The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.
Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
1Department of Neurology, Vanderbilt University Medical Center, 2Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, 3Neurology Service, Veteran Affairs TVHS
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.
We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (http://www.proteinwisdom.org). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.
Published July 25, 2013. Keywords: Genetics, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
1Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, 2Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, 3Department of Pathology, UT Health Science Center at San Antonio, 4Department of Microbiology, UT Health Science Center at San Antonio, 5Cancer Therapy and Research Center, UT Health Science Center at San Antonio
Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.
A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.
Published November 7, 2012. Keywords: Immunology, Molecular Biology, Genetics, Microbiology, directed evolution, thermal behavior, thermostability, endolysin, enzybiotic, bacteriolytic, antimicrobial, therapeutic, PlyC
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
1Language and Genetics Department, Max Planck Institute for Psycholinguistics, 2Donders Institute for Brain, Cognition and Behaviour
Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.
1Akonni Biosystems, Inc.
An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.
Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.
Published March 16, 2011. Keywords: Genetics, random mutagenesis, directed evolution, LB agar drug gradient, bacterial complementation, ColE1 plasmid, DNA polymerase I, replication fidelity, genetic adaptation, antimicrobials, methylating agents
Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains
1Department of General Microbiology, Georg-August University
Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.
Published January 18, 2014. Keywords: Cellular Biology, Bacillus subtilis, evolution, adaptation, selective pressure, beneficial mutation, intraspecies competition, fluorophore-labelling, Fluorescence Microscopy
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
1Department of Life Sciences, National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev
To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.
Published December 18, 2013. Keywords: Biochemistry, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive