Mutagenesis II

Is a pseudo reversion an intragenic suppression?

Yes

Describe how a pseudo reversion occurs?

1. Strain has a mutation

2. Strain acquires a new mutation that masks the phenotype of the first one

3. The strain has suppressed the mutation

What are the 3 types of intragenic suppression?

1. Same codon/same nucleotide

2. Different codon in the same gene

3. Frame shift

What is pseudo reversion?

It is when you have a partially or restored gene product even if the wild type sequence is not restored

What is an example of a pseudo-reversion?

It is when you have a single base deletion in your original sequence which causes a premature stop codon and an altered amino acid sequence. A single base can be added and the proper reading frame is restored but you are still left with an altered sequence

What are tRNA suppressors? Are they a trans or cis acting mutation?

It is when there is a mutation in tRNA (mutation in anti-codon) which allows the anti-codon to pair with the mutant codon. They are a trans acting mutation.

What is an example of a tRNA suppressor?

There is a mutation in the codon which creates a stop codon. A mutation can occur in the tRNA holding glutamine which allows for the anti-codon to pair with the codon. The glutamine is now added and the rest of the amino acid sequence is unaffected.

What is another type of suppressor mutations?

Bypass suppressors

What do bypass suppressors arise from? What type of mutation aside from suppressor? What occurs here?

Genes involved in related pathways. They are compensatory mutations. Functional genetic redundancy occurs here.

What are bypass suppressors?

They allow for a cell to function without a defective gene by using an alternative pathway or protein to yield the same phenotype

What is an example of a bypass suppressor?

argD mutants can suppress many different proB and proA mutants including proBA deletion mutants. argD mutate accumulate high intracellular concentrations of Acetyl-GSA which can be hydrolyzed to GSA by the argE gene product

How can argD mutants grow without added arginine?

This is because they can grow slowly because of other transaminases

What are common phenotypes of mutants in bacteria?

1. Require additional supplements

2. Loss of ability to use a nutrition source

3. Resistance to an antibiotic or small molecule

What is a phototroph? Is it the wild type or mutant? What is an auxotroph? Is it the wild type of mutant?

A prototroph means that it can make the product (can make everything it needs to grow and survive) and it is the wild type. While an auxotroph means it is deficient in one of the metabolites or a biochemical pathway.

Describe what it means by a hisA mutant

The mutant is auxotrophic for histidine (it is unable to make histidine)

Do lacZ mutants grow on lactose? Why?

No they do not because they cannot metabolize it

Is P.aeruginosa resistant to many antibiotics? If yes what is an example? What does it do?

Yes and an example is carbapenems which will prevent bacteria from making peptidoglycan which causes them to die once they get in through porins. P.aeruginosa has gotten around this through a mutation in the porin gene

What is the spontaneous mutation rate of E.Coli? Is this considered low or high? Why?

1/108 (one error in every 108 nucleotides replicated). Rate of spontaneous mutation is low because of the proofreading capability of DNA pol.

What do mutator strands have?

Defects in proofreading and have much higher mutation rates

Is repair important in replication? What can it lead to?

Yes and it can make some mutations more often

What are the genes responsible for mismatch repair? What is the increase in mutation frequency?

mutH, mutS, mutL and 10²-10³

What are the genes responsible for proofreading? What is the increase in mutation frequency?

mutDc and 10³-10⁴

What are the genes responsible for G-A mismatch repair? What is the increase in mutation frequency?

mutT and 10³-10⁴. mutY and >10²

How big is the average gene? How big is a chromosome?

Gene is 10³ bp and chromosome is 5 × 10⁶ bp.

Is there an equal chance of a mutation occurring anywhere? What are 90% of mutations?

Yes there is and silent mutations (no phenotype)

What is mutagenesis used for?

Increase the probability that you will mutate your gene but also increases the frequency of other mutations. You are able to isolate your gene and do something direct with it

What are mutagens used for?

To increase the mutation rate

What are four types of mutagens?

1. Ethidium bromide

2. Nitrosoguanadine (NTG), ethyl methane sulfonate (EMS)

3. Nitrous acid

4. UV irradiation

What is the effect of ethidium bromide? What is it commonly used for?

Inserts between paired bases (is an intercalating agent) (is a carcinogen). Is commonly used to stain gels.

What is the effect of Nitrosoguanadine (NTG), ethyl methane sulfonate (EMS)?

Alkylating agent which modifies and generates O6-methylguanine

What is the effect of nitrous acid?

Deaminates bases

What is the effect of UV irradiation?

Causes crosslinks between adjacent pyrimidines

What does ethyl methanesulfonate react with? What does it make? What happens when polymerase gets to this?

It reacts with guanine to make O-6-methylguanine. Polymerase doesn’t know what to make of this weird base and pairs it with a thymine instead of a cytosine.

What is the result of EMS?

A G:C base pair can become an A:T basepair which can result in a different amino acid

What are thymine-thymine dimers a result of? What type of linkage is between them? What happens when polymerase gets to them? What type of mutation does this result in?

UV irradiation and a covalent linkage between the adjacent bases causes polymerase to see them as one base instead of two. Results in a frameshift mutation

What is an Ames test?

It is a toxicology test to see if it causes cancer or not

There is a strain in salmonella known as salmonella typhimurium, what is it auxotrophic in? Why can’t it?

Histidine meaning it can’t synthesize this amino acid because of a missense mutation

Describe how you would carry out an Ames test with salmonella typhimurium

1. Plate salmonella on to His- media

2. Add suspected carcinogen (DNA altering chemical)

3. Count frequency of revertants (back mutations) that are not His+

Can reversion events occur in salmonella?

Yes and this is creating them in the his allele so it is inducing change