Mutagenesis

Who described the mechanism for evolution based on mutation?

Darwin and Wallace

Who counted peas? What did this show?

Mendel and this showed that inheritance was due to discrete units rather than a blend

What were discrete units referring to from Mendel? What are humans?

Genes and humans are a vessel for genes

What do genes undergo?

Selective pressure

What is variation?

It is difference and difference must arise

What are two possible ways that variation could arise?

1. Directed

2. Spontaneous

Who were the researchers who performed the Fluctuation test? What was the model organism? What is it? Why was it chosen?

Luria and Delbruck. T1 is a phage that kills E.coli and was used because mutants that are resistant are readily isolated

What was the Fluctuation test trying to determine?

If the mutants arised as a consequence of exposure to the phage (directed) or are they selected for (spontaneous)

If directed what would happen? Why?

You should get about the same number of mutants each time you grow a culture because the phage would induce it

If spontaneous what would happen? Why?

The number of mutants isolated would be highly variable each time (it would fluctuate). This is because the mutation could happen at any time in any of the generations and there is no predictable pattern

For the T1 phage acting on E.Coli what is the type of mutation?

It is spontaneous mutation

What are point mutations? Can they sometimes be a deletion?

They are a base substitution that replaces one base with another. Yes

What are the three types of point mutations?

1. Silent

2. Missense

3. Nonsense

What is a silent mutation? What do silent mutations have to do with? What is this?

It is when a base is changed (the DNA is changed) but the protein remains the same (same amino acid). Has to do with the degeneracy of the genetic code meaning that multiple codons encode for the same amino acid.

What is a missense mutation? Is the protein changed?

It is changing the codon of one amino acid for a different amino acid. Yes it is

What are conserved changes? What type of mutation are they associated with? What is the alternative?

It is when the amino acid is switched to another amino acid that has the same properties meaning the protein function is unlikely to be changed (hydrophilic AA switched for a hydrophilic AA). Missense mutation. You could also have the AA switched for a different AA with different properties that will alter protein function.

What are phenotypes driven by?

Proteins

What is a nonsense mutation?

It is when a single change can result in a stop codon (premature stop codon). The message is terminated and you now have a truncated protein

What are the three stop codons?

1. UAG (amber)

2. UAA (ochre)

3. UGA (opal)

What is the simplest AA used in lab? Why isn’t glycine used?

Alanine because glycine has free rotation around the carbon bonds which adds another variable in mutagenesis

What are frameshift mutations?

It is when you add or delete a base (or two) that changes the reading frame

What do deletions and rearrangements arise from? What does this require? What is a full deletion of a gene?

Recombination and RecA is required. It would be null because it removes the entire gene and doesn’t affect downstream genes

What are deletions, rearrangements and insertions ultimately doing?

Changing the nature of the DNA

What are three methods that can be used to perform insertions?

1. Bacteriophage

2. Insertion sequences

3. Transposons

What do bacteriophages do for insertions? What are transposons?

They insert into the chromosome. Transposons are jumping genes and cause a disruption in the open reading frame

Are some genes essential? What would happen if we deleted one?

Yes and null mutations would be lethal

How can essential genes be studied? What type of mutants are these called?

By isolation mutations that are dead under one condition and alive under another. They are called conditional mutants

What is an example of a conditional mutant? Describe how it works

Temperature sensitive mutant. They grow at the permissive temperature and will die at the restrictive temperature

What is another example of a conditional mutant? What happens to the protein? What is it often used for?

Cold sensitive and the protein is nonfunctional when you lower the temperature. It is often used to identify genes that encode for things in a complex (assembly defects)

Describe how replicas plating works to get a conditional mutant (describe the steps)

1. Mutagenize the culture (mutant it)

2. Dilute and plate at the permissive temp

3. The colonies will grow and then you can take a copy using felt and transfer it to a different plate

4. Incubate this replica plate at the non-permissive temperature to see if the colonies will grow or not

5. If they don’t grow then they are conditional mutants

What is a reversion? Is it a low or high frequency event?

It is when there is a change then it can revert back. It is a low frequency event

Describe how a reversion could occur

1. Strain has a mutation

2. Strain acquires a second mutation which is a back mutation

3. The strain has now been changed back to the original

How can insertion mutations be reverted?

By precise excision of the intervening DNA

Can deletion mutations be restored back by true reversion? Why?

No they cannot because template is required to revert back and if a null mutation occurs then there is no template remaining

Does phenotype use normal letters?

Yes

What does amp^r mean?

Ampicillin resistant

What does Trp- mean? Lac-?

Trp- means tryptophan auxotroph and Lac- means inability to ferment lactose

What does auxotroph mean?

The inability to make it (deficient in that process)

What is the nomenclature for proteins? Genes?

Gene: Is italicized and lower case

Protein: Capitalized first letter

How do we write an insertion?

lacZ::Tn10 (indicates a transposon insertion in the lacZ gene)

How do we write a null mutation?

delta symbol(lacZ)

What do better characterized mutant alleles get?

A number

If mutation is mentioned in the genotype what does this mean?

The mutation is implied and it doesn’t need a + or -