Validated ncRNAs with Northern blot analysis.

*<p>ncRNAs have been validated with 5′ and 3′ RACE.</p>†<p>ncRNAs have been previously identified or detected by Wilms et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070720#pone.0070720-Wilms3" target="_blank">[51]</a>.</p><p>F and R at the end of each ncRNA tag denote strand information: Forward and Reverse.</p

Variation of 5′ UTR length.

<p>The distance between TSS and start codon (5′ UTR) varied substantially from 0 to 521 nt, averaging 88 nt and median of 61 nt. Among the 675 protein coding genes, 1.8% (12) were leaderless (≤10 nt), 39% (253) were short (11∼50 nt), while 30% (203) were long (>100 nt). About 51% (345) of 5′ UTRs were longer than 60 nt.</p

Summary of RNA sequencing and alignment^*.

*<p>Short sequence reads were aligned to the <i>A. tumefaciens</i> C58 reference genome using the Bowtie 2 program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070720#pone.0070720-Langmead1" target="_blank">[45]</a>.</p

Transcriptional regulation of <i>thiCOGG</i> operon by a TPP riboswitch (C1_2541934R).

<p>A putative riboswitch at the 5′ UTR of thiamine biosynthesis operon, <i>thiCOGG</i>, transcriptionally regulates gene expression. <b>A</b>) Secondary structure predicted by mFold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070720#pone.0070720-Zuker1" target="_blank">[94]</a>: Δ<i>G</i>  = −35.08 kcal/mol. (<b>B</b>) Northern blot analysis with a probe specific to the riboswitch and (<b>C</b>) a probe specific to the downstream gene, <i>thiC</i> (<b>C</b>). Total RNA was isolated from <i>A. tumefaciens</i> strain C58 grown in YEP medium until mid-log phase (YEP-L), YEP medium until late stationary phase (YEP-S), AB induction medium without AS (AB), AB induction medium with AS (IND), and AB with 100 µg/mL of thiamine (AB+Thi). +RP, RNA samples were treated with RNAprotect Bacteria reagent (Qiagen, USA). *AB and *AB+Thi, RNA samples were not treated. Ethidiumbromide stained 16S rRNA bands were included as loading control.</p

Induction of <i>vir</i> genes with AS.

<p>Expression of 24 <i>vir</i> genes with and without AS was visualized for data validation. Depth of coverage at each nucleotide position from 180,590 to 211,094 of Ti plasmid was plotted for (<b>A</b>) forward strand and (<b>B</b>) reverse strand. A total of 24 <i>vir</i> genes were included: <i>virA</i>, <i>virB</i> (B1∼B11), <i>virG</i>, <i>virC</i> (C1, C2), <i>virD</i> (D1∼D5) and <i>virE</i> (E0∼E3).</p

Validation of selected ncRNAs by Northern blot analysis.

<p>Depth of coverage profiles and Northern hybridization images of 22 <i>Agrobacterium</i> ncRNAs under four growth conditions: YEP medium until mid-log phase (YEP-L), YEP medium until late stationary phase (YEP-S), AB induction medium without AS (AB), AB induction medium with AS (IND). (<b>A</b>) Fifteen ncRNAs encoded on the circular chromosome (C1), (<b>B</b>) five ncRNAs encoded on the linear chromosome (C2), and (<b>C</b>) two ncRNAs encoded on the pAt plasmid (pAt).</p

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Distribution of ncRNAs on four replicons.

<p>Distribution of ncRNAs on four replicons.</p

Expression correlation between <i>cis</i>-antisense RNAs and putative target genes.

<p>Log-transformed RPKM data for 354 <i>Agrobacterium</i> asRNAs were plotted against log-transformed RPKM data of genes encoded on the complementary strand. Pearson product-moment coefficient was given (<i>r</i>² = 0.02).</p

A thi-box riboswitch and thiamine biosynthesis gene operon.

<p>A thi-box riboswitch and thiamine biosynthesis gene operon.</p