Genetic variability among and within domestic Old and New World camels at the α-lactalbumin gene (LALBA) reveals new alleles and polymorphisms responsible for differential expression.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

The ß-casein in camels: molecular characterization of the CSN2 gene, promoter analysis and genetic variability.

The ß-casein is the most abundant protein in camel milk and its encoding gene (CSN2) is considered in other species a 'major' gene for the presence of alleles associated to different level of expression. In the present paper, we report for the first time the characterization of the nucleotide sequence of the whole ß-casein-encoding gene (CSN2) plus 2,141 bp at the 5'-flanking region in Camelus dromedarius. The promoter region and the complete cDNA are also provided for the first time in Camelus bactrianus. The gene is spread over 7.8 kb and consists of 9 exons varying in length from 24 bp (exon 5) to 519 bp (exon 7), and 8 introns from 95 bp (intron 5) to 1,950 bp (intron 1). The composite response element (CoRE) region was identified in the promoter, whereas the presence of mature microRNA sequences improves the knowledge on the factors putatively involved in the gene regulation. A total of 46 polymorphic sites have been detected. The transition g.2126A>G falls within the TATA-box of dromedary CSN2 promoter with a putative influence on the transcription factor binding activity. The frequency of the G allele is 0.35 in a population of 180 she-camels belonging to 4 different ecotypes. In the same population, a conservative SNP (g.4175C>A) was found at the codon 7 of the signal peptide, whereas a comparative analysis with a cDNA sequence available in the database evidenced a missense SNP (g.4180T(Leu)>G(Arg)) at exon 2. Four SNPs were found in the bactrian camel. The SNP c.666G>A is responsible for the amino acid change Met(201)→Ile and it represents the first missense allele at the ß-casein in camels. Finally, five interspersed repeated elements were identified at intronic level, whereas the presence of putative bio-functional peptides belonging to ACE-inhibitor and anti-oxidative families confirms the potential protective role of the camel milk for the human nutrition.

Molecular genetic characterization of ovine CSN1S2 variants C and D reveal further important variability within CSN1S2.

Within this study, the recently identified ovine CSN1S2 variants C and D were characterized at the molecular genetic level. Sequencing of the cDNA and of parts of the DNA identified several sequence differences within CSN1S2*C and D in comparison to CSN1S2*A and B. CSN1S2*C is characterized by two non-synonymous single nucleotide polymorphisms (SNPs) within exon 7 (c.178A>G, c.187G>T) leading to the amino acid substitutions p.Val45Ile and p.Ala48Ser. CSN1S2*D is caused by the SNP c.183G>C, leading to an amino acid replacement at position 46 (p.Arg46Ser). A very common c.527G>A-SNP within exon 15, resulting in the amino acid substitution p.Arg161His and producing the new variant CSN1S2*G, not detectable by isoelectric focusing and previously misidentified as CSN1S2*A, was also identified. On the basis of the identified sequence differences, a new nomenclature is proposed and a possible phylogenetic pathway shown for ovine CSN1S2 variants.

Short communication: molecular genetic characterization of ovine alpha(S1)-casein allele H caused by alternative splicing.

Sequencing of ovine CSN1S1*H cDNA showed an absence of exon 8 in comparison with GenBank sequences; the absence was confirmed by protein sequencing. We demonstrated that this allelic aberration is the result of a deletion of 4 nucleotides, the last 3 of exon 8 and the first 1 of intron 8, which are replaced by an insertion of 13 nucleotides in the DNA sequence. The insertion is a precise duplication of a part of the adjacent intronic sequence of CSN1S1*C''. These sequence differences result in an inactivation of the splice donor sequence distal to exon 8, leading to upstream exon skipping during the serial splice reactions of the ovine CSN1S1*H pre-mRNA, and may affect the specific casein expression as well as protein characteristics.