The C. elegans Rhomboid Genes

<div><p>(A) Dendogram showing the relation between the seven-pass transmembrane domains of Rhomboids from C. elegans (C.e.), Drosophila melanogaster (D.m.), and Homo sapiens (H.s.) calculated with the neighbor joining method using CLUSTAL X (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020334#pbio-0020334-Thompson1" target="_blank">Thompson et al. 1997</a>).</p> <p>(B) Alignment of C. elegans (C.e.) ROM-1 and ROM-2 and Homo sapiens (H.s.) Rho-1 relative to Drosophila melanogaster (D.m.) Rho-1. Residues identical to those of <i>Drosophila</i> Rho-1 are highlighted in black, and similar residues are highlighted in grey. The thick black lines indicate the predicted seven-pass transmembrane domains. The three black triangles point at the residues forming a catalytic triad that forms a charge-relay system to activate the essential serine residue during peptide bond cleavage, and the three open triangles indicate other conserved residues necessary for the enzymatic activity as identified in D.m. Rho-1 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020334#pbio-0020334-Urban3" target="_blank">Urban et al. 2001</a>). The region underlined with a dotted line indicates the extent of deletion in the <i>rom-1</i>(<i>zh18</i>) allele.</p> <p>(C) Intron-exon structure of the <i>rom-1</i> locus and extent of the deletion in the <i>rom-1(zh18)</i> strain. The numbers indicate the position of the deletion break-points relative to the A in the ATG start codon.</p></div

Alternative Splicing of <i>lin-3</i> mRNA

<div><p>(A) RT-PCR amplification of <i>lin-3</i> mRNA from mixed-stage N2 cDNA before (left) and after (right) size fractionation by preparative agarose gel electrophoresis. The lowest band corresponding to LIN-3S is most prominent, and the two upper bands correspond to LIN-3L and LIN-3XL.</p> <p>(B) Intron-exon structure of the <i>lin-3</i> locus. The <i>lin-3L</i> splice variant is generated by the usage of an alternative (more 3′ located) splice donor in exon 6a. The <i>lin-3XL</i> variant contains the additional exon 6b inserted between exons 6a and 7. The regions encoding the EGF repeat in exon 5 and part of 6a and the transmembrane domain in exon 7 are outlined, and the positions of the PstI sites used for the construction of the minigenes are indicated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020334#s4" target="_blank">Materials and Methods</a>). The structure of the <i>lin-3S</i> and <i>lin-3L</i> minigenes is shown in the lower part of the graphic.</p> <p>(C) Sequence alignment of the alternatively spliced region in LIN-3 with the corresponding region in <i>Drosophila</i> Spitz. The 15 and 41 amino acids in LIN-3L and LIN-3XL, respectively, in the juxtamembrane region break the alignment of LIN-3 with Spitz. The C-terminal end of the EGF domain is underlined with a horizontally hatched bar, and the beginning of the transmembrane domain is underlined by a diagonally hatched line.</p></div

A Relay Model for Vulval Induction

<p>The AC initiates vulval development by secreting the LIN-3 growth factor independently of ROM-1. In response to the AC signal, the proximal VPCs up-regulate ROM-1 expression and start secreting LIN-3 in a ROM-1-dependent manner to relay the AC signal.</p

Expression Pattern of <i>rom-1::nls::gfp</i>

<div><p>Expression pattern of the <i>zhIs5[rom-1::nls::gfprom-1::]</i> transcriptional reporter during vulval development. Images on the left (A, C, E, G, and I) show the corresponding Nomarski pictures with the arrows pointing at the Pn.p cell nuclei and the arrowhead indicating the position of the AC nucleus.</p> <p>(B) A mid L2 larva before vulval induction with uniform <i>rom-1::nls::gfp</i> expression in all the Pn.p cells.</p> <p>(D) An early L3 larva in which <i>rom-1::nls::gfp</i> expression was decreased in all VPCs except P6.p (see text for a quantification of the expression pattern). Note that the nuclei of hyp7 and the Pn.p cells that had fused to hyp7 displayed strong <i>rom-1::nls::gfp</i> expression (P1.p, P2.p, P3.p and P9.p in the example shown).</p> <p>(F) A mid to late L3 larva in which P6.p had generated four descendants. Expression of <i>rom-1::nls::gfp</i> occurred only in the 3° descendants of P.4.p and P8.p after they fused to hyp7.</p> <p>(H) An L4 larva during vulval invagination. No <i>rom-1::nls::gfp</i> was detectable in the 1° and 2° descendants of P5.p, P6.p, and P7.p, but the AC and the surrounding uterine cells displayed strong <i>rom-1::nls::gfp</i> expression.</p> <p>(K) A late L2 to early L3 larva following the ablation of the precursors of the somatic gonad. No up-regulation of <i>rom-1::nls::gfp</i> in P5.p, P6.p, or P7.p was observed. The scale bar in (K) is 10 μm.</p></div

