Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Co-Crystal Structures of CBR1–Hydroxy-PP and Hck–PP1

<p>(A, C, and E) show the binding mode of hydroxy-PP in co-crystals with CBR1. The inhibitor is oriented with its <i>t</i>-butyl group partially exposed to solvent and points toward the surface of the protein. The phenolic moiety of the inhibitor binds deeply within the substrate-binding pocket and makes close contacts to Ser193 and Tyr139 of the catalytic triad and the bound cofactor NADP. (B, D, and F) show the binding mode of the kinase inhibitor PP1 in complex with Hck. PP1 occupies the ATP-binding pocket as an adenosine analog. Although both protein structures show different folds (A and B), the morphology of CBR1- and Hck-binding sites are similar, and inhibitors hydroxy-PP and PP1 bind to these sites with similar shape complementarity (C and D). Key H-bond interactions between hydroxy-PP and the Ser193 and Tyr139 of CBR1 are indicated (E). The exocyclic amine of PP1 in complex with Hck makes essential H-bonds with the main-chain carbonyl oxygen of Glu339 and the side-chain oxygen of Thr338 (F). Disruption of this key H-bonding interaction by derivatization of the exocyclic amine destroys kinase affinity. The figure was prepared using the PyMol 2002 graphics system (DeLano Scientific, San Carlos, California, United States).</p

CBR1 Inhibitors Enhance Daunorubicin-Mediated A549-Cell Killing, yet Prevent Apoptosis in Serum-Starved Cells

<div><p>(A) Cell viability as a function of drug treatment. DMSO, PP-L (8 μM), and hydroxy-PP-Me (8 μM) do not have a pronounced effect on cell viability when used alone. Daunorubicin (DR) alone induces a moderate decrease in cell viability that is accentuated by concomitant treatment with hydroxy-PP-Me.</p> <p>(B) Cell viability decreases dose dependently with concomitant daunorubicin (DR) treatment. Daunorubicin treatment induces a moderate decrease in cell viability when used alone. Hydroxy-PP-Me (1–8 μM) induces a dose-dependent decrease in cell viability with concomitant daunorubicin treatment.</p> <p>(C) PI staining of dead cells is appreciably decreased in serum-starved cells treated with CBR1 inhibitors. A high number of cells were PI stained 65 h following serum starvation in both control and PP-L treated conditions (top). Cells treated with the CBR1 inhibitors hydroxy-PP-L or hydroxy-PP-Me during serum starvation show appreciably less staining (bottom).</p> <p>(D) Quantification of PI-stained cells by fluorescence measurement 65 h following serum starvation. Hydroxy-PP-L and hydroxy-PP-Me induce a dose-dependent decrease in PI staining; whereas PP-L does not.</p></div

IC₅₀ Values against CBR1 and Fyn Kinase Are Tabulated for PP Derivatives

<p>IC₅₀ Values against CBR1 and Fyn Kinase Are Tabulated for PP Derivatives</p

Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284–15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 Å; 1 Å=0.1 nm) crystal structure of the FAH·CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 μmol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation

Affinity Purification and Identification of Human CBR1

<div><p>(A) Reactigel beads appended with hydroxy-PP or PP (control) were used for affinity purification of hydroxy-PP protein targets.</p> <p>(B) Hydroxy-PP-binding proteins in A549 cell lysates. Cytosolic fractions of A549 cell lysate (1.7 mg protein each) were incubated with the indicated affinity resin, and bound proteins were resolved by SDS-PAGE (12% acrylamide gel) followed by silver staining. Untreated beads and PP-control resin samples (lanes 1 and 2) indicate little nonspecific binding. Lanes 3, 4, 5, and 6 were loaded using the hydroxy-PP resin incubated with cell lysate and the indicated competitor. Vehicle or competitor compounds (200 μM) were added to the lysate 30 min before incubation with beads (lanes 4–6). Protein of bands B1–B3 did not bind hydroxy-PP beads when pretreated with hydroxy-PP (lane 5).</p> <p>(C) MS/MS peptide sequencing. Two tryptic peptides from bands B1–B3 were used to identify human CBR1.</p></div

Cell Morphology–Based Screen for Biologically Active Small Molecules

<div><p>(A) Steps in the drug-screening process. Five human cell types, including one primary and four cancer cell lines, were treated for 24 h with the screening library that included compounds of known function and related analogs. The Cytometrix (TM) data analysis package was used to analyze microscopy data for each treatment condition.</p> <p>(B) PCA plot of the phenotypic attributes. Colored spheres represent a single compound at one concentration (ranging from 6 nM to 40 μM by 3-fold increases); lines connecting the spheres indicate a single compound's effects over a range of concentrations. Spheres are colored as follows: known protein kinase inhibitors (blue), paclitaxil (green), and novel compounds structurally related to the protein kinase inhibitors (red). The PCA provides aggregate variables termed “components” made up of multiple independent variables, each with a “loading factor.” These values are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030128#st002" target="_blank">Table S2</a>. Structures of each compound are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030128#sg001" target="_blank">Figure S1</a>.</p> <p>(C) Structures of the known kinase inhibitors PP and PP2 (blue), as well as the novel “hit compound” hydroxy-PP (red), are shown. Linker analogs of PP (PP-L) and hydroxy-PP (hydroxy-PP-L) that were used to ascertain the functional tolerance of replacing the <i>t</i>-butyl substituent at N-1 of the “hit compound” hydroxy-PP are shown.</p> <p>(D) Morphological attribute tabulation for cells treated with 129.4 μM PP2 (blue lines) or 0.4 μM hydroxy-PP (red lines) in each of five cell types. Data for the x-axis is grouped by the probe used (α-tubulin antibody, Hoechst dye, and lectin stain). Each of 14 attributes contributing to the magnitude of the response (y-axis) is shown as a red-filled square.</p> <p>(E) Visual morphology of A549 cells treated with hydroxy-PP or PP2. Hoechst dye or α-tubulin antibody was used to stain cells. The PP2-treated cells are more elongated and have more a condensed nuclear structure as compared with hydroxy-PP-treated cells.</p></div