Exon skipping events showed by gel electrophoresis analysis.

<p>Gel electrophoresis analysis of the clones showing exon skipping events. Lines 1–2 shorter (397 bp) and longer (421 bp) mRNA variant showing the splice out the exon 18 for the <i>CSN1S1</i> (<b>a</b>). Lines 4–5 shorter (142 bp) and longer (185 bp) mRNA variant showing the skipping of the cryptic exon 2 for the <i>CSN3</i> (<b>b</b>).</p

Lama glama complete CSN1S1 cDNA (αs1-casein).

<p>Complete cDNA sequence (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/LK999986" target="_blank">LK999986</a>) and exon subdivision (brackets) of <i>L</i>. <i>glama CSN1S1</i> (upper line) and comparative alignment with the homologous αs1-casein cDNA of <i>C</i>. <i>dromedarius</i> (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/AJ012628" target="_blank">AJ012628</a>). Dashes represent identical nucleotides to those in upper lines. In lower cases the 5’- and 3’- Un-Translated Regions (UTR), the polyadenylation signal (Pas) is indicated by a box. The corresponding mature protein is reported in bold, whereas the signal peptide is in italics and asterisk represents the termination stop codon. Putative phosphorylation sites predicted with a score over 90% are indicated with P. Phosphorylated serines reported by Kappeler et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124963#pone.0124963.ref015" target="_blank">15</a>] are underlined, whereas the plusses indicate Ser preferentially phosphorylated by Fam20C.</p

Lama glama complete <i>CSN1S2</i> cDNA (αs2-casein).

<p>Complete cDNA sequence (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/LK999989" target="_blank">LK999989</a>) and exon subdivision (brackets) of <i>L</i>. <i>glama CSN1S2</i> (upper line) and comparative alignment with the homologous αs2-casein cDNA of <i>C</i>. <i>dromedarius</i> (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/AJ012629" target="_blank">AJ012629</a>). Dashes represent identical nucleotides to those in upper lines. In lower cases the 5’- and 3’- Un-Translated Regions (UTR), the polyadenylation signal (Pas) is indicated by a box. The corresponding mature protein is reported in bold, whereas the signal peptide is in italics and asterisk represents the termination stop codon. Putative phosphorylation sites are indicated with P. Phosphorylated serines reported by Kappeler et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124963#pone.0124963.ref015" target="_blank">15</a>] are underlined, whereas the plusses indicate Ser preferentially phosphorylated by Fam20C.</p

Splicing combinations and cryptic spliceosome mechanism at the κ-casein gene (CSN3) Schematic representation of the aligned exon structure of the llama κ-casein gene (<i>CSN3</i>).

<p>The top of the figure shows the two possible splicing combinations with (upper) and without (lower) the cryptic exon. The down part of the figure shows in details the sequence of cryptic exon 2 (in brackets) with the respective 5’- and 3’- intronic flanking regions. The branch point site is fully underlined and the bold adenine represent the main branch point (black triangle), followed by a polypyrimidine tract (Py_n) and the acceptor splice site (AS). The dotted-line and the white triangles represent additional polypyrimidine tracts and alternative branch points, respectively. The donor splice site (DS) for the following intron splicing is normally present.</p

Lama glama complete <i>CSN3</i> cDNA (κ-casein).

<p>Complete cDNA sequence (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/LK999995" target="_blank">LK999995</a>) and exon subdivision (brackets) of <i>L</i>. <i>glama CSN3</i> (upper line) and comparative alignment with the homologous κ-casein cDNA of <i>C</i>. <i>dromedarius</i> (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/Y10082" target="_blank">Y10082</a>). Dashes represent identical nucleotides to those in upper lines. In lower cases the 5’- and 3’- Un-Translated Regions (UTR), the polyadenylation signal (Pas) is indicated by a box. The corresponding mature protein is reported in bold. The chymosin cleavage site (Phe⁹⁷-Ile⁹⁸) is indicated by a flash-arrow, whereas the glycomacropeptide (GMP) 65 amino acids long is reported in bold italics. The signal peptide is in italics and asterisk represents the termination stop codon. P, phosphorylated serine, whereas the plusses indicate Ser preferentially phosphorylated by Fam20C; G, glycosylated threonines.</p

Lama glama complete <i>CSN2</i> cDNA (β-casein).

<p>Complete cDNA sequence (EMBL acc. no.: <a href="http://www.ncbi.nlm.nih.gov/nuccore/LK999992" target="_blank">LK999992</a>) and exon subdivision (brackets) of <i>L</i>. <i>glama CSN2</i> (upper line) and comparative alignment with the homologous β-casein cDNA of <i>C</i>. <i>dromedarius</i> (EMBL acc. no: <a href="http://www.ncbi.nlm.nih.gov/nuccore/AJ012630" target="_blank">AJ012630</a>). Dashes represent identical nucleotides to those in upper lines. In lower cases the 5’- and 3’- Un-Translated Regions (UTR), the polyadenylation signal (Pas) is indicated by a box. The corresponding mature protein is reported in bold, whereas the signal peptide is in italics and asterisk represents the termination stop codon. Putative phosphorylation sites are indicated with P. Phosphorylated serines reported by Kappeler et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124963#pone.0124963.ref015" target="_blank">15</a>] are underlined, whereas the plusses indicate Ser preferentially phosphorylated by Fam20C.</p

Genetic variability detected by the comparison among the casein genes transcripts in llama and the dromedary camel cDNAs reported by Kappeler et al.

<p>Polymorphic nucleotides in the triplets are underlined, when they fall within the peptide leader they are also reported in Italics. Amino acid changes are showed in bold. Insertions/deletions are reported as dashes. Numbering is relative to the llamas cDNAs.</p><p>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124963#pone.0124963.ref015" target="_blank">15</a>].</p

Fluorescence in situ hybridization of an <i>EDNRB</i>-spanning bovine BAC clone on metaphase chromosomes.

<p>Results from a sheep related to hypopigmented lambs (A), and from an unrelated sheep (B). Arrows indicate positions of (expected) hybridization signals.</p

Impact of air pollution from different sources on sperm DNA methylation

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 – 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen.</p

Agarose gel electrophoresis of DNA fragments resulting from duplex PCR.

<p>Lane 3: sample from sheep homozygous for the deletion (<i>EDNRB −/−</i>), lane 4: sample from sheep heterozygous for the deletion (<i>EDNRB +/−</i>), lane 5: sample from sheep without the deletion (<i>EDNRB +/+</i>); lane 1: DNA size marker (Gene Ruler 100 bp Plus DNA Ladder, Fermentas, St. Leon-Rot, Germany); lane 2: no template control.</p