title: Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene.
abstract: The sequence of a protein determines its function by influencing its folding, structure, and activity. Similarly, the most conserved residues of orthologous and paralogous proteins likely define those most important. The detection of important or essential residues is not always apparent via sequence alignments because these are limited by the depth of any given gene's phylogeny, as well as specificities that relate to each protein's unique biological origin. Thus, there is a need for robust and comprehensive ways of evaluating the importance of specific amino acid residues of proteins of known or unknown function. Here we describe an approach called Mut-seq, which allows the identification of virtually all of the essential residues present in a whole genome through the application of limited chemical mutagenesis, selection for function, and deep parallel genomic sequencing. Here we have applied this method to T7 bacteriophage and T7-like virus JSF7 of Vibrio cholerae.
authors: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors: William P
authors: WP
authors: Robins
authors: Shah M
authors: SM
authors: Faruque
authors: John J
authors: JJ
authors: Mekalanos
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics*; D017123:Bacteriophage T7 / Q000235:genetics; D020029:Base Pairing / Q000235:genetics*; D018389:Codon, Nonsense / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D005814:Genes, Viral / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D020125:Mutation, Missense; D017902:Podoviridae / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D014734:Vibrio cholerae / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D018389:Codon, Nonsense; D018388:Codon, Terminator; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase
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