Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions

Abstract

In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5’- and 3’-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species.

The single nucleotide polymorphism g.133A>C in the stearoyl CoA desaturase gene (SCD) promoter affects gene expression and quali-quantitative properties of river buffalo milk

“…The ligation products were transformed into JM109 High-Efficiency Competent Cells (Promega) following the manufacturer's guidelines. White recombinant clones were randomly chosen and screened by PCR according to Pauciullo and Erhardt (2015) using the following combination of primers: M13 For 5′-GTAAAACGAC-GGCCAGT-3′ and M13 Rev 5′-AACAGCTATGAC-CATG-3′.…”

Lama glama αS1-casein: Identification of new polymorphisms in the CSN1S1 gene

“…The mix was set up in a final volume of 20 µL using Verso Reverse Transcriptase (Thermo Fisher Scientific Inc.) according to the standard protocol recommended by the firm. The PCR was performed according to Pauciullo and Erhardt (2015) using primers (forward: 5′-AGTTTGCTGCTTCTTCCC-3′; reverse: 5′-ATGGCAGTTACAGGAGAAG-3′) corresponding to nucleotides 21 to 38 of the first exon and complementary to the nucleotides 295 to 313 of the 21st exon of the llama CSN1S1 cDNA (EMBL ID: LK999986).…”

“…A DNA fragment 153 bp long (estimated according to EMBL Acc. N. KN269544.1) spanning from bp 2 of exon 11 to the 36th nucleotide of exon 12 of the llama CSN1S1 gene was amplified by using the primers (forward) 5′-AAGTTGTTTCCAGTACCAC-3′ and (reverse) 5′-TTGGGAGGGCATATCTT-3′, as well as the same PCR mixture conditions reported by Pauciullo and Erhardt (2015). Thermal conditions were 95°C for 4 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 20 s, with the final extension at 72°C for 5 min.…”

“…In fact, whereas dromedary camel milk has been characterized with regard to its protein fractions (Kappeler et al, 1998;Shuiep et al, 2012;Pauciullo et al, 2013Pauciullo et al, , 2014 and biologically active peptides (Erhardt et al, 2016), very little information is available on milk proteins from South American camelids, including llamas. Early studies on llama milk were focused on the composition in fat, protein, lactose, microminerals, and so on (Morin et al, 1995;Riek and Gerken, 2006), whereas only recently a detailed description of llama milk proteins has been provided (Saadaoui et al, 2014) and the genetic basis of casein synthesis has been elucidated (Pauciullo and Erhardt, 2015).…”

“…Two genetic variants of α S1 -CN have been described in dromedary camels by Kappeler et al (1998) and recently clarified by Erhardt et al (2016), considering a new allele identified by Shuiep et al (2012). Conversely, in llama, protein isoforms have been found for α S1 -CN by Saadaoui et al (2014) and confirmed as an exon-skipping event by Pauciullo and Erhardt (2015), whereas 2 variants (A and B) were reported by Saadaoui et al (2014).…”