Mutational Analysis

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(Ref: Hyde’s Genetics, Chapter 20)

Part08

Essential goals

• Describe the various classes of mutations and how they differ

• Generation of different types of mutants by genetic crosses

• Differences between forward and reverse genetics and their advantages

• The various approaches used to isolate mutants in genetics

• Isolation of recessive mutations in a mosaic screen• Phenocopying: a phenotype that is controlled by the

environment, but looks like a genetically controlled phenotype

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Introduction

• Isolation of specific mutants is important for several reasons– 1) Can be used to study general cellular

processes– 2) Can serve as models for studying diseases– 3) Can be used to identify genes whose

encoded proteins interact with other mutants

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Mutational Classes

• Mutations can be described based on:

– 1) their effect on the encoded protein

– 2) the phenotype produced under different conditions

– 3) how the mutant behaves in certain genetic backgrounds

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Mutations Based on Changes in Protein Sequence

• Missense mutation– Results in a different amino acid

• Nonsense mutation– Introduces a premature translational

termination codon

• Frameshift mutation– Affects all the codons following the nucleotide

insertion or deletion

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Mutations Affected by Environmental Conditions

• Conditional mutants exhibit the wild-type phenotype at the permissive condition and a mutant phenotype at the restrictive condition

• Nutritional mutants– Auxotrophs can grow on rich media

• Temperature-sensitive mutants– Often, the permissive temperature is lower – Example: Himalayan rabbit

Mutations Affected by Environmental Conditions

Figure 20.2

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Mutations Based on Genetic Interactions

• With a recessive mutation, the phenotype of a homozygous mutant is compared to that of a deletion heterozygote– If identical, the mutation is a null mutation

• A null allele (amorph) has no apparent encoded protein activity

– If the heterozygote phenotype is more severe, the mutant allele is called a hypomorph• It encodes a protein with reduced activity

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Mutations Based on Genetic Interactions

– If the heterozygote phenotype is less severe, the mutant allele is called a hypermorph• It encodes increased levels of protein activity

• Neomorphic alleles produce a phenotype that corresponds to a “new” protein activity– The expression of the gene occurs at either a

new location or at a new time in the life cycle

Null allele: loss-of-function allele, a mutant version of a gene that either does not express a protein or the protein entirely lacks any activity

Hypomorphic allele: loss-of-function allele, an allele that encodes a protein with reduced activity relative to the wild-type allele

Hypermorphic allele: gain-of- function allele, an allele that encodes a protein with increased activity relative to the wild-type allele

Fig.20.4: A model of how protein activity produces different mutant phenotypes

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Figure 20.5

Use of a deletion to define the type of mutant allele

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Chemical-Induced Mutations

• Chemicals can generate gain-of-function, loss-of-function, and conditional mutations

– Alkylating agents• Ethyl methanesulfonate (EMS)• N-ethyl-N-nitrosourea (ENU)

– Base analogs• 5-bromouracil• 2-aminopurine

How to generate mutants

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Energy-Induced Mutations

• X-rays transfer high levels of energy into the DNA molecule, which breaks the phosphodiester bond– Single and double breaks in the DNA molecule

can produce inversions, translocations, and deletions

– These chromosomal rearrangements usually result in loss-of-function alleles

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DNA Insertional Mutations

• Mutations can also be produced by inserting a piece of foreign DNA randomly in the genome

• Transposable elements– Introduce nonsense mutations– Block transcription by insertion into the

promoter region

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Forward Genetics

• There are two common ways to isolate mutants

– Forward genetics involves randomly mutagenizing the genome to generate a wide array of mutants

– Reverse genetics involves the targeted mutagenesis of a specific gene

Common ways to isolate mutants

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Figure 20.8

Forward genetics vs reverse genetics

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Genetic Screens Used in Forward Genetics

• The mutation of every single gene in the genome is termed saturation

• Reaching saturation depends on 3 factors– 1) Mutagen must be applied at a high dose– 2) All desired genes must be mutable at

roughly the same efficiency– 3) A very large number of mutagenized

progeny must be generated and screened

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Isolation of Dominant Mutations

• Males are mutagenized with EMS and then mated to wild-type females

• The F1 progeny are examined for dominant mutations in an F1 screen

• To determine if the F1 mutant possesses one or more dominant mutations, it is crossed to a wild-type individual, and the frequency of F2 mutants is determined

