Genes transcribed by T7 RNAP do not interact <i>in vivo</i>.

<p><b>A.</b> Diagram of <i>Bgl</i>II fragments (thick coloured regions), PCR primers (yellow arrows) and ligation products (grey arrows) used in 3C. Ovals denote transcription units under the control of the T7 promoter. We determined the ligation frequency of the <i>Bgl</i>II fragment encoding <i>P_T7-T7gene10</i> (light blue; 24 kb) with (i) the 3-kb fragment encoding <i>P_T7-YFP</i> located 80 kb away (orange; using primers <i>a</i> and <i>c</i>), and (ii) a 5-kb promoter-less control fragment 22 kb away (purple; using primers <i>a</i> and <i>b</i>). As a secondary control for 3C efficiency, we also measured the ligation frequency of two adjacent fragments located at a randomly-chosen genomic site (pink and green; using primers <i>d</i> and <i>e</i>). This genomic map is drawn to scale. <b>B.</b> 3C. The ‘3C strain’ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040207#pone-0040207-g003" target="_blank">Fig. 3A</a> was grown to OD₆₀₀ = 0.4 in arabinose (+T7 RNAP) or glucose (−T7 RNAP), 3C templates prepared using <i>Bgl</i>II, and ligation products detected by PCR using the primer pairs indicated. Images show PCR products resolved on an agarose gel, and stained with SYBR green I. Loading controls (lanes 5–6) show that band intensities are proportional to the amount of ligation product present in the preceding PCR reactions, and thus to the contact frequency of the selected restriction fragments. All PCR products are of the expected size, and depend upon formaldehyde crosslinking (lane 3) and restriction nuclease digestion (lane 4). For each condition, the ‘test gene contact frequency’ (of <i>T7gene10</i> with <i>YFP</i>) was calculated by dividing the intensity of the band produced by the primers <i>a:c</i> by the intensity of the band produced by primers <i>a:b</i>. This adjustment corrected for differences in 3C efficiency, and so allowed ligation frequencies obtained from different samples (i.e., ± T7 RNAP) to be directly compared. Transcription of <i>T7gene10</i> and <i>YFP</i> by T7 RNAP had no effect on their contact frequency (compare test gene contact frequencies in lanes 1 and 2). Normalizing for 3C efficiency using the ligation frequency of the adjacent fragments (i.e., <i>a:c</i>/<i>d:e</i>) yielded the same result (1.9±0.6 a.u. for +T7 vs. 2.7±0.2 a.u. for −T7). a.u. = arbitrary units. *all measurements had standard deviations of less than 1% (n = 3). ^†assessed by qPCR.