The <i>LST1</i> exon 1B sequence inhibits protein expression.

<p>HeLa and HEK-293T cells were transiently cotransfected with expression constructs encoding the red fluorescent protein mCherry and either an EGFP expression vector, Exon1B-EGFP or Exon 1C-EGFP fusion construct. <b>(A, B)</b> Flow cytometry analysis of EGFP intensity in HeLa (A) and HEK-293T (B) transfectants expressing Exon1B-EGFP (yellow line), Exon 1C-EGFP (green line) or the unmodified EGFP vector (blue line). Untransfected cells are displayed as a solid grey curve. The mCherry expression was used to gate and select positive transfectants, which were analysed for the intensity of EGFP expression. <b>(C)</b> Lysates from HEK-293T transfectants were probed by western blot analysis using a GFP-specific antibody (lower panel). To ensure that comparable amounts of protein were loaded, the membrane was additionally probed with a tubulin-specific antibody (upper panel). <b>(D, E)</b> Quantitative flow cytometry analysis of EGFP expression in HeLa (D) and HEK-293T (E) transfectants. The analysis of EGFP expression was performed as described in (A, B) and the mean fluorescence intensity was quantified. A value of 100% was set for cells transfected with the unmodified EGFP vector. Mean values from 5 independent experiments are indicated within the columns +/− s.d. Both HeLa and HEK-293T transfectants expressing Exon1B-EGFP displayed significantly reduced EGFP levels when compared with cells transfected with the empty vector (p = 0.009). <b>(F)</b> Quantitative western analysis of EGFP expression. Lysates were probed as described in (C). The EGFP signal intensity was quantified and normalized for tubulin expression. A value of 100% was set for lysates from cells transfected with the unmodified EGFP vector. Mean values from 3 independent experiments are indicated within the columns +/− s.d. Transfectants expressing Exon1B-EGFP displayed significantly reduced EGFP expression levels when compared with cells transfected with the empty vector (p = 0.049).</p

The <i>LST1</i> 5′-UTR contains several uORFs.

<p><b>(A)</b> Schematic overview of the <i>LST1</i> 5′ exons. All <i>LST1</i> transcripts contain one of the noncoding exons 1A–E, which include transcription initiation sites, followed by the exon 2 sequence, which contains the start codon of the main ORF. The five alternative exons 1A–E and the exon 2 sequence are displayed as grey boxes; introns are represented by black lines. The start codons of uORFs 1–7 are indicated; additionally an uAUG in exon 1E is annotated. <b>(B)</b> List of uORFs in the <i>LST1</i> 5′-UTR and their corresponding lengths in base pairs from start to stop codon. <b>(C)</b> Sequence of the <i>LST1</i> 5′-UTR in transcripts initiated at exon 1B. The exon 1B and exon 2 sequences are labelled; the AUGs of uORF4 and of the main ORF are highlighted in grey. The amino acids encoded are indicated beneath the nucleic acid sequence. <b>(D)</b> Sequence comparison of the exon 1B sequence flanking the start codon of uORF4. The sequence is conserved in pan troglodytes (sequence accession number NW 003457113.1), macaca mulatta (NW 001116486.1), sus scrofa (NW 003610614.1) and bos taurus (NW 003104557.1).</p

Sample size (N), haplogroup counts and diversity in the population groups studied.

a<p>The phylogeny of haplogroups is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053731#pone.0053731.s001" target="_blank">Figure S1</a>.</p><p>Relative haplogroup frequencies are given within parentheses.</p><p>H, haplogroup diversity; SD, standard deviation.</p

Median-joining networks showing phylogenetic relationships of the Moldavian, Romanian and Ukrainian Y-haplotypes within haplogroups I-M423 (A) and R-M17* (B).

<p>Networks were constructed from 17 STRs for haplogroup R-M17* and 15 STRs for haplogroup I-M423 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053731#s2" target="_blank">Materials and Methods</a>. For each network, the smallest circles represent a count of one individual. Branch lengths are proportional to the number of mutational steps separating two haplotypes.</p

Geographical location of the samples analyzed.

<p>Geographical location of the samples analyzed.</p

PCR-RFLP protocols developed for P15, v13, U8 and M423 markers.

<p>F refers to the forward primer, and R refers to the reverse primer for a particular locus.</p>a<p>PCR annealing temperature in C°.</p>b<p>PCR product size in base pairs.</p>c<p>RFLP fragments in base pairs.</p>d<p>Mismatched primer (mismatched bases are underlined).</p

Multidimentional Scaling Analysis (MDS) plot of R_ST values from Y chromosome STR haplotype frequencies showing genetic affinities among 29 populations from eastern, central and southern Europe.

<p>The stress value for the MDS plot is 0.056. Original references, number of samples and haplotypes and name codes for all populations included in the analysis are listed in Table S3. Symbol designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053731#pone-0053731-g002" target="_blank">Figure 2</a>. The ellipses are drawn around the clusters of east/central European (eastern and western Slavic) (I), southeast European (Balkan-Carpathian) (II) and Italian (III) populations. While the Cheg Albanian population is a genetic “outlier” among southeast European populations, geographically it belongs in the Balkan-Carpathian cluster (indicated by an arrow).</p

AMOVA calculation results for 10 Y-STRs.

a<p>Twenty nine populations (the same as in MDS analysis) were pooled into five groups defined according to ethnicity (Moldavians, Romanians, Eastern and Western Slavs, Italians) or geography (Balkans) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053731#pone.0053731.s004" target="_blank">Table S3</a>).</p

Principal Component Analysis (PCA) plot of Y chromosome haplogroup profiles showing genetic affinities among 28 populations from eastern, central and southern Europe.

<p>Original references, number of samples and name codes for all populations included in the analysis and their haplogroup frequencies are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053731#pone.0053731.s002" target="_blank">Table S1</a>. Moldavian populations are indicated by stars; north Mediterranian groups by diamonds; eastern and central European populations including Ukrainians from Moldova by circles, and north Balkan groups including Romanians by squares.</p