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Mutational Dissection http://philos.biol.mun.ca/~b4241/4241_mccr/mutationaldissection.html Chapter 12: Mutational Dissection Meagan Abbott Renee Mulrooney Christa Lewis Colleen Buckley Overview: The purpose of mutational analysis is to understand normal biological function by genetically disrupting normal gene activity , and analyzing mutant phenotypes. We will explore basic approaches used by geneticists to produce mutants. Forward Genetics: identification of heritable differences and description off phenotypes PRECEEDS the molecular analysis. Some cases... A Geneticist wants to study a biological process, so they look through a group of mutagenized individuals, and identify the individuals with phenotypes affecting this specific development. If the phenotype produced by a mutation is known and wants to be studied further, gene location will be mapped, and the study of mutant alleles will be done by various techniques. Reverse Genetics: Start by studying the molecule, and then mutate the gene that encodes it. Ie: You identify a protein or RNA, and now you want to know what the phenotype is when the gene encoding this product is mutated. This chapter will explore classical and recently developed techniques for forward and reverse genetics. Selection of a Mutagen mutagen selection is vital to mutagenesis outcome mutagens can be divided into two types Choice of General Mutagenic Agents Ideally, a general mutagen will mutate all genes at a constant rate producing a broad spectrum of varying mutational events. Reality, gene sizes vary across a genome. Mutational target size is therefore difficult to calculate. Problem is due to the fact that typical mutagenesis identifies those changes which lead to sought after mutant phenotypes. In comparing two genes, the proportion of mutations which create mutant phenotypes can be very different. Some commonly used General Mutagens mutagenicity depends on a multitude of factors: uptake into organism (and germline) in sufficient quantity must be strong enough to withstand being metabolized more rapidly then the time required for it to reach the germline must be able to be delivered at high enough doses to induce high freqency mutation without being severly cytotoxic Four classes of mutagens and their outcomes Direted Mutations and Phenocopies DNA repair, DNA replication, and information transfer during gene expression has enabled direct mutation techinique targeting techniques enable visualization of the phenotype exhibited by individuals lacking function of individual genes Two broad methods of ways to achieve directed muations; 1)inactivate gene using mutagens targeted to the DNA (Targeted Gene Knockouts, Site-Directed Mutagenesis and Related Techniques), or 2)Leave gene intact, blocking the activity of one of the gene products (antisense RNA, dsRNA interference, and chemical library screening.) http://philos.biol.mun.ca/~b4241/4241_mccr/mutationaldissection.html Advantage of gene inactivation mutagens will be passed on from one generation to the next manipulation of the cell must only occur once Advantage of inactivation of gene product can be applied to many organisms despite how well developed the genetic technology is Mutational Assay System The key to mutational analysis is to match the appropriate mutagen with the appropriate system for detecting mutations. Somatic vs Germline Mutations somatic mutation: A mutation that arises in a somatic cell, and consequently is not passed through the germline to the next generation germline mutation: mutations occurring in the cells that are destined to develop into gametes Somatic: If one somatic cell mutates in developing tissue, it becomes the progenitor A population of clones arise from that progenitor, making a patch of mutant cells = Mutant Sector Dominant somatic mutation will show up in the phenotype but recessive will not. If a mutation arises in a post mitotic cell, it will have negligible effect on the phenotype. Somatic mutations are not passed on to the progeny. Plants=exception. Germline: If a mutant gamete undergoes fertilization the mutation will be passed on to the next generation.fig 12.18 Dominant and Recessive Germline Mutations Forward and Reverse Mutations Genetic Selections vs Genetic Screens http://philos.biol.mun.ca/~b4241/4241_mccr/mutationaldissection.html Genetic Selections - a mutational selection technique that enriches the frequency of specific (usually rare) genotypes by establishing environmental conditions that prevent growth or survival of other genotypes. Genetic Screens - a mutagenesis procedure in which essentially all mutagenized progeny are recovered and are individually evaluated for mutant phenotype. Advantages vs Disadvantages Genetic selections - mutational events are quite rare (0.0001%) and therefore being able to kill off the non-mutational progeny (99.9999%), identification of mutants is much easier. Genetic Screens - any imaginable phenotype can be sought out, but very labor intensive therefore few individuals can be examined compared to genetic selection. Analysis of recovered mutations isolated mutations of genes and gene products, can be used to draw conclusions about the genes properties. Mutations: a mini review Counting the genes in a biological process Classification system of mutations Further aspects of Mutation Analysis Summary.
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