Expression of the <i>egl-17::cfp</i> Reporter in <i>rom-1(0)</i> and <i>lin-3(rf)</i> Mutants

<div><p>Photographic images on the left (A, C, E, G, and I) show the expression of the <i>arIs92[egl-17::cfp]</i> reporter in the VPCs of mid-L2 larvae of the different genotypes indicated.</p> <p>Pie graphs on the right (B, D, F, H, and J) show semi-quantitative representations of the expression levels observed in individual VPCs in the different backgrounds. A solid black color indicates the strongest expression of EGL-17::CFP as it was observed in P6.p of many (59%) wild-type animals; dark grey indicates intermediate, light grey weak, and white undetectable expression. The numbers inside the pie charts are the corresponding percentage values, and <i>n</i> refers to the number of animals examined for each case. EGL-17::CFP expression in each VPC of <i>rom-1(0)</i> or <i>lin-3(e1417rf)</i> animals was compared against the same VPC in wild-type animals (considered as expected value) with a Chi² test for its independence; *** <i>p</i> ≤ 0.0001, ** <i>p</i> ≤ 0.001. The row to which a dataset was compared is indicated on the right. All photographs were taken with identical exposure and contrast settings. The scale bar in (I) is 20 μm.</p></div

Additional file 1: Table S2. of Non-typhoidal Salmonella DNA traces in gallbladder cancer

Primer sequences used for detection of Salmonella (PDF 122 kb

Additional file 4: Figure S2. of Non-typhoidal Salmonella DNA traces in gallbladder cancer

Specificity and Sensitivity for detection of Salmonella reads in whole exome sequence of gallbladder samples. (A) Specificity for detection of Salmonella reads in whole exome sequence of gallbladder samples. Exome sequenced reads were reversed (not complement) to maintain the genome complexity and used an input file to detect random Salmonella reads. No Salmonella reads were found in the samples with reversed whole exome sequence. (B) Sensitivity for detection of Salmonella reads in gallbladder samples as a function of increasing genome sequence coverage. Gallbladder tumour sample 16 T with highest number of Salmonella reads was down-sampled to 1x, 5x, 10x, 15x, 25x, 50x, 75x and 100x. Salmonella reads were counted (black line) and plotted against increasing coverage of the genome on x-axis. (PDF 5395 kb

Additional file 3: Table S1. of Non-typhoidal Salmonella DNA traces in gallbladder cancer

Detailed annotation table of read sequences of different Salmonella species identified across gallbladder cancer patient samples. (PDF 348 kb

Additional file 2: Figure S1. of Non-typhoidal Salmonella DNA traces in gallbladder cancer

Abundance and annotation of Salmonella reads found across the 16 of 26 gall bladder cancer samples. Heat map representation of individual Salmonella reads (in rows) identified from 6 different isolates found across the 16 gall bladder cancer samples (in column) is shown. Variable length and number of overlapping reads, each of 150 bp obtained from paired end Illumina sequence for each isolate, were assembled into contigs based on Clustal X2 multiple alignment. The unique total length of contigs generated is shown in second column reflecting the total length of the gene covered in the study. The contigs generated were annotated based on gene annotation database of Salmonella isolates from NCBI database. A representative general class for all genes identified is shown in the third column. (PDF 11190 kb

Additional file 5: Figure S3. of Non-typhoidal Salmonella DNA traces in gallbladder cancer

Sanger validation of Salmonella read sequences in gall bladder cancer samples Individual read sequences were PCR amplified and Sanger sequencing trace of individual read sequence with their blast output is represented in the figure. (PDF 1437 kb