15 research outputs found

    Properties of mutant cells and effect of salt shock on cell growth.

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    <p>(A) Growth of <i>E. coli</i> mutant cells under normal culture conditions. Growth of wild-type, ▵<i>rsgA</i>, ▵<i>rbfA</i>, ▵<i>rimM</i>, ▵<i>rpsF</i> or ▵<i>rrmJ</i> cells at 37°C in LB medium was monitored by measuring OD<sub>600</sub>. (B) Accumulation of 17S RNA in mutant cells. One µg of total RNA fraction prepared from each of the cells was electrophoresed on 1.8% agarose gel. The 3′ truncation product of 16S rRNA is indicated by an asterisk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065747#pone.0065747-Hase1" target="_blank">[11]</a>. The band of slightly lower migration than that of 16S rRNA was confirmed as 17S RNA by northern hybridization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065747#pone.0065747.s001" target="_blank">Figure S1</a>). The ratio of the amount of 17S RNA to that of 16S rRNA is shown below each lane. (C) Defect in subunit assembly of ribosome in mutant cells. Cells were lysed with alumina powder and the cell debris was removed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065747#s2" target="_blank">Materials and Methods</a>. Ten A<sub>260</sub> units of crude cell extracts were fractionated by 5%–20% sucrose density gradient ultracentrifugation. (D) Growth curves of mutant cells after salt shock. Cells were grown at 37°C in LB medium. When OD<sub>600</sub> had reached 0.8, 0.9 M NaCl was added to the medium. The OD<sub>600</sub> value subtracted from that measured immediately after salt shock is plotted.</p

    Premature and transient induction of genes regulated by σ<sup>E</sup>.

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    <p>Cells in which a ribosome maturation factor or a ribosomal protein was removed (A–C) and wild-type cells treated with 80 µg/ml kasugamycin, 1.2 µg/ml chloramphenicol or 2.5 µg/ml streptomycin (D) were used. Levels of <i>rpoE</i> mRNA (A and D), <i>rpoH</i> mRNA (B) and MicA (C) at the indicated time points after salt shock were analyzed by qRT-PCR. Total RNA fraction prepared from each of the cells was used as the template.</p

    Effects of protein synthesis inhibitors on processing of 17S RNA and on subunit association.

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    <p>Wild-type cells were grown at 37°C in LB medium with 80 µg/ml kasugamycin (Kas) or 1.2 µg/ml chloramphenicol (Chl). (A) One µg of total RNA fraction prepared from wild-type, ▵<i>rsgA</i>, or drug-treated cells was electrophoresed on 1.8% agarose gel. The ratio of the amount of 17S RNA to that of 16S rRNA is shown below each lane. (B) Ribosome profiles of wild-type cells, RsgA-deletion cells and wild-type cells treated with protein synthesis inhibitors are shown. Cells were lysed with alumina powder and the cell debris was removed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065747#s2" target="_blank">Materials and Methods</a>. Ten A<sub>260</sub> units of crude cell extracts were fractionated by 5%–20% sucrose density gradient ultracentrifugation.</p

    Effects of inhibitors of DNA synthesis, transcription and translation on salt tolerance.

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    <p>Wild type cells were grown at 37°C in LB medium with the indicated concentration of each drug. When OD<sub>600</sub> reached 0.8, 0.9 M NaCl was added to the medium. Three different concentrations for each antibiotic, which were determined in consideration of the inhibitory effect on the growth of wild type cells in LB medium in the absence of salt shock, were used (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065747#pone.0065747.s004" target="_blank">Table S1</a>). Growth of drug-untreated ▵<i>rsgA</i> cells is shown as the control.</p

    Additional file 3: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

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    Figure S2. GFP expression patterns of 16 Gal4FF;UAS:GFP fish that showed reduced performance of the active avoidance response. A dorsal view, a ventral view, and a schematic side view with positions of coronal sections are shown on the top. Serial coronal sections with position numbers are shown in the bottom. a hspGGFF10C, b hspGGFF20A, c hspGFF38B, d hspGFF55B, e SAGFF36B, f SAGFF70A, g SAGFF81B, h SAGFF120A, i SAGFF226F, j SAGFF228A, k SAGFF231A, l SAGFF233A, m SAGFF234A, n SAGFF234D, o hspGFFDMC12A, p hspGFFDMC56B. Scale bars in whole brain images: 500 Îźm. Scale bars in coronal section images: 200 Îźm. (PDF 3264 kb

