Issues and Prospects of the Library in Japan : From the Answer of the Examination in Asistant Librarian Training

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Purification and characterization of HsCRY1.

<p>A. Western blot confirming the expression of HsCRY1 using the <i>Homo sapiens</i> CRY1 polyclonal rabbit antibody. B. <b>Coomassie-stained</b> SDS PAGE of purified HsCRY1 (66 kDa). C. UV-visible spectrum of purified HsCRY1. Inset: Averaged cwEPR spectrum of a ∼290 µM sample of HsCRY1. The peak-to-peak linewidth is 15.4 G at a g-value of 2.0042, which indicates an anionic semiquinone. D. UV-visible absorbance spectra representing the photoreduction of HsCRY1 in the presence of 20 mM DTT. The sample was exposed to 100 µmol m⁻² s⁻¹ of 440–460 nm light for a maximum of 85 minutes. Black line - before irradiation; blue line - after 85 min irradiation; red, dashed line – reoxidation in dark (within ∼2 min). Inset: the difference spectrum (Light minus dark) showing peak reduction at 450 nm consistent with oxidized flavin.</p

Analysis of developmental time (until eclosion) in HsCRY1 expressing transgenic <i>Drosophila</i>.

<p>For all tests, parent flies were maintained for 24 hours in a 50 ml conical falcon tube containing 10 ml of growth medium in order to lay eggs. Adult flies were then removed and tubes transferred to constant white fluorescent light (200 µmol m⁻² sec⁻¹) or continuous dark (DD) in a temperature controlled and humidity controlled plant growth chamber at 22°C. Time to eclosion was measured every 12 hours and eclosed flies counted and plotted for each time point as a percentage of total eclosed flies. Typically between 100 and 500 flies were counted per <i>Drosophila</i> line. Experiments were performed in triplicate showing qualitatively similar results (peak of eclosion); the data from a representative experiment is shown. A. Time to eclosion was plotted for LL (continuous light). Wild type controls were the non-expressing <i>tim-Gal4</i> parental lines. <i>cry^b</i> mutant flies carried the <i>cry^b</i> mutation in the genetic background of the <i>tim-Gal4</i> parental lines. B. Same as for ‘A’, in constant darkness (DD). C. Relative time to eclosion (x-axis) under differing wavelengths of light at 3 µmol m⁻² sec⁻¹ blue light (B), red light (R) and UV/A light (peak 380 nm) (UV). Y-axis is number of flies pooled from three independent experiments. <i>Hscry1</i>(A) flies expressed HsCRY1 under control of the <i>tim-Gal4</i> promoter, <i>Hscry1</i> (B) flies expressed HsCRY1 under the <i>da-Gal4</i> promoter; <i>cry⁰² i</i>s the null mutant allele (Dolozelova et. al. 2007).</p

Transcriptome analysis of cryptochrome dependent gene expression in developing pupa one day after third instar larval stage.

<p>A, B Venn diagram of genes either upregulated (red) or downregulated (green) in the indicated strains under conditions of complete darkness. Comparison were performed in triplicate biological samples and analysed for statistically different expression patterns according to established programming techniques (see methods). C. Graphical representation of categories of genes either repressed or activated by cryptochromes in the indicated comparisons. All comparisons are for strains maintained in complete darkness (DD) from the time of egg laying. D,E. Venn diagrams of genes up-regulated or down-regulated in the indicated strains (wt, <i>cry⁰</i>, or <i>Hscry1</i> transgenics) after transition from dark to two hours blue light at 60 µmol m⁻² sec⁻¹. F. Graphical representation of categories of genes either repressed or activated by cryptochromes in the indicated comparisons.</p

Verification of expression of indicated genes by qPCR and consensus promoter elements involved in cry regulation.

<p>Solid rectangles represent expression levels in dark, white rectangles expression after two hours of blue light. A: <i>dro3</i>, B: <i>white</i>, C. <i>7506</i>; D. <i>end</i>. E. Transcription factor binding sequence DNA motifs conserved in cryptochrome regulated promoters of <i>Drosophila</i> as obtained using <i>Drosophila</i> cisTargetX (Aerts et al., 2010), the size of the letter indicates the probability to find the corresponding base at the different positions (adenine (<b>A</b>), cytosine (<b>C</b>), guanine (<b>G</b>) and thymine (<b>T</b>)).</p

Heterologous CRY expression does not restore a wild-type arrhythmic phenotype to <i>cry^b</i> mutant flies.

<p>Actogramms of rhythmicity conferred by <i>Hscry</i> constructs in LL and DD. Three day old adult flies expressing HsCRY1, HsDM in both wild type and <i>cry^b</i> mutant genetic backgrounds were entrained for 72 hours to 12 hr day/night cycles and analyzed for free running period in LL and DD for 10 days. Rhythmicity and period length are shown. The mean values of circadian period (h), associated powers (see Methods) and activities (number of events per 0.5 h) are given ± s.e.m.</p