Genetic characterization of the oxytocin-neurophysin I gene (<i>OXT</i>) and its regulatory regions analysis in domestic Old and New World camelids

<div><p>Oxytocin is a neurohypophysial peptide linked to a wide range of biological functions, including milk ejection, temperament and reproduction. Aims of the present study were a) the characterization of the <i>OXT</i> (Oxytocin-neurophysin I) gene and its regulatory regions in Old and New world camelids; b) the investigation of the genetic diversity and the discovery of markers potentially affecting the gene regulation. On average, the gene extends over 814 bp, ranging between 825 bp in dromedary, 811 bp in Bactrian and 810 bp in llama and alpaca. Such difference in size is due to a duplication event of 21 bp in dromedary. The main regulatory elements, including the composite hormone response elements (CHREs), were identified in the promoter, whereas the presence of mature microRNAs binding sequences in the 3’UTR improves the knowledge on the factors putatively involved in the <i>OXT</i> gene regulation, although their specific biological effect needs to be still elucidated. The sequencing of genomic DNA allowed the identification of 17 intraspecific polymorphisms and 69 nucleotide differences among the four species. One of these (MF464535:g.622C>G) is responsible, in alpaca, for the loss of a consensus sequence for the transcription factor SP1. Furthermore, the same SNP falls within a CpG island and it creates a new methylation site, thus opening future possibilities of investigation to verify the influence of the novel allelic variant in the <i>OXT</i> gene regulation. A PCR-RFLP method was setup for the genotyping and the frequency of the allele C was 0.93 in a population of 71 alpacas. The obtained data clarify the structure of <i>OXT</i> gene in domestic camelids and add knowledge to the genetic variability of a genomic region, which has received little investigation so far. These findings open the opportunity for new investigations, including association studies with productive and reproductive traits.</p></div

Polymorphisms detected by the comparison among the complete sequences of <i>OXT</i> gene and the regulatory regions of domestic camelids investigated in the present study (<i>C</i>. <i>dromedarius</i>, <i>C</i>. <i>bactrianus</i>, <i>V</i>. <i>pacos</i>, <i>L</i>. <i>glama</i>).

<p>Polymorphisms detected by the comparison among the complete sequences of <i>OXT</i> gene and the regulatory regions of domestic camelids investigated in the present study (<i>C</i>. <i>dromedarius</i>, <i>C</i>. <i>bactrianus</i>, <i>V</i>. <i>pacos</i>, <i>L</i>. <i>glama</i>).</p

Genetic diversity detected by the sequencing of the <i>OXT</i> gene and its regulatory regions in domestic camelids (<i>C</i>. <i>dromedarius</i>, <i>C</i>. <i>bactrianus</i>, <i>V</i>. <i>pacos</i>, <i>L</i>. <i>glama</i>).

<p>Genetic diversity detected by the sequencing of the <i>OXT</i> gene and its regulatory regions in domestic camelids (<i>C</i>. <i>dromedarius</i>, <i>C</i>. <i>bactrianus</i>, <i>V</i>. <i>pacos</i>, <i>L</i>. <i>glama</i>).</p

Genotyping of the g.622C>G at alpaca <i>OXT</i> promoter.

<p>Genotyping of the SNP MF464535:g.622C>G in the promoter region of <i>Vicugna pacos OXT</i> by <i>Bfo</i> I PCR-RFLP. Line 1, CC homozygous sample; line 3, GG homozygous sample; line 2, heterozygous sample. Line L, Mid Range DNA ladder 100bp-3kb (Jena Bioscience).</p

Primer sequences, annealing temperature (T_a) and amplicon size used for the sequencing and genetic diversity discovery at <i>OXT</i> locus in domestic camelids.

<p>Primer sequences, annealing temperature (T_a) and amplicon size used for the sequencing and genetic diversity discovery at <i>OXT</i> locus in domestic camelids.</p

MicroRNA target sequences affected by SNP at 3’UTR.

<p>The transversion C>G (MF464535:g.1682C>G in alpacas and MF464534:g.1731C>G in llamas) falling 19 bp downstream the stop codon (underlined) affects different microRNA target sequences with 8mer (mir-4651, mir-608), 7mer-m8 (mir-6737-5p reported as example, but also mir-6819-5p) and 7mer-A1 (mir-6747-5p, mir-342-5p and mir-4664-5p). Binding of mature miRNAs are shown, whereas the site of the SNP is indicated in bold.</p

Temporins are effective in vivo when given 7 days after experimental infection.

<p>Mice were infected with a sub-lethal dose (10⁵ CFU/mouse) of <i>S.aureus</i> A170 (A) or a sub-lethal dose(10⁵ CFU/mouse) of <i>S.enterica</i> serovar Paratyphi B (B–C) and 6 days later were treated with the 35/75 pool of temporins. A: kidneys from mice infected with <i>S.aureus</i> A170 (closed box) and kidneys from mice infected with <i>S.aureus</i> A170 and treated after 6 days with TA plus TB-YK (open rhomb); B: gastro instestinal tract from mice infected with <i>S.enterica</i> serovar Paratyphi B (closed box ) and gastro intestinal tract from mice infected with <i>S.enterica</i> serovar Paratyphi B and treated after 6 days with TA plus TB-YK (open rhomb); C: liver from mice infected with <i>S.enterica</i> serovar Paratyphi B (10⁵ CFU/mouse) (closed box) and liver from mice infected with <i>S.enterica</i> serovar Paratyphi B (10⁵ CFU/mouse) and treated after 6 days with the TA plus TB-YK (open rhomb).</p

Temporins initiate their antibacterial activity by drillings holes in the bacterial membrane.

<p>A: <i>S.aureus</i> A170, untreated; B: <i>S aureus</i> A 170 treated for 10 min with TA (8 µg/ml) plus TB-YK (5 µg/ml) (B); C: <i>S.enterica</i> serovar Paratyphi B untreated; D: <i>S.enterica</i> serovar Paratyphi B treated for 10 min with TA (100 µg/ml) plusTB-YK (4 µg/ml).</p

<i>OXT</i> gene in domestic camelids.

<p>Comparative alignment of the complete nucleotide (nt) sequences of oxytocin-neurophysin I encoding (<i>OXT</i>) gene in domestic camelids. Numbering is relative to the first nucleotide of the first exon (+1) and dashes represent nt identical to those in the first line. In lower cases the 5’- and 3’- Un-Translated Regions (UTR), the polyadenylation signal is dot-underlined. The coding region corresponding to the signal peptide is underlined, whereas the sequence coding for the nonapeptide hormone is indicated in bold, and the neurophysin I is in bold italics. The tripeptide processing signal (GKR) is double underlined and asterisks indicate the stop codon. The duplication event of 21bp in <i>C</i>. <i>dromedarius</i> is wave-underlined. Polymorphic sites within the investigated samples are indicated with R = A/G, S = C/G and Y = C/T.</p

Mice infected with GFP-labelled bacteria were analysed using the Leica macrofluo instrument (Wetzlar, Germany) equipped with the Leica application suite 3.1.0 software.

<p>A: liver from mice infected with <i>S. enterica</i> serovar Paratyphi B; B: liver from mice infected with <i>S. enterica</i> serovar Paratyphi B and immediately treated with TA plus TB-YK; C: gastro instestinal tract from mice infected with <i>S. enterica</i> serovar Paratyphi B; D: gastro instestinal tract from mice infected with <i>S. enterica</i> serovar Paratyphi B and immediately treated with TA plus TB.</p