The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules-3

<p><b>Copyright information:</b></p><p>Taken from "The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules"</p><p>http://www.biomedcentral.com/1741-7007/5/33</p><p>BMC Biology 2007;5():33-33.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC2048497.</p><p></p>ydin RNAi-induced cells (black squares) compared to non-induced controls (triangles). Cells were maintained in log phase by diluting the culture every 24 h. (B and C) Trajectories of individual cells over the course of a 40 s time-lapse. While non-induced control cells (B) are motile, cells locomotion is severely compromised in TbHydin RNA-induced cells (C)

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules-2

<p><b>Copyright information:</b></p><p>Taken from "The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules"</p><p>http://www.biomedcentral.com/1741-7007/5/33</p><p>BMC Biology 2007;5():33-33.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC2048497.</p><p></p>(A) and TbHydin RNAi-induced (B and C) cells. In non-induced cells, the two central pair tubules are nucleated simultaneously (A, arrows), however only a single central pair tubule is present in the TbHydin RNA-induced cell (B, arrow). (C) serial thin sectioning of TbHydin RNA-induced cells from the basal plate through to where the flagellum exits the flagellar pocket demonstrates that the central pair mispositioning defect also originates at the basal plate. Central pair position is determined relative to the PFR (arrow). Note how this position is invariant throughout the series. Scale bars = 200 nm

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules-0

<p><b>Copyright information:</b></p><p>Taken from "The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules"</p><p>http://www.biomedcentral.com/1741-7007/5/33</p><p>BMC Biology 2007;5():33-33.</p><p>Published online 7 Aug 2007</p><p>PMCID:PMC2048497.</p><p></p>ydin RNAi-induced cells (black squares) compared to non-induced controls (triangles). Cells were maintained in log phase by diluting the culture every 24 h. (B and C) Trajectories of individual cells over the course of a 40 s time-lapse. While non-induced control cells (B) are motile, cells locomotion is severely compromised in TbHydin RNA-induced cells (C)

Origin Recognition Complex architecture in the eukaryotes <i>S. cerevisiae</i>, <i>T. brucei</i> and <i>N. gruberi</i>.

<p>The architecture of the Origin Recognition Complex (ORC; composed of Orc subunits numbered 1–6), bound to the Orc1-related factor Cdc6 and to DNA (black line), is shown for <i>S. cerevisiae</i> based on work by Chen et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032674#pone.0032674-Chen1" target="_blank">[22]</a>; the specific arrangement of Orcs 2–5 is inferred from Moreno del-Alamo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032674#pone.0032674-MorenodelAlamo1" target="_blank">[58]</a>. In <i>T. brucei</i>, recognisable ORC subunit orthologues are identified, while subunits that are absent or highly diverged are shown as dotted circles containing question marks. The <i>T. brucei</i> ORC subunit indicated as Orc1 appears to be a bi-functional Orc1-Cdc6 protein, and it is unknown if it therefore occupies a distinct architectural position in the ORC or adopts a distinct structure. Putative ORC subunits identified bioinformatically in <i>N. gruberi</i>, a free-living relative of <i>T. brucei</i>, are shown for comparison; here again, Orc1 appears to be an Orc1-Cdc6 fusion.</p

MCM helicase subunits in <i>T. brucei</i> and co-expression with ORC1/CDC6 as epitope tagged variants.

<p><b>A.</b> An unrooted phylogenetic tree is shown, detailing the homology between predicted MCM helicase subunits in <i>T. brucei</i> (Tbr) relative to orthologues in <i>H. sapiens</i> (Hsa), <i>S. cerevisiae</i> (Sce) and <i>A. thaliana</i> (Ath). Complete protein sequences were aligned with ClustalX, using default settings, and the phylogenetic tree was displayed using TreeView (Page, 1996); the distance corresponding to 10 amino acid changes per 100 positions is indicated (0.1) <b>B.</b> A diagrammatic representation of the MCM helicase subunits in <i>T. brucei</i>. The length of the predicted polypeptides is shown (in amino acid residues), and the position of conserved functional motifs are indicated: an N-terminal Zinc Finger (Zn, blue box); and Walker A and B boxes (A and B, red boxes), an Arginine finger (R, orange box) and sensor 1 and 2 motifs (S1 and S2, green boxes), all involved in nucleotide binding and hydrolysis. <b>C.</b> Western blots of procyclic form TREU 927 <i>T. brucei</i> cells co-expressing C-terminally HA-tagged TbMCM subunits (MCM-HA) and C-terminally Myc-tagged TbORC1/CDC6. The upper panel shows TbMCM-HA expression in whole cell extracts, detected using anti-HA antibody, and the bottom panel shows TbORC1/CDC6-Myc from the same whole cell extracts detected using anti-Myc antibody. Single clones are shown for TbMCM4-HA and TbMCM6-HA, two clones for TbMCM2-HA and TbMCM7-HA, and three clones for TbMCM3-HA. Size markers (kDa) are indicated.</p

Western blot analysis of TbORC1/CDC6-Myc and TbMCM-HA immunoprecipitations.

