The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes

AbstractBackground: Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects.Results: We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre–lox site-specific recombination to catalyze plasmid fusion between the univector – a plasmid containing the gene of interest – and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3′ end gene fusions using homologous recombination in Escherichia coli.Conclusions: Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small and a facile technology to characterize them is lacking. Using a strategy combining Global Protein Stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Using CRISPR screening, we established that the stability of many proteins is regulated through degrons located at their C-terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complexes adaptors that regulate C-terminal degrons including six CRL2 and two CRL4 complexes and computationally implicate multiple non-CRLs in end recognition. Human proteome analysis revealed that the C-termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3 ligase-dependent modulation of proteome composition. Thus, we propose that a series of ‘C-end rules’ operate to govern protein stability and shape the eukaryotic proteome

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs

Application of a synthetic human proteome to autoantigen discovery through PhIP-Seq

In this study, we improve on current autoantigen discovery approaches by creating a synthetic representation of the complete human proteome, the T7 “peptidome” phage display library (T7-Pep), and use it to profile the autoantibody repertoires of individual patients. We provide methods for 1) designing and cloning large libraries of DNA microarray-derived oligonucleotides encoding peptides for display on bacteriophage, and 2) analysis of the peptide libraries using high throughput DNA sequencing. We applied phage immunoprecipitation sequencing (PhIP-Seq) to identify both known and novel autoantibodies contained in the spinal fluid of three patients with paraneoplastic neurological syndromes. We also show how our approach can be used more generally to identify peptide-protein interactions and point toward ways in which this technology will be further developed in the future. We envision that PhIP-Seq can become an important new tool in autoantibody analysis, as well as proteomic research in general

Development of the (d,n) proton-transfer reaction in inverse kinematics for structure studies

Transfer reactions have provided exciting opportunities to study the structure of exotic nuclei and are often used to inform studies relating to nucleosynthesis and applications. In order to benefit from these reactions and their application to rare ion beams (RIBs) it is necessary to develop the tools and techniques to perform and analyze the data from reactions performed in inverse kinematics, that is with targets of light nuclei and heavier beams. We are continuing to expand the transfer reaction toolbox in preparation for the next generation of facilities, such as the Facility for Rare Ion Beams (FRIB), which is scheduled for completion in 2022. An important step in this process is to perform the (d,n) reaction in inverse kinematics, with analyses that include Q-value spectra and differential cross sections. In this way, proton-transfer reactions can be placed on the same level as the more commonly used neutron-transfer reactions, such as (d,p), (9Be,8Be), and (13C,12C). Here we present an overview of the techniques used in (d,p) and (d,n), and some recent data from (d,n) reactions in inverse kinematics using stable beams of 12C and 16O.Comment: 9 pages, 4 figures, presented at the XXXV Mazurian Lakes Conference on Physics, Piaski, Polan

CRK9 contributes to regulation of mitosis and cytokinesis in the procyclic form of Trypanosoma brucei

<p>Abstract</p> <p>Background</p> <p>The <it>Trypanosoma brucei </it>cell cycle is regulated by combinations of cyclin/CRKs (cdc2 related kinases). Recently, two additional cyclins (CYC10, CYC11) and six new CRK (CRK7-12) homologues were identified in the <it>T. brucei </it>genome database <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr></abbrgrp>.</p> <p>Results</p> <p>Individual RNAi knockdowns of these new proteins in the procyclic form of <it>T. brucei </it>showed no apparent phenotype except for the CRK9 depletion, which enriched the cells in G2/M phase. But a similar CRK9 knockdown in the bloodstream form caused no apparent phenotype. CRK9 lacks the typical PSTAIRE motif for cyclin binding and the phenylalanine "gatekeeper" but binds to cyclin B2 <it>in vitro </it>and localizes to the nucleus in both forms of <it>T. brucei</it>. CRK9-depleted procyclic-form generated no detectable anucleate cells, suggesting an inhibition of cytokinesis by CRK9 depletion as well. The knockdown enriched cells with one nucleus, one kinetoplast and two closely associated basal bodies with an average distance of 1.08 mm in between, which was shorter than the control value of 1.36 μm, and the cells became morphologically deformed and rounded with time.</p> <p>Conclusion</p> <p>CRK9 may play a role in mediating the segregation between the two kinetoplast/basal body pairs prior to cytokinetic initiation. Since such a segregation over a relatively significant distance is essential for cytokinetic initiation only in the procyclic but may not be in the bloodstream form, CRK9 could be specifically involved in regulating cytokinetic initiation in the procyclic form of <it>T. brucei</it>.</p

Platinum-(IV)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21(waf1/cip1)-independent pathway in human colorectal cancer cells

Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells.Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells

Tumor markers in breast cancer - European Group on Tumor Markers recommendations

Recommendations are presented for the routine clinical use of serum and tissue-based markers in the diagnosis and management of patients with breast cancer. Their low sensitivity and specificity preclude the use of serum markers such as the MUC-1 mucin glycoproteins ( CA 15.3, BR 27.29) and carcinoembryonic antigen in the diagnosis of early breast cancer. However, serial measurement of these markers can result in the early detection of recurrent disease as well as indicate the efficacy of therapy. Of the tissue-based markers, measurement of estrogen and progesterone receptors is mandatory in the selection of patients for treatment with hormone therapy, while HER-2 is essential in selecting patients with advanced breast cancer for treatment with Herceptin ( trastuzumab). Urokinase plasminogen activator and plasminogen activator inhibitor 1 are recently validated prognostic markers for lymph node-negative breast cancer patients and thus may be of value in selecting node-negative patients that do not require adjuvant chemotherapy. Copyright (C) 2005 S. Karger AG, Basel

Cancer proliferation gene discovery through functional Genomics

Retroviral short hairpin RNA (shRNA) - mediated genetic screens in mammalian cells are powerful tools for discovering loss- of- function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half- hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost- effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells