUP models. Random variation owing to sampling effect or size and composition of the reference population may explain the difficulty in finding a defined pattern in the results
One-pot synthesis of hydroxamic acids from aldehydes and hydroxylamine
No full textA one-pot oxidative transformation of aldehydes into hydroxamic acids by the use of an aqueous solution of hydroxylamine is reported. The methodology gives high yields and makes use of cheap, abundant and easily available reagents
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