Molecular basis for the reproductive division of labour in a lower termite-1

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>bold line: cDNA strand; grey bar: open reading frame; numbers; II fragment; yellow bar: II fragment obtained from the first RDA; orange bar: II fragment obtained from the second RDA; turquoise bar: signal peptide; green bar: poly-adenylation-signal; red bar: poly-A-tail; asterisk: II restriction site; italic numbers: number of individual sequenced fragments

Molecular basis for the reproductive division of labour in a lower termite-4

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>Cloned inserts of randomly picked clones were PCR-amplified, denatured and dot-blotted onto two duplicate nylon transfer membranes and hybridized to radioactively labelled female neotenic (A) and worker (B) representations

Molecular basis for the reproductive division of labour in a lower termite-2

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>sured by qRT-PCR. The Y-axis is on negative logscale indicating the gene expression levels and the calculated errors (SD), for female neotenics (NF), male neotenics (NM), female primary reproductives (PF), male primary reproductives (PM), female alates (AF), male alates (AM) and workers (W) of both sexes

Molecular basis for the reproductive division of labour in a lower termite-5

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>bold line: cDNA strand; grey bar: open reading frame; numbers; II fragment; yellow bar: II fragment obtained from the first RDA; orange bar: II fragment obtained from the second RDA; turquoise bar: signal peptide; green bar: poly-adenylation-signal; red bar: poly-A-tail; asterisk: II restriction site; italic numbers: number of individual sequenced fragments

Molecular basis for the reproductive division of labour in a lower termite-0

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>Cloned inserts of randomly picked clones were PCR-amplified, denatured and dot-blotted onto two duplicate nylon transfer membranes and hybridized to radioactively labelled female neotenic (A) and worker (B) representations

Molecular basis for the reproductive division of labour in a lower termite-3

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis for the reproductive division of labour in a lower termite"</p><p>http://www.biomedcentral.com/1471-2164/8/198</p><p>BMC Genomics 2007;8():198-198.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1988835.</p><p></p>easured by qRT-PCR. The Y-axis is on negative logscale indicating the gene expression levels and the calculated errors (SD). cpt: caput, thx: thorax and abd: abdomen

Chromatin-specific termination at the homotypic cluster of TTF-I.

<p>(A) Overview of the murine rRNA gene and the location of the TTF-I binding sites. A homotypic cluster of TTF-I sites is located in the terminator region. The distances between TTF-I binding sites, their orientation and the gene promoter are indicated. A comparison of the TTF-I binding sites T₀ and the termination sites T₁ to T₁₀ is depicted. (B) Increasing amounts of TTF-IΔN348 were incubated with 50 nM of either a fluorescently labelled 30-mer oligonucleotide containing a Sal-box motif (T₂) or a control oligonucleotide of the same length. Protein-DNA interactions are quantified by microscale thermophoresis. Curve fitting with a Hill coefficient of 1 resulted in a K_D of 500 nM+/−120 nM for the T₂ sequence. (C) Transcription reaction using the circular rDNA minigene plasmid pMr-SB containing a single termination site, a partially purified nuclear extract lacking most of the nuclear TTF-I (DEAE280), performed in the presence or absence of recombinant TTF-I. The positions of the long read-through and the terminated transcripts are indicated. (D) Transcription on free DNA and chromatin, using the pMrWT-T DNA containing the promoter with the TTF-I binding site T₀ and the full terminator with the 10 termination sites. DNA (lanes 1–8) and chromatin (lanes 9–16) were incubated with increasing concentrations of TTF-I as indicated and the DEAE280 extract. The position of the long, non-terminated read-through transcript (RT) and the terminated transcripts are indicated.</p

Multiple termination sites enable cooperative binding of TTF-I to chromatin.

