Prokaryotic genetics- 47 Flashcards
Mutagenesis and mutations
Why are we interested in mutants?
-Finding and characterising mutants has been essential for developing our understanding of biochemistry and cell biology.
-Mutants are essential for our biotech exploitation of bacteria and an issue for controlling disease.
-Mutation in bacteria are caused by the same mechanisms as in our cells i.e. we can find mutagens, which are likely to be carcinogenic.
-Mutants can be spontaneous or result of exposure to mutagens, compounds which increase likelihood of mutagens.
How can bacteria protect their DNA?
Bacteria have different enzymes that can repair their DNA.
Bacteria can produce melanin and other pigments to protect from radiation damage.
How often does DNA polymerase make a substitution?
Approximately once every 10^7 bases- more often than eukaryotic DNA polymerase.
Genome is 5.4 x 10^6 base pairs. After 2 generations 3.2 x 10^7 base pairs synthesised and probably 3 substitutions made.
There can be millions of E. coli in a ml of culture. But DNA repair will ensure some are corrected.
What is a replication error?
When the wrong base is inserted by DNA polymerase.
What is a tautomer?
Isomers that exist in equilibrium.
Tautomer of the bases have different H-bonding pattern. If it is in a tautomer form in DNA replication then the tautomer base pairing will occur:
-minor tautomer of T and G leads to GT base pairing.
-minor tautomer of A and C leads to AC base pairing.
What is base pair slipping?
Repeat nucleotides can lead to frameshift mutations.
What are mutagens? Give examples.
Mutagens are chemical or physical agents causing damage to DNA.
Mutagens can increase mutation rate by orders of magnitude.
E.g. nitrous acid, reactive oxygen species, alkylating agents, intercalating agents (inserting themselves between base pairs), UV light.
What are intercalating agents?
Insert themselves between base pairs.
Usually have flat multiple ring structures.
Binds between base pairs.
DNA tries to bind with intercalating agent.
Distorts the helix.
Can also lead to frameshift mutations.
E.g. Ethidium bromide. Notice extended flat ring system- flatness allows it to be inserted between base pairs. Used to be added to DNA gels.
What is point mutation?
A change to one base pair. They have different effects at a protein level, therefore varying consequences.
E.g. Substitution, deletion and insertion.
Insertions and deletions can also be called indels.
What can the segregation of mismatched base pairs result from?
Could result from the error in replication, tautamerisation or damage, such as deamination.
E.g. Hypoxanthine- deamination of adenine, H base pairs with C.
Therefore, culture will be a mixture of different genotypes and potentially phenotypes.
What are the consequences of point mutations?
Some mutations are lethal, cells will die and hence not inherited.
Non-coding: may have no consequence at all, but non-coding DNA can have other functions e.g. promoters and other regulatory sequences.
Promoter: can effect transcription (up or down) but may have no consequence at all.
Genes under control of promoter: transcribed to mRNA- depends, can affect sequence of protein or regulation of translation.
What can be the effect of point mutations in protein coding sequence?
Substitutions in coding region leads to:
-silent
-missense
-nonsense
What are the consequences of mutations?
Third base pair substitution often, but not always silent.
Most first and second base pair substitution result in missense. Some third base pair substitutions also do.
Some substitution in 1st, 2nd and 3rd position can result in nonsense mutations. No translation continued, due to stop codon.
What do insertions and deletions result in?
Frameshift mutations.
Frameshift mutations are better tolerated when at the end, because less is affected.
What happens to the protein after silent, missense and nonsense & frameshift mutations?
Silent: nothing. Genotype changed but not phenotype.
Missense: often nothing but can be detrimental. Genotype changed and phenotype may be changed.
Nonsense and frameshift: usually detrimental (because you lose everything after the mutation) but can be tolerated close to C-terminus. Genotype and phenotype changed.
What is the effect of a large scale mutation- deletion?
Can remove kilobases and lose several genes or be just a single base pair.
Can result in frameshift mutations one of few bases are lost within a gene affecting coding or regulation.
What is the effect of large scale mutations- inversions?
Can flip kilobases and several genes or be much shorter.
Can disrupt genes (no longer functional) or just invert them (not an issue).
What is the effect of large scale mutations- tandem repeats?
Part of genome is duplicated.