    Additional file 2: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

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    Figure S1. Performance of two-way active avoidance response of double transgenic (Gal4FF;UAS:zBoTxBLC:GFP) fish. The following Gal4FF transgenic fish lines were crossed with UAS:zBoTxBLC:GFP effector fish, and analyzed for two-way active avoidance fear conditioning. a hspGGFF10C (n = 6), b hspGGFF20A (n = 10), c hspGFF38B (n = 10), d hspGFF55B (n = 6), e SAGFF81B (n = 12), f SAGFF226F(n = 9), g SAGFF233A (n = 9), h SAGFF234A (n = 11), i hspGFFDMC12A (n = 12), j hspGFFDMC56B (n = 10), k hspGGFF19B (n = 9), l hspGGFF19C (n = 9), m hspGFF62A (n = 5), n gata6SAGFF94A (n = 6), o SAGFF27C (n = 6), p SAGFF38A (n = 8), q SAGFF87C (n = 5), r SAGFF92A (n = 8), s SAGFF183A (n = 5), t SAGFF195A (n = 6), u SAGFF212C (n = 5), v SAIGFF170B (n = 10), w hspGFFDMC76A (n = 10), x hspGFFDMC85C (n = 11). Mean ± SEM and avoidance (%) for individual fish are plotted. Performance of wild type fish (n = 28) described in Fig. 2 is shown in dotted lines. Two-way ANOVA, fish groups (wild type fish treated by CS-US, wild type fish treated by CS only and double transgenic fish including fish described in Figs. 2 and 3 × training session days (day 1, day 5), was performed (F = 7.236, P < 0.0001). Dunnett’s multiple comparison post-hoc tests were performed between avoidance percentage of wild type fish and double transgenic fish on session day 1 and day 5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant (P > 0.05). a–j Reduced performance of the active avoidance response was observed. k–x Performance of the active avoidance response was not significantly different between wild type and the double transgenic fish. (PPTX 8767 kb

    Additional file 8: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

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    Figure S4. GFP expression patterns in SAGFF120A;UAS:GFP and SAGFF120A;UAS:GFP;UAS:zBoTxBLC:GFP fish. a Dorsal views of the brains from eight SAGFF120A;UAS:GFP (~10 months old) fish and eight SAGFF120A;UAS:GFP;UAS:zBoTxBLC:GFP (~10 months old) fish are shown. Scale bars: 1 mm. b Areas having more intensity than background (the maximum intensity measured in the posterior part of the telencephalon) were identified by using ImageJ [57] and shown in red. Scale bars: 500 Îźm. c Immunohistochemistry using anti-GFP (green) and anti-NeuN (a neuronal marker, magenta) of coronal sections of the telencephalon and hypothalamus of brain samples from these transgenic fish. The fish numbers correspond to the numbers of the dorsal view images. Scale bars, 200 Îźm. (PPTX 6544 kb

    Additional file 4: of Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

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    Figure S3. GFP expression patterns in SAGFF120A;UAS:GFP fish at embryonic stages. Bright field and fluorescent images of frontal and lateral views of SAGFF120A;UAS:GFP fish at 24, 48, 72, and 96 hpf. Scale bar, 200 mm. (PPTX 1417 kb

    The <i>darl</i> Mutant Shows Retinotectal Mapping Deficits

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    <p>(A and B) The nasal-dorsal quadrant of the retina was labeled with DiO (green), and the temporal-ventral quadrant was labeled with DiD (magenta). In <i>darl<sup>s327</sup>,</i> the ventral branch of the optic tract is missing (arrow). Scale bar is 100 μm.</p> <p>(C and D) Dorsal view of the tectum in the same larvae as in A and B. The ventral half of the <i>darl<sup>s327</sup></i> tectum is not innervated by the dorsal-nasal RGC axons. Anterior is to the left and ventral is to the bottom. Tectal neuropil is demarcated by the dotted line, based on DAPI counterstaining (blue). Scale bar is 50 μm.</p

    Example of a Mutant with Abnormal Morphology of Cone Photoreceptors

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    <p>Photoreceptors in a retinal section stained with DAPI (A and B) and a marker for double cones, zpr1 (C and D) at 7 dpf in WT larva (A, C, and E) and <i>yoi<sup>s121</sup></i> mutant retina (B, D, and F). Merged images of DAPI (in green) and zpr1 (in magenta) are also shown (E and F). Both zpr1-positive and zpr1-negative cone photoreceptors in the mutant are “stumpy” when compared to those in the control retina (arrows). B, bipolar cells; C, cone photoreceptor cells; H, horizontal cells; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar is 10 μm.</p
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