genet 270- lecture 11- mutation
What is a mutation?
A mutation is any heritable change in the DNA sequence of an organism.
It provides the raw material for genetic variation and evolution.
🧠 Tip: “Mutation = heritable DNA change.”
What is a mutant?
A mutant is an organism whose DNA base sequence has been altered compared to the wild-type (normal) version.
🧠 Tip: “Mutant = changed DNA.”
Differentiate between genotype and phenotype.
Genotype: the actual DNA sequence of an organism.
Phenotype: the observable characteristics or traits that result from that DNA.
🧠 Tip: “Genotype writes it; phenotype shows it.”
What is an allele, and how does it relate to mutation?
An allele is a different form of the same gene, caused by variation in DNA sequence.
A mutation creates a new allele that can produce an altered or identical phenotype.
🧠 Tip: “Mutation makes new alleles.”
What is reversion, and what are its consequences?
Reversion restores a mutant phenotype back to wild type, often by restoring the original DNA sequence (true reversion).
It can also occur through compensatory (suppressor) mutations elsewhere in the genome.
🧠 Tip: “Reversion = undoing the change.”
What must happen for a mutation to affect an organism?
The mutation must alter the DNA, RNA, or protein sequence in a way that changes the message or function, such as:
Changing one or more codons, or
Shifting the reading frame (ORF).
🧠 Tip: “Change the code → change the protein.”
How can mutations alter protein structure and function?
Mutations that change amino acids can alter:
α-helices, β-sheets, or turns (secondary structure),
Protein folding and activity (function).
Replacing an amino acid with one of different chemical properties (e.g., charge, polarity) often disrupts shape and function.
🧠 Tip: “Structure drives function — mutate the structure, lose the function.”
What is the overall consequence of many mutations?
Most mutations lead to an alteration in protein structure, which in turn changes or reduces protein function.
This can cause loss, gain, or modification of activity.
What are silent mutations?
A silent mutation changes a DNA codon but does not alter the protein’s structure or activity, often because of codon redundancy in the genetic code.
🧠 Tip: “DNA change, protein stays the same.”
What are leaky mutations?
A leaky mutation partially disrupts a protein’s activity — the protein retains some function but not full wild-type performance.
🧠 Tip: “Not broken, just weakened.”
What are non-conditional mutations?
Mutations whose phenotype is expressed under all conditions, regardless of the environment.
Example: A frameshift that completely inactivates an enzyme.
🧠 Tip: “Always visible, always mutant.”
What are conditional mutations, and what are common examples?
Conditional mutations only show their mutant phenotype under specific conditions, such as:
Temperature-sensitive mutants,
Suppressor-sensitive mutants,
Auxotrophic mutants (require specific nutrients).
They all result from changes in the wild-type DNA sequence, but expression depends on external conditions.
🧠 Tip: “Mutant only when the environment says so.”
What are the five main mutation types classified by changes to the DNA?
Point mutation
Deletion
Insertion
Inversion
Frameshift mutation
What defines a point mutation and what are its characteristics?
Change: Alteration of a single base pair (can include transitions, reversions, missense, or nonsense).
Characteristics:
Can be leaky (partial function retained)
Can revert (back-mutation possible)
🧠 Tip: “Smallest change, biggest flexibility.”
What defines a deletion mutation and what are its properties?
Change: Removal of a segment of DNA.
Characteristics:
Not leaky (loss of sequence = total loss of function)
NEVER reverts (sequence is physically gone)
🧠 Tip: “Delete = gone for good.”
What defines an insertion mutation, and what are its characteristics?
Change: Addition of new DNA into the sequence.
Characteristics:
Not leaky
Can revert (if the insertion is precisely excised)
🧠 Tip: “Add in → jammed function → maybe removable.”
What is an inversion mutation, and how does it behave?
Change: A segment of existing DNA is flipped (inverted) within the chromosome.
Characteristics:
Not leaky
Can revert (if the inversion flips back to original orientation)
🧠 Tip: “Flip once = broken; flip twice = fixed.”
