DNA Mutations,

Damage & Repair

Lecture 1

Mutations

An inherited change in genetic information

DNA susceptible to change

Plus factor driving force in evolution

Minus factor too much change is lethal

Genetic variation in bacteria

Rapid growth

increase from 1 109 in 12 h

Approx 1 in every 109 cells is a spontaneous

mutant (no external agent involved)

Can see 1 mutation event in 12 h

Changes easily seen (single chromosome)

Increase mutation rate using external agents

www.youtube.com/watch?v=gEwzDydciWc

Consequences of mutations
1.

General value

Bad: Antibiotic resistance

Good: Industrial processes (biotechnology)

Evolution

2. Scientific value

Useful tools

gene regulation

assign function to genes

Understand biological processes

Definitions

Genotype:

Phenotype:

Genetic information on the genome

Physical characteristics in that environment

Wildtype:

Not altered with respect to the particular

genotype or phenotype being examined
Genotype + Environment = Phenotype

Causes of Mutations
Induced Mutations

How Do Induced Mutations Occur?

Induced errors

External factors
Contact with damaging agent (mutagen) in the
environment
- Chemical
- Physical
- Infectious Agent
The mutagen directly damages DNA, alters its chemistry or
interferes with its functioning

Causes of Mutations
Spontaneous Mutations

How Do Spontaneous Mutations

Occur?

Spontaneous errors

Natural errors - normal cell processes

Metabolism
By-products of metabolism (eg: free radicals)
DNA Replication errors
Incorrect proofreading by DNA polymerase

eg DNA replication = 1 in 109

Action of mobile genetic elements

eg Transposons

Examples of Types of Mutants

1. Auxotrophic mutants
Identify bacteria that are unable to grow under
certain nutrient circumstances

need particular growth element to be supplied in order

to grow ( eg: amino acid)
cannot use particular C source (eg: lactose)
- Select on MacConkey medium plus C source

Lac positive

Lac negative

Types of mutants
2.

Drug resistant or sensitive mutants

Direct selection on Nutrient Agar plus drug
(for resistance)

3.

Conditional lethal mutations

Temperature sensitive mutants (ts)

Gene not entirely inactive

Functions at certain temp but not others

Used for studying regulatory genes

Mutant Identification: Replica

plating

Mutant enrichment

Mutants at low level vs wild type

Use enrichment to improve isolation of
auxotrophic mutants

Action of Penicillin

Attacks only growing cells

Inhibits transpeptidation enzymes thereby prevents
cross-linking of cell wall peptidoglycan
Cell wall weakens, cell bursts
www.youtube.com/watch?v=UjLmf-cVcMw

Penicillin Enrichment Method to Isolate

Auxotrophic mutants
- Wild-type Bacteria grow
in MM + penicillin most killed
- Mutants cant grow - survive

Bacteria in
Bacteria in
Minimal Medium broth
Nutrient
+ penicillin
Broth
(Wt & Mutants) or add
Mutagen
Minimal medium agar (no aa)
Remaining wt grow
auxotrophic mutants cant grow

Nutrient Agar master plate

(all grow)
Replica plate

Minimal medium agar + aa

WT grows
Auxotroph grows

Ames Test for Mutagenecity

Method to identify environmental mutagens (carcinogens).

Mutational reversion assay using special Salmonella enterica
strains which have permeable membranes, lacks DNA Repair
mechanisms and are histidine auxotrophs auxotrophs.

Ames Assay

Mix mutagen + liver cell extract + Salmonella his- mutant

Control Liver cell extract + Salmonella his- mutant

Liver extract converts potential carcinogens into derivatives that

react with DNA
Plate onto minimal media (no his) and compare mutants that
have reverted (his- to his+) to the control
The more reverted colonies as compared to the control the
greater the mutagenicity of the mutagen.

Ames Test for Mutagenecity