Can lead to overproduction of proteins encoded in the duplicated region.
Also leads to evolution of proteins.
What is the effect of large scale mutations- transposons?
Transposons are nucleotide sequences that are able to move themselves around.
Can disrupt genes.
What is reversion of mutations?
A point of mutation resulting in restoration of the original sequence.
What is intragenic supression?
2 mutations in the same gene and causes a change in each gene.
What are suppressor mutations?
A second mutation happens that results in the original phenotype being restored.
E.g. original salt bridge
Detrimental mutation, protein destabilised Suppression mutation, salt bridge reversed, protein is stabilised.
What happens in the suppression of frame shift?
Wild type, then addition occurs. Then intragenic suppression- downstream removal of base pair.
What is intergenic suppression?
A 2nd mutation occurs in a different gene to the one which the first mutation occurs.
The effect is to suppress the phenotype of the first mutation.
E.g. nonsense suppression.
What is the difference between intragenic and intergenic suppression?
Intragenic- occurs in the same gene.
Intergenic- occurs in another gene.
What are anticodons?
tRNA contains the complementary sequence to the codon of the amino acid they carry.
E.g. codon = GAU (Asp)
anticodon = AUC
remember to write 5’ to 3’
Why might stains with nonsense suppressors not function correctly?
Strains with nonsense suppressors are normally sick because cells tends to translate past normal stop codons and produce longer proteins than normal.
These may not function correctly because they may fold incorrectly.
How can we select mutants?
Replica plating:
-permissible conditions e.g. lower temperature (-Amp)
-normal conditions (+Amp)
What are selectable phenotypes?
-Drug resistance
-Phage resistance
How can we select for other selectable phenotypes? e.g. mutants with replication errors
Conditional lethal mutants
How do you make a histidine auxotroph?
-Take out E. coli and expose to mutagen.
-Grow in complex medium to allow expression of phenotype.
-Penicillin enrichment: change medium to minimal medium, add penicillin, penicillin will kill any growing bacteria. Therefore auxotrophs will not grow.
-Plate on minimal medium + histidine and grow.
-Replica plate to double check.
What is a phenotype lag?
Certain phenotypes don’t express straight away.
Phenotype is not seen for several generations.
How does phenotype lag work? e.g. resistance to phage T1
Phage binds to a protein spanning the periplasm and outer membrane. Protein encoded by gene tonB.
Mutation to tonB = no more protein made, but up to 1000 copies of TonB already there.
Second generation has 500 copies. 250 -> 125 —»> 1. Next generation some cell have 0. These are now resistant.
What is cross feeding?
Metabolic pathway is ‘blocked’.
-Metabolites immediately before block will accumulate.
-If metabolite can diffuse from cell, another bacteria can take it up.
Will not work if feedback regulation means metabolite do not accumulate
-Both mutants can now grow, but are dependent on each other.
-Phenotype only obvious once isolated.
What is the Ames test used for? Why? What is the assumption?
-Used to identify chemicals that are mutagenic and therefore carcinogenic.
-Much quicker than testing in mice or rats (days rather than years).
-Assumption: if chemical is mutagenic to bacteria it is also mutagenic to humans.
What is the Ames test?
It is a biological assay- analytical method to determine the effect of a substance.
Give an example of Ames test.
Uses a His auxotroph of Salmonella typhimurium.
-Plate same number of bacteria on plates with and without chemical to be investigated.
-Incubate overnight.
-Strain shouldn’t be able to grow- this is an indicator that mutation has occurred.
Grow strain + chemical = if chemical is mutagenic, then lots of reversions.
Grow strain - chemical = if not mutagenic, only few reversions.
What are the limitations of the Ames test?
-Chemical itself may not be mutagenic, but a metabolite might be.
e.g. liver oxygenases that normally detoxify sometimes create reactive forms of chemical.
-Uptake into eukaryotic organisms may differ.
What is an autotroph, phototroph, prototroph and auxotroph?
Autotroph- can produce all its food from inorganic chemicals.
Phototroph- is an autotroph that uses sun light for energy source.
Prototroph- can synthesis most nutrients but needs a carbon source, such as glucose. E.g. E.coli
Auxotroph- has more requirements but in this case only histidine.
What are the minimal growth requirements for an E. coli prototroph?
Minimal medium with glucose.
