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Chapter_12_outline - Chapter 12 Mutational Dissection I....

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Chapter 12 – Mutational Dissection I. Chapter Overview A. The purpose of mutational analysis is to understand normal biological function by genetically disrupting normal gene activity and then analyzing the phenotypes of the resulting mutant organisms. B. Forward Genetics – the identification of heritable differences and description of phenotype precedes the molecular analysis of the products encoded by the wild- type and mutant genes C. Reverse Genetics – starting by studying a molecule and then mutating the gene that encodes it. II. The Components of Mutational Dissection A. Several factors are crucial in the recovery of mutations. 1. The choice of mutagen is important because different mutagens produce different arrays of mutations 2. The phenotypes that are used to identify mutant-bearing individuals are also important. B. Techniques of mutational analysis 1. The selection of mutagen according to the kinds of lesions that are desired 2. The assay system that identifies the relevant mutations according to the kinds of processes that one wishes to study 3. The first phase of genetic and phenotypic characterization of the recovered mutations III. Selection of Mutagen A. Advantages of random and direct approaches 1. Directed approaches work much better with reverse genetics. 2. For random mutageneses, general mutagens are required B. Choice of General Mutagenic Agents 1. Ideally, general mutagens should mutate all genes at a constant frequency and produce a broad array of different mutational events. 2. The physical size of genes can differ by several orders of magnitude, making the mutational target size harder to calculate. 3. The mutagenicity of a compound depends on several factors: a. It has to be taken up by the organism and the germ line in sufficient quantity to cause mutation b. It cannot be metabolized rapidly in the body before it reaches the germ line c. Most mutagens are cytotoxic. Effective ones are those that can be delivered at sufficient dosages without be severely cytotoxic. 4. Types of mutagens a. Base-substitution mutagens – Produce base substitutions. Cause transitions at much higher rates than transversions. b. Indel mutagens – Produce insertions or deletions of single base pairs of DNA. c. Insertional mutagens: natural and engineered transposable elements – Transposition events can be manipulated
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experimentally to occur only in a single generation of a series of controlled crosses. This leads to new transposition events that produce insertions at new sites in the genome. d. Mutagens that induce chromosomal rearrangements – chemical agents and certain kinds of high-energy radiation that results in deletions, duplications, inversions and translocations of chromosomes. C. Directed Mutations and Phenocopies
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This note was uploaded on 04/23/2008 for the course BY 214 taught by Professor Woodworth during the Spring '07 term at Clarkson University .

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Chapter_12_outline - Chapter 12 Mutational Dissection I....

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