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Figure 20.9

Isolation of Dominant Mutations

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Isolation of Recessive Viable Mutations

• Males are mutagenized with EMS and then mated to wild-type females

• The F1 progeny are individually mated to wild-type individuals to generate independent lines

• The F2 progeny are randomly mated with their siblings

• About 25% of the F3 progeny will exhibit the recessive mutant phenotype

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Isolation of Recessive Viable Mutations

Figure 20.10

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Isolation of Mutants Resulting from Transposable Elements

• The Drosophila P element is the transposon

• Recombinant P element cannot transpose on its own because the transposase gene is replaced with the wild-type white gene

• Continually mating the P[w+] flies to w– flies and selecting the offspring with red eyes (w+) identifies flies with the mutant chromosome

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Isolation of Mutants Resulting from Transposable Elements

• Genes that are disrupted by insertion of the P element can be easily cloned through two different methods– 1) Generation of a genomic library from the

progeny of these mutant flies• Screening with the P element as probe

– 2) Using inverse circular PCR amplification

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Figure 20.14

(DNA fragment adjacent to the inserted P[w+] element)

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Approaches Used to Screen for Mutants

• The genetic screen is a method used to identify the rare individuals with the desired phenotype in the large population

• Two general approaches– 1) Selection involves segregating the desired

mutants from the remainder of the population (Ex: Identifying revertant from the mutagenized auxotrophs: Ames test)

– 2) Detection improves the identification of the desired mutants from the remainder of the population (ex: lac- mutant in E. coli using IPTG & X-gal)

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Visible Mutant Phenotypes

• Mutants exhibiting a visible mutant phenotype are the most obvious to select– Example: Coat color in mice

• Detection of mutants with an abnormal behavior can be more difficult– Example: Escape response in zebrafish

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Figure 20.15

The escape response measures a zebrafish visual behavior

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Enhancer Trap Expression Screens

• Expression screens identify genes based on when and where the genes are expressed in the organism

• The enhancer trap screen uses a modified P element with the white+ gene, the lacZ gene, and a polyadenylation signal (pA)– When and where the -galactosidase is

expressed reveals the expression pattern that is normally controlled by the enhancer

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Figure 20.16Enhancer trap mutagenesis

Transcription of lacZ increases and is expressed in the same pattern as the gene that is normally controlled by the enhancer

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Figure 20.17

• Enhancer screens have revealed many different expression patterns in Drosophila

Figure 20.18

Mouse gene trap mutagenesis

-LacZ gene trap that inserted in the mouse aquaporin gene

Embryo day12-13

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Reverse Genetics

• One of the most direct methods is to generate specific mutations in a desired gene– Create mutations in a cloned gene in vitro and

reintroduce mutant gene into the organism• Knockout mouse: Contains disrupted gene

–Expresses a loss-of-function phenotype• Transgenic mouse: Retains wild-type alleles

–Expresses a dominant gain-of-function phenotype

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• It is also possible to generate a transgenic animal that expresses a dominant loss-of-function phenotype

Figure 20.19

Dominant negative mutations exhibit a loss-of-function (negative) phenotype in the presence of

the wild-type allele (dominant effect)

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Screening Mutations Uncovered by Deletions

• Wild-type males are randomly mutagenized and then crossed to females with a deletion chromosome

• The F1 progeny are then examined – If sperm contained a mutation in the deleted

region, individual will express a recessive phenotype due to pseudodominance

– If sperm contains a mutation outside the deleted region, individual will express the wild-type phenotype

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Figure 20.20

Genetic crosses to screen for a recessive mutation over a deletion

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Screening Mutations Uncovered by Deletions

• This approach is limited by several factors– 1) A deletion must exist for targeted gene– 2) The deletion must be relatively small– 3) Mutagenized male must be mated to a

female with the correct deletion– 4) It might be difficult to predict the recessive

mutant phenotype

• To get around these limitations, a new screening method was developed

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TILLING• Targeting-induced local lesions in genomes

Figure 20.21: Zebrafish, C. elegans, Arabidopsis

This method relies on the ability to detect F1 that are heterozygous for the mutation of interest and mate to generate homozygote progeny

-The F1 progeny (heterozygous for random mutations) is collected for genomic DNA isolation

-The desired gene is PCR amplified from the genomic DNA and analyzed for a mutation

-The F1 progeny with the desired mutation are either mated or their frozen sperm are used for in vitro fertilization

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TILLING• Advantage: the generation of a series of missense and

nonsense alleles without any preconceived idea about the corresponding phenotypes

• How do we analyze the genomic DNA for the desired mutations?– PCR amplification and analysis of exons

– CODDLE (codons optimized to discover deleterious lesions) software

• The less negative the score, the greater the likelihood of producing a deleterious mutation

• The mutagen will most likely produce nonsense and missense mutations• PCR primers are then designed to amplify the selected exons

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Tetracycline-Regulated Systems

• Provide a method to control the expression of a transgene through environmental conditions

• The systems employ:– A hybrid protein consisting of the bacterial

tetracycline repressor fused to the VP16 transcriptional activator domain of the herpes virus

– A specific DNA-binding sequence, the tetracycline response element (TRE)

Conditional gene mutations

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Tetracycline-Regulated Systems

• Tet-On system

– Transgene expression is activated in the presence of doxycycline

• Tet-Off system

– Transgene expression is repressed in the presence of either tetracycline or doxycycline

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Figure 20.24

Tet-OFF and Tet-ON systems

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Isolating Suppressors and Enhancers

• A suppressor is a mutation in one gene that makes the phenotype of another gene “less mutant”

• An enhancer is a mutation in one gene that makes the phenotype of another gene “less wild-type” or “more mutant”

• They usually encode proteins that either directly interact with the original mutant protein or act in the same biological pathway

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Figure 20.25

Suppressors and enhancers can act

through direct protein-protein interactions

Suppressors Enhancers

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Figure 20.26 Suppressors and enhancers function through a biochemical pathway

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Isolating Suppressors and Enhancers

• Males that are homozygous for the first mutation are mutagenized and then mated to homozygous mutant females – In the F1 and subsequent generations, dominant

enhancers and suppressors in the m2 gene can be identified

– In the F3 generation, recessive m2 suppressors or enhancers can be isolated

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Figure 20.27: Genetic crosses used to isolate either a recessive suppressor or enhancer

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Mosaic Expression Analysis

• A mosaic is an individual composed of more than one genotype and may express different phenotypes in different tissues

• There are 2 general methods for producing a homozygous recessive genotype– Irradiation-induced mitotic recombination– Site-specific recombination

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Phenocopying

• Phenocopy is the production of a mutant phenotype in a genetically wild-type individual by introducing molecules or altering the environment

• Two general methods are employed– Phenocopying via RNAi– Phenocopying via chemical genetics

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Phenocopying Via RNAi

• RNAi (RNA interference) employs the same Dicer enzyme and RISC machinery as the miRNAs described in Chapters 10 and 17

– Double-stranded foreign RNAs are processed into single-stranded, short interfering RNAs

– siRNAs can affect protein expression in three different ways

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Figure 20.33

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Phenocopying Via RNAi

• Three major methods are used to generate siRNAs– 1) Two complementary RNA sequences are

synthesized in vitro– 2) A region of the gene is cloned between two

bacterial or phage promoters– 3) A region of the gene is cloned in an inverted-

repeat orientation downstream of a promoter

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Figure 20.34

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Morpholinos

• Morpholinos are modified deoxyribonucleotides that are 20-25 nucleotides long

– More stable and less toxic than standard oligonucleotides

– Bind to the 5′ UTR of mRNA and prevent its translation

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Figure 20.35

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Phenocopying Via Chemical Genetics

• Chemical genetics is the identification and use of small molecules that interact with specific proteins to generate altered phenotypes– Small organic molecules – Peptide aptamers (short amino acid oligomers)

• Pharmaceutical companies have libraries of millions of natural and synthetic compounds– Can be screened in two basic ways

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Phenocopying Via Chemical Genetics

• Phenotype-based screen– Small molecules are added to cells or organism

to generate a specific phenotype– Similar to forward genetics

• Target-based screen– Small molecules are tested for ability to bind to a

single desired target protein– Similar to reverse genetics

Figure 20.36

Phenocopying Via Chemical Genetics

• Chemical-genetic analysis has two main advantages over mutational analysis

– 1) Different molecules may interact with different portions of a single protein

– 2) Molecules may function as either suppressors or activators of the target protein

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Figure 20.37: Comparison of a mutational and a chemical-genetic analysis