Mutagenesis and mutations

Importance of mutants

• mutations in bacteria are caused by same mechanisms as in or cells

• We can find mutagens, which are likely to be carcinogenic

Bacteria response to mutants

• enzymes attempt to repair DNA

• Can produce pigments to protect from radiation

Errors of DNA pol

• substitution mutations

• Will be inherited and spread if not lethal

• DNA repair will correct some

• Mutations spread rapidly in bacteria pop

Replication errors

• wrong base inserted by DNA pol

Tautomers

• isomers that exist in equi

• Tautomers of bases have different H bonding patterns

• Minor tautomers of the bases Leads to incorrect base pairings

Base pair slipping

• repeat nucleotides

• Lead to far shift mutations

• Incorrect number of repeats added in synthesis

Mutagens

• chemical or physical agents

• Cause damage to DNA

Interchelating agents

• flat, multiple ring structures

• Binds between base pairs

• Distorts helix

• Read as a base during synth, additional nuc added and leads to frameshift mutations

Point mutations

• a change to one base pair

Indels

• insertion or deletion mutations

• Can be considered point mutations

Transition substitutions

• purine to purine

• Pyrimadine to pyrimadine

Transversion substitutions

• pyrimadine to purine

Segregation of mismatched base pairs

• damage either repaired or goes onto be present in one of the daughter strands

• Daughter cells of the mutated daughter strand will both be mutated

Hypoxanthine

• from de-amination of adenine

Point mutations in non coding region

• maybe nothing

• Could be a promoter or other reg sequence and affect transcription

• If in UTR, could affect tailing and hence mRNA stability

Silent mutations

• common if 3rd base pair is substituted

• Same protein produced

Missense mutations

• often 1st and 2nd base pair mutations

• Different AA encoded

Nonsense

• substitution in any base pair

• Premature stop codon encoded

When are nonsense mutations tolerated

• close to c TERMINAL

Inversions

• genes flipped

• Can split genes up

Tandem repeats

• part of genome is duplicated

• Parts may be non function if only part of a gene is duplicated

• Can lead to overproduction of proteins, may be toxic in high amounts

• Leads to protein evolution

Transposons

• jumping genes

• Insert at random

• Can insert within genes and split them

Reversions

• point mutations which restores original sequence

Suppressor mutations

• second mutation which restores original phenotype

• Original genotype not restored

Intragenic suppression

• 2 mutations within same gene, leading to original phenotype

Intergenic suppression

• second mutation

• Occurs in different gene from 1st mutation

• 1st mutation phenotype suppressed

Nonsense suppression

• mutant tRNA

• Overrides stop codon

• Continues translation

SupF

• suppresses amber mutations

• Inserts tyrosine at stop codon

Effect of nonsense suppression

• normally sick

• Cell often translates past stop codons, proteins are longer than normal

• May fold incorrectly and so not function correctly

Increasing likelihood of finding mutants

• expose bacteria to mutagen

• Grow in complex medium to allow mutant phenotype expression

• Change medium to minimal , mutants wont grow

• Add penicillin to kill growing bacteria

• Auxotrophs wont have grown so wont be killed

• Grow on plate w supplement needed

• Replica plate to check

Phenotype lag

• when a phenotype isn’t seen for several generations

• Eg an outer membrane protein no longer encoded for

• Still present on bacterial cell membranes from divisions

• Takes many generations to dilute out the protein

Cross feeding

• metabolic pathway blocked

• Metabolites from previous pathway accumulate

• Diffuse out of cell to another mutant bacteria which can’t produce said metabolite , which provides first cell with metabolite which it can’t produce

• They feed each other, so can’t live independently

• Phenotype only obvious in isolation

• Only works if feedback regulation prevents accumulation of metabolites

Ames test

• identifies mutagenic chemicals

• Rapid due to fast generation time of bacteria

• Assumes if a chemical is mutagenic to bacteria then it must be carcinogenic to us

Ames test limitations

• chemical itself may not be mutagenic but a metabolite might bbe

• Uptake into eukaryotes may differ than prokaryotes

Ames test steps

• grow auxotroph

• Plate on 2 plates, one w chemical being investigated and one without

• Grow

• Lots of reversion mutations (bacteria grow) = mutagenic chemical

• Few reversions = not mutagenic