2 March, 2005

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Chapter 12
Mutational dissection

• 2 March, 2005

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Overview

• Mutational analysis is a way to discover genes
  involved in a biological process.
• Process usually involves application of a mutagen
  followed by screening or selection for mutants
  with variant phenotypes.
• Mutations that reduce or eliminate gene function
  are called loss-of-function.
• Mutations that increase gene activity or create
  novel activity are called gain-of-function.

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Mutational analysis

• Powerful tool for studying biological processes
• forward genetics identification of mutants and
  descriptions of their heritable phenotypes
  precedes molecular analysis of products
• reverse genetics based on genome sequences, gene
  of potential interest is mutated and the
  phenotype of mutated gene is studied
• In classical genetics, mutagens were widely used
• In a neo-classical approach, insertional mutagens
  (e.g., transgenic elements) both disrupt gene and
  tag it for isolation

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Components of mutational dissection

• Not all possible mutations of gene can be
  recovered for analysis
• both mutagen and nature of gene contribute to its
  target size, the ability to produce useful
  mutations
• goal is to saturate for mutations, identifying
  all genes that affect the biological process
• Steps in mutational analysis
• selection of mutagen
• assay system
• genetic and phenotypic characterization of
  mutations

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Target Size
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Selection of mutagen

• Random vs directed approaches
• Choice of mutagen depends upon ability of mutagen
  to give rise to mutations with effect on
  phenotype, whether in coding region or in
  regulatory region
• Mutagenicity depends on many factors
• uptake and toxicity to cells
• sex and species differences
• prokaryote vs eukaryote

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Targeted gene knockouts

• Replacement of endogenous gene with ectopic
  (introduced) genetically engineered DNA that
  inactivates gene
• homologous recombination replaces normal gene
  with inactive one
• heritable change, commonly done in bacteria,
  yeast, and mice
• Site-directed mutagenesis
• alteration of specific sites in cloned DNA
  molecule

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Phenocopy

• Specifically interfere with mRNA or gene product
• Antisense RNA
• introduce into target cells RNA complementary
  (antisense) to mRNA
• hybridizes to endogenous mRNA, resulting in loss
  of protein product
• Double-stranded RNA interference
• introduction of dsRNA that leads to destruction
  of mRNA
• Chemical compounds (aka, chemical genetics)
• small molecules tested for ability to affect
  protein
• can be done in automated systems

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Mutational assay

• Somatic mutations
• occur in somatic cells
• mutant sectors of tissue result from mutated
  somatic cell clone
• usually dominant
• usually not passed to offspring
• exception plants in which reproductive tissue
  grows from mutant somatic tissue
• Germinal mutation
• in germ-line set aside during development
• mutations detected in progeny of mutagenized
  individual

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Germ-line mutations

• Dominant mutations appear in F1
• Autosomal recessive mutations require F2 or F3 or
  special backcrosses
• Special techniques for autosomal recessive
  mutations
• induction of development in unfertilized eggs
  (e.g., zebrafish)
• induction of mutated sectors produced by mitotic
  crossover (e.g., Drosophila)
• procedure accelerates screening, but still
  requires crosses to recover mutation

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Forward and reverse mutations

• Unrelated to forward and reverse genetics
• Forward mutation change away from wild-type
  allele
• a ? a
• D ? D
• Reverse mutation change back toward wild-type
  allele (reversion, back mutation)
• a ? a
• D ? D

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Genetic selection vs genetic screen

• Genetic selection
• mutagenesis scheme kills off all individuals
  which do not have trait
• especially useful in microbial systems for
  detecting rare mutations
• Genetic screen
• individuals carrying mutation identified because
  they or some of their progeny display phenotype
  of interest
• must examine every individual
• especially useful in study of development

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Genetic screens (1)

• Can be applied to any problem, depending upon
  ingenuity and resources
• Biochemical mutations
• screening for auxotrophs from mutagenized
  prototrophs
• supply various substrates required for growth
• Morphological mutations
• change in shape or form
• Lethal mutations
• premature death
• recessive lethals are more
• useful than dominant lethals
• that are difficult to maintain

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Genetic screens (2)

• Conditional mutations
• display wild-type under permissive
  (nonrestrictive) conditions
• display mutant phenotype under restrictive
  conditions
• e.g., temperature-sensitive
• mutations
• Behavioral mutations
• Secondary screens
• search for mutations
• that alter mutant phenotype
• modifier mutations
• application of recombinant DNA technology

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Analysis of mutations

• Single genetic differences (dominant, recessive,
  multiple alleles) can characterized and mapped by
  standard genetic means
• Crosses between recessive mutants with the same
  phenotype reveal whether the mutations are in the
  same gene (alleles) (complementation test)
• Genes can be cloned and molecular differences
  identified
• Eventually, functional sites and domains can be
  identified

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Diagnostics

• Both gain-of-function and loss-of-function can be
  dominant or recessive
• Sometimes extensive analysis is needed to
  distinguish between gain-of-function and
  loss-of-function
• Loss-of-function
• partial or complete elimination of activity of
  genes encoded product
• Gain-of-function
• hypermorph more gene activity
• neomorph novel gene activity

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• Assignment Concept map, Solved Problem 1, Basic
  problems except 9, Challenging problems except
  20, 21.

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