Mutagenesis Flashcards
Why do we study mutants?
- Aid in developing biochem + cellular knowledge.
- Useful in biotech
- Bacterial mutations = same mechanisms in humans for e.g. cancer.
How do bacteria repair/fight against mutations?
- Enzymes repair DNA
- Melanin + other pigments = protect from radiation.
How can bacterial DNA be mutated?
- DNA polymerases make mistakes when polymerising DNA
- Causes spontaneous mutations.
What are 3 examples of spontaneous mutations?
- Replication errors
- Tautomers
- Base pair slipping
What are replication errors?
Wrong base substitution by DNA Pol
What are tautomeric mutations?
DNA base pairs w spontaneous change in structure (become tautomers) = mismatched in DNA replication.
e.g. Adenine normally w Thymine instead tautomeric A = pair w Guanine.
What is base pair slipping?
Repeat nucleotides cause frameshift mutations - no longer in groups of 3 by adding/removing nucleotides.
How can increase mutation rate?
Mutagens: - chemical/physical agent that damages DNA
How do intercalating agents affect mutation rate?
Intercalating => inserting substance into DNA + binding btwn BPs.
- Distort double helix + cause frameshift mutations
What are point mutations?
One BP changes e.g. substitution CATG -> AATG
Is inserting/removing a BP a point mutation?
Some say = “indel” instead.
Different substitutions in DNA
- Transitions
- Transversions
What is a transition?
Point mutation - either subs 2 ring purines (A <-> G) or 1 ring pyrimidines (C <-> T).
What is a transversion?
Point mutation - DNA changing 2 ring purine (A/G) for 1 ring pyrimidine (C/T)
How does mismatched base pairs occur?
- Replication errors
- Tautamerisation
- Damage e.g. deamination
Example of deamination causing mismatching:
e.g. deamination of adenine -> hypoxanthine.
1st daughter cell may get repaired via DNA pol
2nd daughter cell = carries mutation:
- H acc base pairs w C not T so C inserted instead.
- This means next division = one cell => C pairs w G so changes whole sequence, other cell => still mutated w H.
What is the result of mismatched base pairs?
Causes culture to be mixture of different genotypes (genes + genetics) and sometimes phenotypes (observable characteristics)
What are the consequences w point mutations?
Depends on where it occurs in bacterial genome
Do all point mutations lead to inherited characteristics?
No some mutations = lethal so not inherited.
What is consequence of point mutation on non-coding regions of bacterial DNA?
- Maybe no consequences and isn’t significant
- If part of regulation or promoter sequences = can be detrimental
What is consequence of point mutation on a promoter region of bacterial DNA?
- Maybe insignificant.
- May cause up/down regulation of transcription of certain proteins causing imbalances and potentially detrimental effects.
What is consequence of point mutation on genes under promoter control in bacterial DNA?
- Depends on mutation and where in coding region occurs.
- Could affect sequence of DNA or regulator of translation
How much DNA in bacteria + archaea is non-coding?
6-14%, much less than eukaryotes = more chance for mutation to affect protein coding sequence.
What are types of mutations in protein coding?
Silent -> e.g. 3 genes to code for Arginine = swap one for other doesn’t affect anything.
Missense -> mutation changes whole amino acid coded for.
Nonsense -> mutation changes amino acid code into STOP codon, halting synthesis of protein in that location.
Effect of silent mutation in coding sequence on protein
Silent -> Genotype change not phenotype.
i.e. overall no net change in protein.
Effect of missense mutation in coding sequence on protein
Missense -> Genotype changes + potentially phenotype changed.
i.e. overall often no effect but potentially detrimental.
Effect of nonsense + frameshift mutation in coding sequence on protein
Nonsense -> Genotype + phenotype changes.
i.e. usually detrimental but potentially tolerated near C-terminus (happens near end so most of code already there)
What is a deletion mutation?
Removing chunk of genome, either base pairs or kilo-bases (removes whole genome areas)
Consequences:
- can cause frameshift mutation
- or affect coding/regulatory regions of gene.
What is an inversion mutation?
Chunk of genome flips and is turned around.
Consequences:
- depends on where and what part affecting.
- No issues if gene turned round but issue if gene is cut in half (can’t work).
What is a tandem repeat mutation?
Part of genome duplicates, potential to evolve proteins.
Consequences:
- Can cause overproduction of proteins if producing 2 copies.
What are transposon mutations?
Transposons = “JUMPING GENES” nucleotide sequences insert themselves + move independently around DNA.
Consequences:
- Disruption of genes.
How can mutations be reversed?
- Point mutation restores original sequence.
What is a suppressor mutation?
Second mutations that occurs causing restoration of original phenotype.
Intragenic suppression = suppression in same gene as original mutation
What is intergenic suppression?
Second mutation suppressing another occurring in a different gene to the one in which first mutation occurs.
e.g. nonsense suppression
Overview of translation
tRNA w anticodon binds to codon on mRNA and amino acids add in in right order.
tRNA w complementary sequence to codon of AA sequence they carry.
e.g. AAA for Lys -> anticodon = UUU
How is nonsense suppression e.g. of intergenic suppression ?
STOP codon mutation added into mRNA => so tRNA anticodon is mutated to understand the STOP codon and instead inserts another AA into site enabling translation to continue to occur.
What are amber mutations ?
UAG mutations that signal the end of translation.
What is the mutant tRNA known as and functions?
supF = suppressor of amber (UAG) mutation via insertion of tyrosine at stop codon site.
How do nonsense suppressors affect bacterial strains?
Normally unhealthy strains;
Cells translate past normal stop codons causing longer proteins w incorrect functions or folding.
What does a triangle/delta symbol in genetics mean?
Deletion in gene or genetic information
How can we select mutants in populations?
For selectable phenotypes e.g. drug resistance, plate w and without certain drugs in media.
For others: grow via replica plating under permissible and normal conditions.
How to make a histidine auxotroph?
- Expose to mutagen
- Grow in complex medium.
- Transfer to minimal media w penicillin (only kills grown bacteria not auxotrophs which don’t grow)
- Plate on minimal media w histidine
- Check on replica plate is His auxotroph.
What is phenotype lag?
The time taken for genetic change to manifest as a visible or measurable characteristic.
How long does phenotype lag often take?
Often phenotypes not seen for multiple generation.
Example of phenotype lag w T1 phage and EC?
Phage binds to protein encoded by tonB.
tonB mutation = stops protein synthesis, (>1000 copies already present).
2nd generation = 500 copies, 3rd = 250, 4th = 125 –> eventually cells w 0 copies leads to resistant EC to phage.
What is cross feeding?
Different bacterial strains or species exchange metabolites or nutrients, to supporting each other’s growth or survival.
- Occurs when metabolites accumulate before block in metabolic pathway.
(only works if feedback regulation means metabolites can accumulate)
What is the Ames test?
Widely used test that identifies mutagenic (therefore carcinogenic) chemicals.
Assumption of the Ames test?
If chemical is mutagenic to bacteria it is also mutagenic to humans.
Steps of Ames test
- Strains of S. typhimurium are picked that carry mutations to His biosynthesis, so cannot produce His.
- Plate these on minimal media, one w chemical other without.
Results:
If mutagenic, reversions should be abundant (mutagen induced DNA mutations)
If non-mutagenic reversions minimal.
What are the limitations of the Ames test?
Chemical = maybe not mutagenic but metabolite could be and could alter chemical.
