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Temperature-dependent dynamic deactivation of pol V Mut E38K/ΔC17.
No full text(A) Sketch showing dynamic deactivation of pol V Mut E38K/ΔC17 (200 nM) with ATPγS /ATP (500 μM) at 37°C and 30°C in the presence of saturating concentration of dNTP’s (mix of dTTP, dCTP, dGTP 500 μM each). Representative DNA synthesis gels for pol V Mut E38K/ΔC17 with ATP/ATPγS at 37°C and 30°C are presented in (B-E). Each experiment was repeated 3 times, and the average % PE (percent p/t DNA extended) with the SD (standard deviation) for each reaction time point is graphed in panels (F-G). Pol V Mut E38K/ΔC17 deactivates in about 1.5 h at 37°C and completes only one round of DNA synthesis with ATP/ATPγS (B-C and F-G black circles in the graphs). In contrast, at 30°C, pol V Mut E38K/ΔC17 deactivates in about 3h, which allows the enzyme to complete 4 rounds of DNA synthesis with ATPγS and 3 rounds with ATP (D-E and F-G white triangles in the graphs). Deactivated pol V Mut E38K/ΔC17 is not “dead” and is reactivated by adding RecA* (200 nM; RecA* in B-G). % PE refers to percent p/t DNA extended and was calculated as the integrated gel band intensities of extended hairpin DNA over total DNA intensity.</p
<i>Taco1</i><sup><i>mut/mut</i></sup> mice generate a normal immune response.
No full text(A) Body weights from uninfected and MCMV infected Taco1wt/wt and Taco1 mut/mut mice. Spleens and livers from uninfected and MCMV infected 30 week old Taco1wt/wt and Taco1 mut/mut mice were prepared for flow cytometry to determine (B) the number of total spleen lymphocytes, (C) the number of spleen T cells, (D) the number of spleen NK cells, (E) the number of total liver lymphocytes, (F) the number of liver T cells, (G) the number of liver NK cells, and (H) the number of m38+ CD8+ T cells in the spleen and liver. The data are representative of results obtained from at least 5 mice from each genotype and each infection group. Error bars indicate SEM; *pt-test.</p
Pol V Mut binding to p/t DNA visualized at single-molecule resolution in real-time.
No full text(A) Sketch of smFRET experimental setup. An AF555 donor-labeled p/t DNA linked to streptavidin-biotin is attached to a glass slide surface. AF647 acceptor-labeled pol V Mut is then added, and DNA binding is observed as an increase in acceptor fluorophore emission that counter-correlates with a drop of a donor emission. (B) A representative smFRET trajectory showing multiple binding and unbinding events of ATPγS-activated pol V Mut E38K/ΔC17 (green = donor, red = acceptor, blue = FRET efficiency). ATPγS-activated pol V Mut was added at t = 30 s after the start of image acquisition. Data were collected for up to 3 min, prior to the onset of photobleaching. (C) Histogram representing smFRET efficiencies corresponding to the binding of ATPγS-activated pol V Mut E38K/ΔC17 to AF555-labeled p/t DNA. FRET efficiency is calculated as E = IA / (ID+IA), where IA and ID represent acceptor and donor emission respectively. (D-F) Representative smFRET images are shown along with representative individual FRET trajectories of ATPγS-dependent binding of pol V Mut E38K/ΔC17 to p/t DNA. AF555-labeled p/t DNA is shown as green spots, and unbound AF647-labeled pol V Mut E38K/ΔC17 is shown as red spots. The pol V Mut E38K/ΔC17-p/t DNA binding events are shown as colocalized pol V Mut E38K/ΔC17 and p/t DNA signals (yellow/orange spots). Pol V Mut E38K/ΔC17 (D-E) or ATPγS activated pol V Mut E38K/ΔC17 (F) is added at t = 30 s after the start of image acquisition, followed by addition of ATPγS (t = 60 s, middle panel). Pol V Mut does not bind p/t DNA in the absence of ATPγS (D and S1 Movie). The addition of ATPγS activates pol V Mut E38K/ΔC17, resulting in binding to p/t DNA (E and S2 Movie) and pol V Mut E38K/ΔC17-p/t DNA binding events are indicated by the arrows. If pol V Mut is activated by ATPγS prior to addition to p/t DNA (F and S3 Movie), multiple and rapid p/t DNA binding events occur, indicated by arrows. The images shown in (D-F) are smFRET data integrated over 1 min following pol V Mut addition (left panel) or first binding events (middle and right panels). Scale bar is 150 mm.</p
<i>Foxg1</i><sup><i>MUT/WT</i></sup> mice show impaired learning on the conditioned fear task.
No full textA) FoxG1MUT/WT mice (MUT, n = 15) show increased baseline freezing (on training day before stimulus) compared to WT mice (n = 15) at 14 weeks old (F[1,27] = 13.365, p = 0.001, corrected p = 0.012, η2 = 0.331). B) MUT mice have decreased freezing (over baseline freezing) to context (F[1,27] = 25.568, p2 = 0.486). C) MUT mice have decreased freezing (over baseline freezing) to cue (F[1,27] = 26.588, p2 = 0.496). Uncorrected p-values: ***p<0.001.</p
Dreimal Mut zum Aufwerfen und Bearbeiten einer soziologischen Frage
No full textHenkel A. Dreimal Mut zum Aufwerfen und Bearbeiten einer soziologischen Frage. In: Schöneck-Voß N, Wenzelburger G, Wolf F, eds. Promotionsratgeber Soziologie. VS Verlag; 2011
<i>Mrps34</i><sup><i>mut/mut</i></sup> mice have hypertrophic hearts and increased lipid accumulation in their livers.
