1. Part08
Mutational Analysis
(Ref: Hyde’s Genetics, Chapter 20)
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2. 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
Are the hyphens needed in addition to the numbers?
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3. 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
Are the hyphens needed in addition to the numbers?
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4. 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
Should the font be smaller in the sub categories as in previous slide?
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5. 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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6. 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
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7. Mutations Affected by Environmental Conditions
Figure 20.2
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8. 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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9. 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
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10. Fig.20.4: A model of how protein activity produces different mutant phenotypes
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
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11. Use of a deletion to define the type of mutant allele
Figure 20.5
Use of a deletion to define the type of mutant allele
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12. Chemical-Induced Mutations
How to generate mutants
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
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13. 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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14. 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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15. 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
In both points, should involves be involve?
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16. Forward genetics vs reverse genetics
Figure 20.8
Forward genetics vs reverse genetics
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17. 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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18. 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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19. Isolation of Dominant Mutations
Figure 20.9
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20. 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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21. Isolation of Recessive Viable Mutations
Figure 20.10
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22. 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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23. 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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24. (DNA fragment adjacent to the inserted P[w+] element)
Figure 20.14
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25. 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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26. 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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27. The escape response measures a zebrafish visual behavior
Figure 20.15
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28. 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 b-galactosidase is expressed reveals the expression pattern that is normally controlled by the enhancer
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29. Enhancer trap mutagenesis
Figure 20.16
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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30. Enhancer screens have revealed many different expression patterns in Drosophila
Figure 20.17
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31. Mouse gene trap mutagenesis
-LacZ gene trap that inserted in the mouse aquaporin gene
Embryo day12-13
Figure 20.18
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32. 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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33. Dominant negative mutations exhibit a loss-of-function (negative) phenotype in the presence of the wild-type allele (dominant effect)
It is also possible to generate a transgenic animal that expresses a dominant loss-of-function phenotype
Figure 20.19
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34. 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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35. Genetic crosses to screen for a recessive mutation over a deletion
Figure 20.20
Genetic crosses to screen for a recessive mutation over a deletion
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36. 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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37. TILLING Targeting-induced local lesions in genomes 20-37
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
Figure 20.21: Zebrafish, C. elegans, Arabidopsis
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38. 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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39. Tetracycline-Regulated Systems
Conditional gene mutations
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)
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40. 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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41. Tet-OFF and Tet-ON systems
Figure 20.24
Tet-OFF and Tet-ON systems
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42. 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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43. Figure 20.25
Suppressors and enhancers can act through direct protein-protein interactions
Suppressors
Enhancers
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44. Suppressors and enhancers function through a biochemical pathway
Figure 20.26
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45. 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
In second line, should “the” be deleted for sense?
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46. Figure 20.27: Genetic crosses used to isolate either a recessive suppressor or enhancer
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47. 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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48. 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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49. 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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50. Figure 20.33
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51. 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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52. Figure 20.34
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53. Morpholinos
Morpholinos are modified deoxyribonucleotides that are 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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54. Figure 20.35
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55. 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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56. 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
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57. Phenocopying Via Chemical Genetics
Figure 20.36
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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58. Figure 20.37: Comparison of a mutational and a chemical-genetic analysis
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