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Cellular and Molecular Biology > Lecture 47: Mutagenesis and mutations > Flashcards

Lecture 47: Mutagenesis and mutations Flashcards

Tuesday 18th February 2025

1
Q

Why are we interested in mutants?

A

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

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2
Q

Is it true that ‘Mutants can be spontaneous or a result of exposure to mutagens ‘?

A

Yes

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3
Q

Is it true that bacteria have different enzymes that can repair their DNA?

A

Yes

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4
Q

Is it true that bacteria can produce melanin and other pigments to protect from radiation damage?

A

Yes

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5
Q

Is it true that DNA polymerase makes a lot of mistakes in bacteria?

A

Yes

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6
Q

How often does DNA make a substitution (mutation)?

A

E. coli DNA polymerase makes a substitution app once every 107 bases

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7
Q

E. coli DNA polymerase makes a substitution app once every 107 bases

Genome is 5.4 x 10^6 base pairs

After two generations 3.2 x 107 base pairs synthesised and probably 3 substitutions made

A

E. coli DNA polymerase makes a substitution app once every 107 bases

Genome is 5.4 x 10^6 base pairs

After two generations 3.2 x 107 base pairs synthesised and probably 3 substitutions made

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8
Q

What happens to mutations that aren’t repaired/fixed?

A

They’ll be inherited by future n

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9
Q

Give 3 ways that spontaneous mutation may occur

A
  • Replication errors
  • Through Tautomers (isomers that exist in equilibrium)
  • Because of base pair slipping
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10
Q

What is a replication error?

A

Where the wrong base is inserted by DNA polymerase

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11
Q

How do tautomers cause mutation?

A
  • DNA bases exist in common (“keto” or “amino”) and rare (“enol” or “imino”) tautomeric forms. These rare forms have different hydrogen-bonding properties, causing mispairing during replication.
  • If a DNA base shifts to its rare tautomeric form right before replication, it pairs incorrectly.
  • If the mispaired base is not corrected before the next round of replication, the incorrect base pairing becomes permanent.
  • This leads to a point mutation (base substitution)—usually a transition mutation, where a purine is swapped for another purine (A ↔ G) or a pyrimidine for another pyrimidine (C ↔ T).

-

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12
Q

What is a frameshift mutation?

A

A frameshift mutation is a type of gene mutation that occurs when nucleotides (A, T, C, or G) are inserted or deleted from the DNA sequence in a way that shifts the reading frame. Since DNA is read in groups of three bases (codons), shifting the frame changes how the entire sequence is interpreted, leading to a completely different protein.

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13
Q

Describe base pair slipping

A
  • This is a spontaneous mutation that occurs when DNA polymerase slips during replication, particularly in regions with repeating sequences (like AAAA or TTTT).
  • This leads to frameshift mutations, which can disrupt the entire protein sequence.
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14
Q

Is it true that mutagens can increase mutation rate by orders of magnitude?

A

Yes

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15
Q

What are mutagens?

A

chemical or physical agents causing damage to DNA

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16
Q

What different things can mutagens be?

A
  • Nitrous acid
  • Reactive oxygen species
  • Alkylating agents
  • Intercalating agents
  • UV light
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17
Q

Describe the structure of interchelating agents

A
  • They usually have flat, multiple ring structures
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18
Q

What do interchelating agaents do?

A
  • They bind between base pairs
  • They distort the DNA helix
  • They can cause frameshift mutations
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19
Q

What is an example of an interchelating agent?

A

Ethidium bromide

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20
Q

Draw the structure of ethidium bromide?

A
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21
Q

Take care to distinguish between what happens to DNA and to proteins

A

Take care to distinguish between what happens to DNA and to proteins

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22
Q

What is a point mutation?

A

A change to one base pair i.e substitution, deletion, insertion.

Can have different effects at the protein level

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23
Q

Apart from a point mutation, what can insertions and deletions also be called?

A

Indels

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24
Q

What are the different types of substituion that can occur?

