A SspI PCR-RFLP detecting a silent allele at the goat CSN2 locus

The comparison between the cDNA sequence obtained and the published sequences of the goat CSN2 alleles showed a new single nucleotide polymorphism (SNP) (transition C-T) at the 180th nucleotide of the ninth exon. This mutation, which took place at 124 nt from the polyadenylation site, identifies a silent allele at the CSN2 locus named CSN2 A1. Since the 9th exon C-T transition creates a SspI endonuclease restriction site, the SspI digestion of a PCR product of 360 bp spanning the 9th exon and flanking regions, would allow carriers for the presence of thymine to be identified. The allelic frequency of the CSN2 A1 allele, determined in 170 goats belonging to an undefined genetic type reared in the province of Naples (Italy), was 0.23 It has been observed that the sequences in the 3’ untranslated regions (UTR), proximal to the polyadenylation site, can affect the mechanism of mRNA deadenylation and degradation. Therefore, it is reasonable to hypothesize that the C-T transition might, directly or indirectly, influence the stability of the mRNA and, consequently, the amount of protein produced

Identification and characterization of the donkey CSN1S2 I and II cDNAs

The αs2 casein, encoded by the CSN1S2 gene, is one of the three Calcium sensitive caseins present in the milk of ruminants of zootechnical interest and in the milk of Equidae species (horse and donkey). In the present study, we cloned, sequenced and analysed two different donkey CSN1S2 cDNAs that we called CSN1S2 I and CSN1S2 II. The first, which spans over a fragment of 1016 nt, is constituted by 19 exons and encodes for a predicted protein (called αs2-I) of 221 aminoacids; the second, of which we determined the entire sequence (16 exons), encodes for a predicted peptide (called αs2-II) of 168 aminoacids. Alternative splicing and genetic markers are reported for both genes

A point mutation in the splice donor site of intron 7 in the as2-casein encoding gene of the Mediterranean River buffalo results in an allele-specific exon skipping

The CSN1S2 cDNA of 10 unrelated Mediterranean River Buffaloes reared in Southern Italy was amplified by RT-PCR, while the region from the 6th to the 8th exon of the CSN1S2 gene was amplified from genomic template. cDNA sequence comparisons showed that five individuals had a normal transcript only (named CSN1S2A), one had a deleted transcript only (named CSN1S2B), because of the splicing out of the 27-bp of exon 7, and the remaining four had a heterozygous pattern. Analysis of the genomic sequences revealed a FM865620: g.773G>C transversion that caused inactivation of the intron 7 splice donor site and, consequently, the allele-specific exon skipping characteristic of the CSN1S2B allele. The g.773G>C mutation creates a new AluI restriction site enabling a PCR– RFLP rapid genotyping assay. The cDNA sequences showed three additional exonic mutations forming an extended haplotype with the g.773G>C polymorphism: FM865618: c.459C>T, c.484A>T and c.568A>G homozygous and heterozygous respectively in the CSN1S2BB and CSN1S2AB buffaloes. The first is silent, while the remaining two are non-conservative (p.Ile162Phe and p.Thp200Ala respectively). The genotype frequencies (37 CSN1S2A/A, 15 CSN1S2A/B and one CSN1S2B/B) are in agreement with Hardy–Weinberg equilibrium, with the frequency of the deleted B allele being 0.16. The predicted bubaline as2B protein is 198 aa long instead of 207 aa and would also be characterized by the presence of Phe at position 147 and Ala at 185

Characterization of two new alleles at the goat CSN1S2 locus.

Two novel alleles at the goat CSN1S2 locus have been identified: CSN1S2(F) and CSN1S2(D). Sequence analyses revealed that the CSN1S2(F) allele is characterized by a G --> A transition at the 13th nucleotide in exon 3 changing the seventh amino acid of the mature protein from Val to Ile. The CSN1S2(D) allele, apparently associated with a decreased synthesis of alpha s2-casein, is characterized by a 106-bp deletion, involving the last 11 bp of the exon 11 and the first 95 bp of the following intron. Methods (PCR-RFLP and PCR) for identification of carriers of these alleles have been developed

Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2) plus 1,476 nt at the 5' flanking region and 51 nt at the 3' flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5) to 498 bp (exon 7) and 8 introns from 92 bp (intron 5) to 22 59 bp (intron 1). Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene) were also identified at 3 different locations of the sequenced region: 5' untranscribed region, intron 1, and intron 4

Use of multivariate factor analysis to characterize the fatty acid profile of buffalo milk

The suitability of multivariate factor analysis (MFA) to extract a small number of latent variables able to explain the correlation pattern among fatty acids (FA) in buffalo milk was evaluated. FA profile of milk samples from 214 Italian water buffaloes was analysed by gas chromatography. MFA, performed on the correlation matrix of 52 FA, was able to extract 10 latent factors with specific biological meaning related to a common metabolic origin for FA associated with the same factor. Scores of the factors were treated as new quantitative phenotypes to evaluate the effect of age, month of calving and lactation stage. MFA approach was effective in describing the FA profile of buffalo milk by using a low number of new latent variables that clustered FA having similar metabolic origin and function. The new variables were also useful to test the effect of environmental and individual animal factors on milk FA composition