What is a frameshift mutation, and what are its key features?
Change: Addition or deletion of bases not in multiples of three, shifting the reading frame.
Characteristics:
Not leaky (usually abolishes protein function)
Can revert (if another mutation restores the frame)
🧠 Tip: “Shift the frame, lose the game — unless restored.”
What key question did Lederberg & Lederberg (1952) investigate?
They tested whether mutations arise spontaneously or are induced by exposure to selective agents (phage infection).
🧠 Tip: “Do mutations happen by chance or by challenge?”
What experiment did Lederberg & Lederberg perform?
They used replica plating:
Grew E. coli colonies on non-selective medium (no phage).
Replica-plated these colonies onto multiple plates containing phage T1.
🧠 Tip: “Master plate → replicate plates → look for survivors.”
What did the replica plating results show?
T1-resistant colonies appeared in the same positions on all replica plates.
When these colonies were traced back to the original master plate, they were already resistant before phage exposure.
🧠 Tip: “Same spot = mutation came first.”
What was the conclusion of the Lederberg & Lederberg experiment?
Mutations (like T1 resistance) occur spontaneously, before exposure to the selective agent — not as a direct response to it.
🧠 Tip: “Mutation is chance, not choice.”
Define mutation rate.
The probability that a gene will mutate during a single generation — or the chance of mutation to a specific phenotype.
It varies depending on:
Gene size,
Nucleotide sequence,
Number of genes involved in a phenotype.
🧠 Tip: “Mutation rate = odds per generation.”
What are the three main mechanisms that generate spontaneous mutations?
Replication errors
Alteration of nucleotides
Recombination errors
🧠 Tip: “Replicate, modify, recombine — three ways DNA mutates naturally.”
How do replication errors cause spontaneous mutations?
Through misincorporation of tautomeric bases — rare forms of nucleotides with altered base-pairing properties.
These cause base-pair transitions (purine↔purine or pyrimidine↔pyrimidine swaps).
Example: G:T(enol) mispair →
If tautomer is in the daughter strand → GC→AT transition.
If tautomer is in the template strand → AT→GC transition.
🧠 Tip: “Tautomers trick the polymerase — one base flip at a time.”
What happens when a G:T(enol) mispair forms during replication?
If T(enol) (rare tautomeric form of thymine) pairs with guanine, it creates a G:T mismatch.
On the next round of replication, this mismatch can convert:
GC → AT if T(enol) was in the daughter strand, or
AT → GC if T(enol) was in the template strand.
🧠 Tip: “Tautomer location decides the transition direction.”
What does it mean when we say T(enol) in the template strand recruits guanine?
It means that the tautomeric form of thymine (T*) behaves like cytosine, so during replication it pairs with guanine (G) instead of adenine (A).
When replication continues, this pairing leads to an AT → GC transition in the new DNA.
🧠 Tip: “T(enol) pretends to be C — attracts G, swaps AT for GC.”
Why can tautomeric transitions escape DNA repair mechanisms?
Because tautomeric bases form normal hydrogen bonds, they often pass proofreading undetected.
They can also revert to normal structure before mismatch repair occurs, making detection unlikely.
Only if the tautomer is in the unmethylated daughter strand might mismatch repair remove it.
🧠 Tip: “They look correct, so repair ignores them.”
What is slipped-strand mispairing, and when does it occur?
Occurs when one DNA strand slips relative to the other during replication, especially in short, repeated sequences.
Daughter strand slippage → insertion (extra base added)
Template strand slippage → deletion (base lost)
🧠 Tip: “Slip forward = lose; slip back = gain.”
What kind of mutations does slipped-strand mispairing cause?
It causes frameshift mutations, since the number of inserted or deleted bases is not a multiple of three, altering the reading frame.
🧠 Tip: “Slippage shifts the frame.”
What is deamination, and how does it generate mutations?
Deamination is the spontaneous removal of an amino group from a base.
About 5% of cytosines are methylated (MeC) in bacterial DNA.
When MeC or C are deaminated:
C → U, which pairs with A → GC → AT transition.