<p><b>A.</b> Input (I) and eluate (E) samples from immunoprecipitations (IPs) from procyclic form whole cell extracts using antibody against HA are shown from cells co-expressing TbORC1/CDC6-Myc (ORC-myc) and TbMCM3-HA, TbMCM6-HA or TbMCM7-HA, as well as from control cells expressing only Myc-tagged TbORC1/CDC6. Samples were separated on a 10% SDS-PAGE gel, transferred to a membrane and probed with anti-HA antibody (upper panel) or with anti-Myc antibody (lower panel). <b>B</b> shows the reciprocal experiment in which IP was performed with anti-Myc antibody from cells co-expressing TbORC1/CDC6-Myc and TbMCM6-HA or TbMCM7-HA, and from control cells expressing only HA-tagged MCM6 or MCM7. Size markers (kDa) are indicated.</p

Effect of TbORC1/CDC6, TbORC4 and Tb7980 RNAi on bloodstream form <i>T. brucei</i>.

<p><b>A.</b> Analysis of nuclear (N) and kinetoplast (K) DNA configurations in bloodstream form <i>T. brucei</i> cells at time points following RNAi induction (induced by tetracycline; Tet+) against <i>TbORC1/CDC6</i>, <i>TbORC4 (13380)</i> and <i>Tb7980</i>; for comparison, N and K configurations are shown in cells without RNAi induction (Tet−). Graphs depict the proportion of cells (derived by counting >200 DAPI-stained cells) with conventional 1N1K, 1N2K, or 2N2K configurations, or with any aberrant configuration (grouped as others). <b>B.</b> FACS profiles of propidium iodide (PI)-stained cells after RNAi induction (Tet+) are shown as histograms after FACS sorting, sampled at the time points post-induction (control cells, without RNAi induction (Tet−), are shown sampled at the time shown, corresponding to growth from an equivalent starting density to the RNAi- induced cells). Peaks corresponding with cells containing 2C and 4C DNA content are indicated, as is the peak position for cells with 8C content (C represents haploid DNA content).</p

RNAi of TbORC1/CDC6, TbORC4 and Tb7980 in bloodstream form <i>T. brucei</i> cells results in rapid growth arrest.

<p>Growth curves are shown for bloodstream form <i>T. brucei</i> cells in the absence or presence of tetracycline (tet−, shown as solid line, and tet+, dashed line, respectively), which induces RNAi, targeting either <i>TbORC1/CDC6</i>, <i>TbORC4 (Tb13380)</i>, or <i>Tb7980</i> mRNA. For each factor, cell density over time was examined in two clonal RNAi cell lines (identified by C).</p

Mass spectrometric characterisation of <i>T. brucei</i> TbMCM-HA immunoprecipitates and yeast two hybrid analysis reveals MCM subunit interactions.

<p><b>A.</b> Eluates are shown from immunoprecipitations (IP) using anti-HA antibody from <i>T. brucei</i> cell lines co-expressing TbORC1/CDC6-Myc and TbMCM3-HA, TbMCM-6HA or TbMCM7-HA; as a control an IP eluate from a cell expressing TbORC1/CDC6-Myc, but no HA-tagged protein, is also shown. Proteins in the IP eluates were separated by SDS-PAGE and visualised by colloidal commassie staining; size markers are shown and Ig indicates immunoglobulin polypeptides. Bands that were excised and analysed by mass spectrometry are numbered; the results of this analysis are shown in <b>B,</b> where the number of unique peptides identified for each band is shown, as well as the <i>T. brucei</i> gene ID and MCM subunit. <b>C.</b> Inter-MCM subunit interactions were examined by yeast 2 hybrid analysis. Growth of yeast clones co-expressing individual TbMCM subunits (numbered 2–7, indicating TbMCM2–7) as fusions with the Gal DNA binding domain (pGBK-MCM) and with the Gal activating domain (pGAD-MCM) is shown; as a control, growth of the fusion protein-expressing plasmids are shown when co-transformed with pGBK or pGAD vectors without any <i>MCM</i> gene insert (V). Growth on minimal medium lacking tryptophan, leucine and histidine (-T-L-H) indicates weak interaction, while growth on the same media supplemented with Aureobasidin A (-T-L-H+AbA) or 3′ aminotriazole (-T-L-L+3-AT), indicates strong interaction; growth on medium lacking only tryptophan and lecuine (-T-L) shows that the cells that cannot grow through interaction are viable. <b>D</b> shows a model for the assembly and subunit architecture of the MCM hexamer in eukaryotes; a putative subunit complex identified by IP in this analysis is indicated (dashed box), while intersubunit interactions revealed by yeast 2 hybrid analysis are shown in the putative heterohexamer (single- and doubled-headed arrows denote one- and bi-directional interactions, respectively, and strong and weak interactions are distinguished by solid and dashed lines, respectively).</p

Co-immunoprecipitation demonstrates interaction between TbORC1/CDC6 and two novel factors.

<p>Input (I) and eluate (E) samples from immunoprecipitations (IPs) using antibody against HA (anti-HA) are shown from procyclic <i>T. brucei</i> whole cell extracts of cells co-expressing TbORC1/CDC6-Myc (ORC-myc) and either Tb7980-HA (IP labelled ORC1/CDC6-7980) or Tb3120-HA (IP labelled ORC1/CDC6-3120); a control anti-HA IP is shown from cells expressing only Myc-tagged TbORC1/CDC6. Samples were separated by SDS-PAGE, transferred to a nylon membrane and probed with anti-HA antibody (upper panel) or with anti-Myc antibody (lower panel). Bands corresponding with immunoglobulin heavy chain (Ig), an anti-HA cross-reacting band (*) and with either HA-tagged Tb7980 or Tb3120 are indicated in the HA IP samples; size markers (kDa) are indicated.</p