A

Transitions and transversions

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25
What is a transition substitution?
A purine (A or G) is replaced with another purine. or A pyrimidine (C or T) is replaced with another pyrimidine.
26
What is a transversion substitution?
A purine (A or G) is replaced with a pyrimidine (C or T) or vice versa. Example: A → C or G → T.
27
What can mismatched base pairs result from?
Could result from error in replication, tautamerisation or damage such as deamination
28
What are the consequences of point mutations?
Can have both lethal and non lethal consequences. Lethal mutations are not inherited, because the organism will die.
29
What is the effect of mutations occuring in the non-coding region?
May have no consequence at all, but non-coding DNA can have other functions e.g. promoters and other regulatory sequences
30
What is the effect of point mutations in the promoters?
Can affect transcription (up or down) but may have no consequence at all
31
What is the effect of mutations in genes that are under the control of promoters?
- These are trasncribed to mRNA, so it depends. - Can affect sequence of protein or regulation of translation
32
Don’t confuse type of mutation with consequence of mutation
Don’t confuse type of mutation with consequence of mutation
33
What percentage of DNA is non-coding?
6-14% of DNA non-coding in bacteria and archaea, more in eukaryotes
34
What do substitutions in the coding region lead to?
- silent mutations - missense mutations - nonsense mutations
35
Third base pair substitution....
Third base pair substitution often, but not always silent
36
Most first and second base pair substitution result in....
Most first and second base pair substitution result in missense Some third base pair substitutions also do
37
Some substitution in 1st, 2nd and 3rd position can result in ...
Some substitution in 1st, 2nd and 3rd position can result in nonsense mutations
38
What is a missense mutation
A missense mutation is a DNA change that substitutes one amino acid for another in a protein
39
What is a nonsense mutation?
A nonsense mutation is a type of point mutation in which a codon that originally codes for an amino acid is changed into a stop codon (UAA, UAG, or UGA). This results in premature termination of protein synthesis, leading to a truncated (shortened) and usually nonfunctional protein.
40
Why are insertion and deletions frameshift mutations?
Because they change the consecutive amino acid sequence
41
Why is it better if insertions and deletions happen closer to the 3' end?
Because they will be tolerated better
42
What happens to the genotype and phenotype in silent mutations?
Genotype changed but not phenotype
43
What happens to the genotype and phenotype in missense mutations? (just one amino acid changed)
Genotype changed and phenotype may be changed
44
What happens to the genotype and phenotype in nonsense and frameshift mutations?
Both genotype and phenotype changed. (usually detrimental but can be tolerated close to C-terminus)
45
Describe deletions
- Can remove kilobases and lose several genes or be just a single base pair - Can result in frameshift mutations one or a few bases are lost within a gene affecting coding or regulation
46
Describe inversions
- Can flip kilobases and several genes or be much shorter - Can disrupt genes (red) or just invert them (blue)
47
Describe tandem repeats
- Part of gene is duplicated - Can lead to overproduction of proteins encoded in the duplicated region - Also leads to evolution of proteins
48
Describe transposons mutations
- Transposons are nucleotide sequences that are able to move themselves around - Can disrupt genes
49
What is a reversion?
Reversion is a point mutation resulting in restoration of the original sequence
50
What is a suppressor mutation (intragenic suppression)?
A second mutation happens that results in the original phenotype being restored. But original genotype is not restored.
51
What is intragenic suppression?
A second mutation occurs in a different gene to the one in which the first mutation occurs. The effect is to suppress the phenotype of the first mutation An example is nonsense suppression
52
what is the meaning of this symbol?▲
A chunk of DNA has been deleted
53
what is an amber mutation?
An amber mutation is a type of nonsense mutation where a codon is mutated into the UAG stop codon. This results in premature termination of translation, leading to a truncated and likely nonfunctional protein.
54
How does SupF suppress an amber mutation ?
SupF suppresses an amber mutation usually by inserting tyrosine at the stop codon site. (Anti-codon change GUA to CUA)
55
A bio (biotin) mutation requires...
Requires biotin supplementation.
56
A ara (arabinose metabolism defect) mutation...
Cannot use arabinose as a carbon source. Which is usually fine if glucose is included in the minimal media
57
Nonsense suppression
- Strains with nonsense suppressors are normally sick because cell tends to translate past normal stop codons and produce longer proteins than normal. - These may not function correctly because they may fold incorrectly.
58
Use moodle quizzes for tests on mutants
Use moodle quizzes for tests on mutants
59
How can we make a histidine autotroph? (lacks histdine)
- Expose bacteria to a mutagen - Grow bacteria in complex medium to allow expression of phenotype - Penicillin enrichment: - Change medium to minimal medium - Add penicillin - Penicillin will kill any growing bacteria - Auxotrophs will not grow - Plate with minimal media and histidine and grow - Finally, replica plate to double check
60
What is phenotype lag?
Where a phenotype is not seen for several generations
61
What is cross feeding
Cross-feeding (also called syntrophy or mutual feeding) is a phenomenon where one organism produces a metabolic byproduct that another organism requires for growth. This occurs frequently in microbial communities, including bacterial cultures. (codependent on each other for growth)
62
What is the Ames test used for?
Used to identify chemicals that are mutagenic and 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
63
Describe the process of the Ames test
- Use a histidine auxotroph of salmonella - Grow, plate, and wait overnight - Strain shouldn't grown becayse there's no histidine present on the media. - Growth will be due to reversions if mutagenic
64
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
65
Key messages
Chemicals and radiation can alter the chemistry of the bases which often results in altered base pairing and hence mutations Point mutations involve single base pairs. Large scale mutations can involve a few to several kbp Reversions: restoring the original genotype Suppression: another mutation happens to suppress the phenotype Selecting for mutations based on phenotype and studying these mutations has taught us much of the fundamental biochemistry and cell biology Ames test is an example of how bacterial mutants can be exploited. In this case to assess whether chemicals are carcinogenic
66
Which codons can tolerate a change to another base in 2nd position without changing the amino acid?
None
67

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