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

The complete nucleotide sequence of goat (Capra hircus) mitochondrial genome.

The goat mtDNA sequences reported to date are fragmentary. By using both in silico cloning procedure and conventional molecular biology techniques we have determined the complete nucleotide sequence of the goat (Capra hircus) mitochondrial genome. The length of the sequence was 16.640bp. Genes responsible for 12S and 16S rRNAs, 22 tRNAs and 13 protein-coding regions are found. The genome organization is conformed to those of other mitochondrial genomes. Comparison between the 13 protein coding genes of goat, cow and sheep reveals that the difference range from 1.2 to 12.2% with a mean of 7.3% between goat and cow and from 0 to 15.6% (mean 4.7%) between goat and sheep

Tracing the origin of raw milk from farm by using automated ribosomal intergenic spacer analysis (ARISA) fingerprinting of microbiota

The aim of the study was to distinguish the raw milk from different farms in relation to their geographical sites within a narrowed territorial district. The goal was achieved by applying a molecular based system for traceability that uses microbial DNA barcodes present in milk. Microbiota of milk were fingerprinted by PCR of the 16Se23S intergenic transcribed spacer using the Automated Ribosomal Intergenic Spacer Analysis (ARISA). A total of 64 markers within the range 279e756 bp were detected on the thirty-eight bulk milk samples, none of which was common to all the patterns. Overall samples did not show relevant differences across the two years of sampling. In fact, every farm maintained a specific core profile over time, thus demonstrating that the interaction between site and year of sampling is not significant and that the variability between years does not affect the distinction between grouping of farms. The system was able to trace the geographical origin of raw milk with a resolution of less than 5 km. According to the European regulations for the protection of the geographical names of foodstuffs which have a tangible link to the territory, the ARISA system described here may represent a suitable analytical tool for tracing the origin of milk integrating and reinforcing traceability processes of the dairy chain

Characterization of the major whey proteins from milk of Mediterranean water buffalo

In this work, the whey protein fractions from 120 Mediterranean water buffalo individual milks were analysed by microchip electrophoresis (MCE), reverse-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (ESI-MS). Validation procedures were carried out for both MCE and HPLC. The chromatographic analysis allowed the complete separation of the whey protein fractions, resulting in a well-defined peak structure; the adopted RP-HPLC and ESI-MS protocols provided identification of b-lactoglobulin (18,266 Da), a-lactoalbumin (14,236 Da) and serum albumin (66,397 Da). The calculated mean concentrations were 4.04 g/l, 2.45 g/l and 0.35 g/l, respectively