No full text<p>(A) Echocardiographic parameters of <i>Mrps34</i><sup><i>wt/wt</i></sup> (n = 5) and <i>Mrps34</i><sup><i>mut/mut</i></sup> (n = 5) mice. LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening; LVDPW, left ventricular posterior wall in diastole; LVSPW, left ventricular posterior wall in systole; IVDS, intraventricular septum in diastole; IVSS, intraventricular septum in systole. Values are means ± standard error. *p<0.05 compared with <i>Mrps34</i><sup><i>wt/wt</i></sup>. Liver sections cut at 8–12 μm thickness were stained with Haematoxylin and Eosin, oil red O and Haematoxylin or Gomori trichrome from young (B) and aged (C) <i>Mrps34</i><sup><i>wt/wt</i></sup> (n = 9) and <i>Mrps34</i><sup><i>mut/mut</i></sup> (n = 9) mice and visualized at 40X magnification. (D) Quantitative measurement of oil red staining using Image J. Data are means ± SEM of four different mice; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test. Serum ALT levels in young (E) and aged (F) <i>Mrps34</i><sup><i>wt/wt</i></sup> (n = 12) and <i>Mrps34</i><sup><i>mut/mut</i></sup> (n = 12) mice.</p
Analysis of motor function and anxiety in <i>FoxG1</i><sup><i>MUT/WT</i></sup> mice.
No full textA) Foxg1MUT/WT mice (MUT) do not show any changes on the accelerating rotating rod task compared to wild-type (WT) littermate controls (10 weeks old, n = 15 for each genotype). B-E: Open Field Assay (OFA, 8–11 weeks, WT n = 26, MUT n = 31). MUT animals have decreased overall distance traveled (B, F[1,54] = 7.885, p = 0.007, corrected p = 0.037, η2 = 0.127) and vertical counts (C, F[1,54] = 6.592, p = 0.012, corrected p = 0.040, η2 = 1.09). MUT animals spend less percentage time in center area (D, F[1,54] = 6.991, p = 0.011, corrected p = 0.046, η2 = 0.115) and travel less percentage distance traveled in the center area (E, F[1,54] = 53.193, p2 = 0.496) in OFA. F-G: Elevated Zero Maze (EZM, 20wks, WT n = 11, MUT = 14). MUT animals did not show any difference in the percent time spent in the open arm (F, F[1,22] = 1.027, p = 0.322, corrected p = 0.446) but traveled more percentage distance in the open arm (G, F[1,22] = 9.838, p = 0.005, corrected p = 0029, η2 = 0.322). Uncorrected p-values: *p<0.05, *p<0.01, ***P<0.001.</p
Conformational regulation of pol V Mut.
No full textThe first step in pol V Mut regulatory pathway requires transfer of a molecule of RecA from the 3'-tip of a RecA* nucleoprotein filament to pol V (UmuD'2C) to form pol V Mut (UmuD'2C-RecA). Pol V Mut exists in 3 conformational states determined by the orientation of RecA on pol V. When first assembled pol V Mut (UmuD'2C-RecA) is catalytically inactive (State 1) and cannot bind to p/t DNA. A subsequent addition of ATP (or ATPγS) activates pol V Mut = UmuD'2C-RecA-ATP (State 2) due to ATP induced reorientation of RecA relative to UmuC, termed “ATP induced RecA switch”. Once activated, pol V Mut can bind to p/t DNA and synthesize DNA, including TLS (X denotes a DNA template lesion). Pol V Mut can either statically deactivate in the absence of DNA synthesis (State 3) with a rate of about 0.02/min at 37°C or deactivate dynamically (State 3) in the presence of DNA synthesis (0.023/min at 37°C). Deactivated pol V Mut can be activated (i.e., reactivated) via transfer of a “new” molecule of RecA from the 3'-tip of a RecA* nucleoprotein filament, in which the new RecA replaces the old RecA [11], followed by binding of ATP/ATPγS. Deactivated pol V Mut cannot be reactivated by binding to ATP or ATPγS. Both deactivation processes occur more rapidly at 37°C compared to at 30°C. Deactivation is inhibited in the presence of bound ATP and accelerated in the presence of bound p/t DNA – see text.</p
Distinct regions of MUT-16 recruit each of the other <i>Mutator</i> proteins.
No full text(A) Table indicates whether mut-16 deletions disrupt MUT-2, RDE-8, NYN-1, MUT-14, RRF-1, and RDE-2 foci. Yes indicates foci present in the majority of animals, No indicates foci absent or severely disrupted, and ND indicates that strain was not constructed or scored. (B-D) MUT-16::mCherry and MUT-2::GFP expression and localization for control strain (B) or when ΔC (C) or ΔK (D) deletions have been introduced into the mut-16::mCherry strain. Scale bars, 5μm. (E) Immunoprecipitation and western blot of MUT-16::mCherry::2xHA (expected sizes between 135–141 kD for MUT-16 deletions and 148 kD for MUT-16 full length) and MUT-2::GFP::3xFLAG (83 kD). Left panels are total lysate from strains indicated above, and right panels are following HA immunoprecipitation. (F) Immunoprecipitation and western blot of MUT-16::mCherry::2xHA (expected sizes between 132–142 kD for MUT-16 deletions and 148 kD for MUT-16 full length) and GFP::3xFLAG::RRF-1 (219 kD). Top two panels are total lysate from strains indicated above, and bottom two panels are following HA immunoprecipitation. The equivalent of ~0.5% of starting material for the input fractions and ~20% of starting material for the IP fractions were loaded onto the gels.</p
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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