2 March, 2005

Chapter 12

Mutational dissection

Normal gene

Altered gene with altered phenotype

mutagenesis

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.

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

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

Target Size

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

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

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

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

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

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+

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

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

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

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

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 gene’s encoded

product

• Gain-of-function– hypermorph: more gene activity

– neomorph: novel gene activity

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