🧠 Tip: “Deamination swaps partners by turning C into U.”
What is oxidative damage, and what mutation does it cause?
Reactive oxygen species (ROS) like peroxides and free radicals modify DNA bases.
Example: Guanine → 8-oxo-guanine, which pairs with adenine instead of cytosine → GC → TA transversion.
🧠 Tip: “Oxygen rusts guanine — swaps GC for TA.”
How can recombination cause spontaneous mutations?
Between direct repeats → deletions
Between inverted repeats → inversions
Both rearrange DNA sequence order and can disrupt gene function.
🧠 Tip: “Direct = delete, inverted = invert.”
Summarize the three major spontaneous mutation mechanisms.
Replication errors (tautomers, slipped-strand mispairing)
Alteration of nucleotides (deamination, oxidation)
Recombination errors (between repeats)
🧠 Tip: “Replicate, modify, rearrange.”
What is mutagenesis?
Mutagenesis is any treatment that alters DNA by exposing an organism or DNA to mutagens — physical or chemical agents that increase mutation frequency.
🧠 Tip: “Mutagens make mutations.”
What are base-analog mutagens?
Compounds structurally similar to normal DNA bases that can be incorporated during replication.
They hydrogen bond correctly (so escape proofreading) but can exist in two forms (tautomeric modes), allowing mis-pairing → mutations.
🧠 Tip: “Base imposters that flip their pairing.”
Give an example of a base-analog mutagen and describe its effect.
5-Bromouracil (5-BU) — analog of thymine.
In normal keto form, pairs with A.
In enol form, pairs with G.
Result: Causes transition mutations:
AT → GC (if tautomerizes after incorporation)
GC → AT (if misincorporated in enol form).
🧠 Tip: “5-BU swaps A↔G through tautomers.”
What type of mutation do base analogs typically cause?
They always cause transition mutations (purine↔purine or pyrimidine↔pyrimidine).
🧠 Tip: “Analogs = transitions only.”
What are chemical mutagens, and how do they act?
Chemical mutagens modify existing DNA bases to change their hydrogen-bonding properties, leading to incorrect base pairing during replication.
🧠 Tip: “They don’t replace bases — they corrupt them.”
What is the common result of most chemical mutagens?
Most chemical mutagens cause transitions (swaps between purine↔purine or pyrimidine↔pyrimidine) by altering base-pairing properties of DNA.
🧠 Tip: “Chemicals tweak bases to trade partners.”
How does nitrous acid (HNO₂) cause mutations?
Action: Converts amino groups to keto groups by oxidative deamination.
Effects:
C → U, which pairs with A → GC → AT transition
A → hypoxanthine, which pairs with C → AT → GC transition
🧠 Tip: “HNO₂ deaminates — swaps C with U and A with hypoxanthine.”
How does hydroxylamine (NH₂OH) cause mutations?
Action:
In vitro: converts cytosine to N⁴-hydroxycytosine
In vivo: causes oxidative DNA damage
Effect: N⁴-hydroxycytosine pairs with adenine, producing GC → AT transitions
🧠 Tip: “Hydroxylamine tricks C into loving A.”
How does ethylmethane sulfonate (EMS) cause mutations?
Action: Alkylates the hydrogen-bonding oxygens of guanine and thymine.
Effect: Impairs base pairing, leading to G:T mispairing → causes GC ↔ AT transitions.
🧠 Tip: “EMS adds alkyl groups — forces base pair swaps.”
What are intercalating agents?
Planar, heterocyclic molecules roughly the same size as a purine–pyrimidine base pair that insert (intercalate) between stacked bases in DNA.
🧠 Tip: “Flat chemicals that slip between DNA bases.”
How do intercalating agents cause mutations?
They distort the DNA helix and, during replication, cause insertion or deletion of base pairs, leading to frameshift mutations.
🧠 Tip: “Intercalate → insert or delete → frameshift.”
What is the mechanism behind frameshift generation by intercalating agents?
They likely stabilize looped-out bases that arise during slipped-strand mispairing in replication:
DNA polymerase slips forward → base deleted.
