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Mutagenesis MUPGRET Workshop Mutation Heritable change in the DNA sequence. Naturally occurring Induced Types of mutations Chromosomal Point Insertion/Deletion DNA repair Mutagens Environmental Chemical Mutations as a tool Associating a phenotype with a gene. Understanding gene function. Studying protein interactions. Understanding cell lineage and organ development. Associating a phenotype with a gene Changes in the DNA sequence that nonfunctional or reduced function proteins often cause a visible change in the appearance of the organism. Some changes do not give visible phenotypes. Often identified as an “off-type” in plant species. Associating a phenotype #2 This is often the first step towards understanding the function of a gene or to dissecting a biochemical pathway. The mutation can be either a naturally occurring one or an induced one. Can be targeted or random. Understanding gene function “You don’t know how something really works until you have to fix it.” Disruptions of the gene can be either non-functional or “leaky”. Often the “leaky” phenotypes will really help you understand how to gene works. Understanding gene function In the case of targeted mutagenesis where you know what the other genes in that would/could be co-regulated with the mutant are you can understand the pathway better by looking at expression of the coregulated genes. Understanding gene function In the case of site directed mutagenesis where you can target particular sequences, you can dissect the part of the protein that is important for function. Can help to ID the catalytic site or a site involved in protein-protein interactions or a site involved in transport, etc. Studying protein-protein interaction Often use a series of alleles that have defects in different parts of the gene to identify the site that is required for protein-protein interaction. The series can be insertions, deletions, or point mutations and may come from nature or be induced or a combination of the two. Understanding cell lineage Usually used with transposon mutagenesis. Transposons are mobile pieces of DNA that can insert into a gene and disrupt its function. Insertion can happen throughout development and can be used to track where cells came from with visible marker. Ac/Ds in Maize Corn example of cell lineage Methods for detection mutations Alteration in electrophoretic mobility Mismatch cleavage methods Mismatch recognition methods Sequencing Protein trunctation test Blazing a Genetic Trail It tells the story of how mutations are involved in several different diseases. http://www.hhmi.org/genetictrail/ Association Genetics Usually used for medical genetics. Recently applied to plant genetics. Which genes were involved in domestication? Is this gene responsible for part of the difference we see in a particular trait such as plant height? Dwarf 8 Mutagenesis and trait analysis suggested that d8 might influence flowering time and plant height. D8 study Sequenced D8 in many ~100 maize lines. Measured flowering time and plant height in the same material. Compare DNA sequence to flowering time and plant height. D8 summary Found several polymorphisms that are associated with changes in flowering time. Data also indicate that D8 has undergone selection. Compare synonymous vs. nonsynonymous substitutions.