<p>(A) Electrophoretic mobility shift assays (EMSA) were performed with a single TTF-I binding site (T₁, lanes 1–4), two binding sites (T_1–2, lanes 5–9) and an array of five binding sites (T_1–5, lanes 10–14) and increasing concentrations of TTF-I as indicated. Nucleoprotein complexes are resolved on native polyacrylamide gels and detected by autoradiography. The positions of the free DNA molecules and the TTF-I-DNA complexes (triangles) are indicated. (B) Monitoring TTF-I binding to the chromatinized terminator by DNase I footprinting. The pMr-T plasmid containing the full terminator was reconstituted into chromatin with <i>Drosophila</i> embryo extract. Chromatin was incubated with increasing concentrations of TTF-I as indicated and partially digested with DNase I. Footprints were analysed by a primer extension reaction using a radioactively labelled oligonucleotide and resolving the DNA on 6% sequencing gels. The marker was generated by partial digestion of the plasmid with the restriction enzyme <i>Sal</i>I and analysed by the same primer extension reaction. The <i>Sal</i>I sites (T₁ to T₁₀) represent the TTF-I binding sites and the triangles indicate sites of DNase I protection. (C) Comparative footprinting of TTF-I binding to the promoter and terminator of free DNA and chromatin. Identical amounts of pMrWT-T were used as free DNA (lanes 1 to 4 and 8 to 11) or chromatin (lanes 5 to 7 and 12 to 14) and incubated with increasing amounts of TTF-I as indicated. DNA was partially digested with DNase I and the purified DNA was analysed by primer extension reactions, either using a radiolabelled oligonucleotide binding close to the promoter (lanes 1 to 7) or binding close to T₁ in the terminator region (lanes 8 to 14). DNA was separated on 8% sequencing gels, dried and analysed after autoradiography. The TTF-I binding sites T₁, T₂ and T₀ and the protected DNase I cutting sites (triangles) are indicated.</p

Distribution of histone modifications at the murine rDNA.

<p>(A) Enrichment of histone modifications at the rDNA locus in 3T3-L1 cells. The whole rDNA repeat is plotted from position +1 (the TSS) to position 45.309. The terminator track indicates TTF-I binding sites by black vertical lines. The black box highlights the clustered terminator elements at the 3′ end of the gene. ChIP-Seq tracks of histone modifications display relative enrichments compared to input. (B) Model depicting the order of binding events at the rRNA gene. The promoter is coloured in blue, a right-headed arrow marks the TSS and the clustered termination sites are depicted in red.</p

Multiple termination sites enhance transcription <i>in vivo</i>.

<p>(A) Reporter plasmids containing the rDNA promoter, <i>Firefly</i> luciferase and either no (pTΔ), one (pT₁), two (pT₂), ten (pT₁₀) termination sites and T₁ and T_1–10 in reverse orientation (pT_1r and pT_10r) were co-transfected with a <i>Renilla</i> luciferase encoding plasmid (pRL-TK) into CHO cells. As a control, empty pBluescript vector was co-transfected. Transcriptional activities were analysed using a dual luciferase reporter assay. The ratio <i>of Firefly</i>/<i>Renilla</i> relative light units (RLU) of three independent experiments is given. Error bars indicate standard deviations. The functional elements and the sizes of the reporter plasmids are depicted. (B) Reporter plasmids were co-transfected with a GFP-TTFΔN348 expression vector and analysed as described in (A). (C) Reporter plasmids were co-transfected with a GFP-TTFΔN470 expression vector and analysed as described in (A). (D) RNA FISH using CHO cell lines with stably integrated rDNA minigenes. CHO-pT₁₀ cells containing an rDNA minigene with a full terminator, were stained with DAPI (in blue in the middle panel), with α-B23 antibody staining the nucleoli (left panel; shown in red in the middle panel), and integrated reporter gene transcripts were visualized by FISH (right panel; shown in green in the middle panel). Bar: 5 µm. (E) Transcription levels of genomically inserted pT₁ and pT₁₀ constructs were assayed using RT-qPCR. Comparative quantitation was performed and RNA levels of the <i>Firefly</i> luciferase sequence were normalized to β-actin expression. Relative transcript levels of three independent experiments are given in relation to non-transfected CHO Flp-In cells (control), error bars denote standard deviations.</p