DNA polymerase slips backward → base inserted.
🧠 Tip: “Slip forward lose; slip back gain — both shift the frame.”
What are mutator genes?
Genes whose normal products function in DNA repair or proofreading.
When mutated, they increase the overall mutation rate throughout the chromosome — a mutator phenotype.
🧠 Tip: “When repair genes break, all mutations spike.”
What happens when a cell acquires a mutator phenotype?
Defective repair proteins cause a high mutation frequency in other genes, destabilizing the genome.
Often seen when proofreading or mismatch repair genes are mutated.
Give examples of key mutator genes and their functions:
mutD / dnaQ → proofreading subunit of DNA polymerase III
dam → DNA methylation (directs mismatch repair to correct strand)
mutH, mutL, mutS → enzymes of the mismatch repair system
🧠 Tip: “MutD proofreads; Dam marks; MutHLS fix.”
Why is it often necessary to use mutagens when isolating mutants?
Because the natural (spontaneous) mutation rate is very low — typically 1 in 10⁶ (10⁻⁶) or less.
Using chemical or physical mutagens increases the mutation frequency, making it easier to find mutants.
🧠 Tip: “One in a million? Use a mutagen.”
How are mutants generated for isolation experiments?
Expose bacteria, phage, or isolated DNA to a mutagenic treatment that increases the mutation rate.
After treatment, the cells are allowed to grow so that mutations can be expressed phenotypically.
🧠 Tip: “Mutate → grow → express.”
Why must bacteria or phage be grown after mutagenesis before identifying mutants?
Because initially, only one DNA strand is altered by the mutagen, and the cell still has wild-type gene product present.
Growth allows time for the mutated gene to replicate and express the mutant phenotype.
🧠 Tip: “Mutate the DNA, then let the cell reveal it.”
Under what conditions are isolation experiments performed?
They are done under conditions that selectively allow mutant growth, so that only the mutants of interest survive and can be identified.
🧠 Tip: “Grow only what mutates right.”
What are the four main experimental approaches to isolate mutants?
Screens
Selections
Enrichments
Isolation of conditionally lethal mutants
🧠 Tip: “S-S-E-C — four ways to find a mutant.”
What is a screen in mutant isolation?
A screen identifies mutants by visually distinguishing them from wild-type on media where both can grow.
Examples:
Color indicator plates
Replica plating to find auxotrophic mutants (unable to grow without a required nutrient).
🧠 Tip: “Everyone grows — you just spot the difference.”
What is a selection, and why is it so powerful?
A selection identifies mutants based on their ability to grow under conditions where the parent cannot.
Extremely efficient — can detect 1 in 10¹⁰ mutants on a single plate.
Examples: antibiotic resistance or phage resistance mutants.
🧠 Tip: “Only the mutant survives.”
How does a screen differ from a selection?
Screen: Both wild-type and mutant grow — researcher must examine colonies to find mutants (e.g., color change).
Selection: Only the mutant grows — researcher directly selects survivors.
🧠 Tip: “Screen: see the mutant. Selection: only mutant left.”
What are enrichment methods in mutant isolation?
Enrichments increase the proportion of mutants in a population.
They work by killing growing (wild-type) cells while non-growing mutant cells survive.
Example: Penicillin enrichment — kills actively dividing cells, sparing non-dividing mutants (e.g., auxotrophs).
🧠 Tip: “Don’t select — enrich by killing the fast growers.”
What are conditionally lethal mutations?
Mutations that cause death or loss of function only under restrictive (non-permissive) conditions, but allow normal growth under permissive conditions.
Example: Host mutants that survive without T1 phage but die with phage.
🧠 Tip: “Alive in comfort, dead under stress.”
How are conditionally lethal mutants isolated?
By replica plating:
Grow colonies under permissive conditions.
Transfer replicas to plates under non-permissive conditions (e.g., with phage or at high temp).
Identify colonies that fail to grow under non-permissive conditions.
🧠 Tip: “Replicate, challenge, find who can’t handle the stress.”