Reciprocal crosses between individuals carrying (+) and not carrying (−) <i>bobt</i> and individuals carrying <i>bobt_KR</i> and <i>bobt _MV</i> alleles.

<p>The presence of <i>bobt</i> was determined using a PCR assay with allele specific primers.</p

Transcript levels of <i>bobt_MV</i> and <i>bobt_KR</i> in flower buds estimated by qRT PCR.

<p>Expression is presented as a ratio of the accumulation of PCR product for <i>bobt</i> and the mt 18S rRNA reference gene. Ratios for <i>bobt_MV</i> are multiplied by 100 to allow visualization of difference on the same scale as <i>bobt_KR</i>. The standard deviation was calculated on the basis of 3–5 sibling pairs F and H. The differences in <i>bobt</i> expression between genders were significant in both MV and KR plants (t-test P<0.05).</p

Southern hybridization results with <i>bobt</i>-specific and <i>atp1</i>-specific probes.

<p>Genomic DNA of <i>Silene</i> from various sites was digested with EcoRI and hybridized with probes derived from <i>atp1</i> and <i>bobt_MV</i>. The same membrane was hybridized with both (<b>A</b>) an <i>bobt_MV</i> probe and (<b>B</b>) an <i>atp1</i> probe. Lanes 1–5 <i>S. vulgaris</i> Mt. View; 6–8 <i>S. vulgaris</i> Krasnoyarsk; 9–11 <i>S. vulgaris</i> Beagle; 12 <i>S. vulgaris</i> Krasnoyarsk; 13 <i>S. vulgaris</i> Beagle; 14–16 <i>S. latifolia</i> Prague. Individuals from the Beagle population differ in Southern-RFLP pattern of <i>atp1</i> region, which is very common in <i>S. vulgaris</i> populations. Faint, but visible, bands visible in Beagle and Krasnoyarsk DNA hybridized with a <i>bobt_MV</i> probe may correspond to weakly homologous regions in nuclear or mt DNA. The bands corresponding to <i>bobt_KR</i> are a bit weaker than <i>bobt_MV</i> bands due to nucleotide divergence between the two variants. One band detected by <i>bobt</i> probes suggests the existence of only one <i>bobt</i> copy in the mt genome, whereas two or three <i>atp1</i>-specific bands reflect the existence of two or three <i>atp1</i> copies. Marker sizes are shown along the right hand side of each blot. The results of PCR with <i>bobt</i> specific primers are shown below (+,−).</p

Structure and genomic context of the two variants of <i>bobt</i>, <i>bobt_KR</i> (A) and <i>bobt_MV</i> (B).

<p><i>bobt_KR</i> and <i>bobt_MV</i> differ in 43 nucleotide sites, but share the same general <i>atp1-cox2</i>-unknown ORF structure between the start and stop sites. They share a segment of <i>atp1</i> at the 3′ end downstream of the stop codon, a segment of <i>cox2</i>, and a segment of unknown origin. The fourth segment, located just downstream of stop codon corresponds to atp1. Its size differs between both copies, because <i>bobt_KR</i> is adjacent to <i>cob</i>, whereas <i>bobt_MV</i> is not. Horizontal arrows designate the locations of the primers (atp1 lo, atp1 up) originally used to discover <i>bobt</i> via PCR co-amplification with the functional copy of <i>atp1</i>. A full-length <i>atp1</i> gene is shown below the <i>bobt</i> variants, the homologous regions are indicated by dotted lines. A scale in bp is given above.</p

Relative transcript levels in flower buds assessed at different regions of the <i>bobt_KR</i>-<i>cob</i> co-transcript.

<p>(Relative) transcript level at the <i>atp1-cob</i> junction and <i>cob</i> is more than twice that in the unknown region of <i>bobt_KR</i>. No signal was detected when RNA instead of cDNA was added to qPCR reaction mixture, which excluded contamination of RNA samples with genomic DNA. (A) Expression is presented as the ratio of the accumulation of PCR product for the specific region and the mt 18S rRNA reference gene and (B) copy numbers of different regions of <i>bobt_KR</i>-<i>cob</i> DNA as a ratio of the accumulation of PCR product for the specific region and the mt 18S rRNA reference gene. Whereas target gene and reference copy numbers are approximately equal, <i>atp1-cob</i> cotranscript and <i>cob</i> transcript levels are more than one order of magnitude lower than mt 18S rRNA. The black lines below the gene indicate the positions of PCR products used for quantitation. The standard deviation was calculated on the basis of 5 sibling pairs F and H. The significant differences (t-test P<0.05) between F and H are marked by **.</p

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