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.
1Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 2Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa
Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.
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.
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
1Department of Biological Sciences, Rensselaer Polytechnic Institute
Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.
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
1Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre
De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.
Published August 6, 2014. Keywords: Genetics, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
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.
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.
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
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