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

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

Molecular cloning, promoter analysis and SNP identification of Italian Nicastrese and Saanen lactoferrin gene

Lactoferrin (Lf) is an iron-binding glycoprotein found in exocrine secretions including milk. High levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. In this report we sequenced and characterized goat lactoferrin cDNA and its promoter region in two different breeds of goat. The complete cDNA comprised 2356 nucleotides, including 38bp at the 5'-UTR and 194bp at the 3'-UTR. The open reading frame is 2127bp long and it encodes a mature protein of 689 aminoacids. A total of 19 nucleotide differences, 11 of them being responsible for 8 aminoacid changes, were identified through the comparison with French, Korean and Tibetan goat lactoferrin cDNAs. About 1700bp of the lactoferrin gene promoter were sequenced. Sequence analysis revealed a non-canonical TATA box, multiple SP1/GC elements, and other putative binding sites for transcription factors, such as NF-kappaB, STAT3 and AP2. Two SNPs were identified, one of which would seem to create a new putative AP2 consensus sequence. The presence of an additional AP2 binding site could be associated with quantitative differences of such protein fraction, which could enhance all the activities related to such protein, and improve mammary gland defence against bacterial infections

Genetic variability detected at the lactoferrin locus (LTF) in the Italian Mediterranean river buffalo

Lactoferrin (LTF) is multi-functional protein belonging to the whey protein fractions of the milk. The gene LTF encoding for such protein is considered a potential candidate for body measurement, milk composition and yield. This study reports on the genetic variability at LTF locus in the Italian Mediterranean river buffalo and its possible association with milk yield. Eleven polymorphic sites were found in the DNA fragment spanning the exons 15-16. In particular, the intron 15 was extremely polymorphic with 9 SNPs detected, whereas the remaining 2 SNPs were exonic mutations (g.88G>A at the exon 15 and g.1351G>A at the exon 16) and both synonymous. The genotyping of the informative samples evidenced 3 haplotypes, whose frequencies were 0.6; 0.3 and 0.1 respectively, whereas the analysis of the exonic SNPs showed a perfect condition of linkage disequilibrium (g.88A/g.1351G and g.88G/g.1351A). The association study carried out by using the SNP g.88G>A showed that buffalo LTF gene has no statistically significant influence on daily milk yield. This study adds knowledge to the genetic variability of a species less investigated than the other ruminant species, that may serve as a useful tool for large-scale screening of buffalo populations

Mediterranean river buffalo oxytocin-neurophysin I (OXT) gene: structure, promoter analysis and allele detection

Oxytocin (OXT) is a very abundant nonapeptide neurohypophysial hormone implicated in several aspects of reproduction, including social, sexual and maternal behaviour, induction of labour and milk ejection. The nucleotide sequence of the whole OXTneurophysin I encoding gene (OXT) in Mediterranean river buffalo was determined, plus 993 nucleotides at the 5’ flanking region. Buffalo oxytocin gene sequence analysis showed two transitions in the promoter region (C→T in position – 966 and G→A in position – 790) and one transversion G→T at the 170th nucleotide of the second exon, responsible for the Arg97→Leu aa substitution which identifies an allele named OXT B. A PCR-RFLP based method for a rapid identification of carriers of these alleles has been developed

Mediterranean River Buffalo CSN1S1 gene: search for polymorphisms and association studies.

The aim of the present work was to study the variability at CSN1S1 locus of the Italian Mediterranean river buffalo and to investigate possible allele effects on milk yield and its composition. Effects of parity, calving season and month of production were also evaluated. Three SNPs were detected. The first mutation, located at position 89 of 17th exon (c.628C>T), is responsible for the amino acid change (p.Ser178Leu). The other two polymorphisms, detected at the positions 144 (c.882G>A) and 239 (c.977A>G) of 19th exon respectively, are silent (3’ UTR). Associations between the CSN1S1 genotypes and milk production traits were investigated using 4,122 test day records of 503 lactations from 175 buffalo cows. Milk yield, fat and protein percentages were analyzed using a mixed linear model. A significant association between the c.628C>T SNP and the protein percentage was found. In particular, the CC genotype showed an average value of about 0.04% higher than the CT and TT genotypes. The allele substitution effect of the cytosine into the thymine was -0.014, with a quite low (0.3%) protein percentage (PP) contribution on total phenotypic variance. A large dominance effect was detected. Furthermore, a characterization of the CSN1S1 transcripts and a method based on MboI-ACRS-PCR for a rapid genotyping of c.628C>T were provided

A novel approach to study laser induced void array formation in fused silica

This study investigated multi-void formation in fused silica using high resolution Finite-Difference-Time-Domain (FDTD) simulations. Despite extensive research dedicated to understanding the mechanisms behind multi-void formation in materials, the fundamental aspects and mechanisms governing self-void array formation in dielectrics and polymers remain poorly understood. By modeling the voids as concentric spheres with densified shells and simulating the laser interaction with the voids, we showed that void array generation in fused silica is a linear mechanism. This study provides valuable insight into the mechanism behind the formation of void arrays in fused silica.Comment: arXiv admin note: substantial text overlap with arXiv:2305.0297