Mutagenesis - Experiments 4 and 5 Flashcards
What is the difference between a spontaneous mutation and an induced mutation?
Spontaneous mutations are completely random and happen without any exposure to environmental agents. Induced mutations are cause by an environmental agent
What is a mutation?
A change in the DNA that affects a gene and its function
What is a forward mutation?
Wild-type -> mutant. Results in a mutant phenotype
What is a reverse mutation?
Mutant -> wild-type. Results in the restoration of the wild-type phenotype
Why are reverse mutations more rare than forward mutations?
Reverse mutations require a precise change in the genetic code to get it to go back to wild-type
Why do we spread millions of auxotrophic cells to measure the mutagenic effect of UV light?
Because reverse mutations are so rare, it takes millions of cells to have decent odds of getting a revertant
Why isn’t it sufficient to only count the number of revertants to determine the rate of reversion?
Mutagens also have a killing effect, and the killing effect needs to be factored in
What medium is used to measure the mutagenic effect of UV?
Minimal media. Any revertants will grow, any that don’t revert won’t grow
What medium is used to measure the killing effect of UV?
Complete media. All cells should grow on complete media, so it is easy to measure how many cells are dying
Why do we only spread a few hundred cells on the complete media plates to measure the killing effect? (3 reasons)
All cells should grow on complete media, so we need to have a small enough number that we can still count. We also need to minimize statistical errors with the small sample size and avoid overcrowding the colonies
Why do we use serial dilutions?
Eliminates the need for very large amounts of the diluent so we get a much smaller and more manageable volume
What parameters are used to calculate Cv in Cv=S0/(Ds x V)? Why is this being calculated?
Cv: cell concentration of the original culture
S0: number of cells on the unirradiated plate
Ds: the dilution used for the supplemented plates
V: the volume plated (usually 0.1 ml)
We need to know how many cells were in the culture before dilution
What parameters are used to calculate Ns in Ns = Cv x Ds x V? Why is this being calculated?
Ns: Number of viable cells plated on the YEPD plates
Cv: cell concentration in the original culture
Ds: dilution used on the supplemented plates
V: volume plated
This tells us how many cells were spread on each YEPD plate
What parameters are used to calculate Nm in Nm = Cv x Dm x V? Why is this being calculated?
Nm: number of viable cells spread on the YM plates
Cv: cell concentration of the original culture
Dm: the dilution used on the YM plates
V: the volume plated
This tells us how many cells were spread on each YM plate
What parameters are used to calculate Pt in Pt = St/Ns? Why is this being calculated?
Pt: the proportion of survivors at time t
St: the number of cells on the supplemented plate irradiated for t seconds
Ns: the number of viable cells spread on the YEPD plates
This tells us the killing effect, tells us what the proportion that should survive the dose of UV light at that amount of time
What parameters are used to calculate Nt in Nt = Nm x Pt? Why is this being calculated?
Nt: the number of cells on each YM plate that are estimated to survive t seconds of UV light
Nm: the number of viable cells on each YM plate
Pt: the proportion of survivors at time t
This is to factor in the killing effect of UV light when determining the reversion frequency
Why does M0 = Rs? What are those parameters?
M0: the number of cells on the unirradiated YM plate
Rs: the number of spontaneous revertants
This plate is not exposed to any mutagens, so any cells on here are spontaneous revertants
What are the parameters used to calculate Fs in Fs = Rs/Nm? Why is this being calculated?
Fs: the frequency of spontaneous reversion
Rs: the number of spontaneous revertants
Nm: the number of viable cells plated on the Ym plates
This is to get the frequency of spontaneous reversions
Why do we have to factor in the number of spontaneous revertants when calculating the number and frequency of induced reversions?
Spontaneous mutations still occur when exposed to a mutagen, so some of the cells on the irradiated plates will be spontaneous revertants
What parameters are used to calculate Rst in Rst = Pt x Rs? Why is this being calculated?
Rst: the number of spontaneous revertants at time t
Pt: the proportion of survivors at time t
Rs: the number of spontaneous revertants on the unirradiated plate
This is to determine how many cells on each irradiated plate are spontaneous revertants
What parameters are used to calculate Rit in Rit = Mt - Rst? Why is this being calculated?
Rit: the number of induced revertants
Mt: the number of cells on the plate at time t
Rst: the number of spontaneous revertants at time t
This is to determine how many cells on each irradiated plate are induced revertants
What parameters are used to calculate Fit in Fit = Rit/ Nt? Why is this being calculated?
Fit: the frequency of induced mutations
Rit: the number of induced revertants at time t
Nt: the number of cells that should survive the UV dose at time t
This shows the mutagenic effect of UV light
What sort of damage does UV light cause to DNA?
Creates photodimers between adjacent pyrimidines
What happens if damage from UV light isn’t fixed?
The DNA structure gets distorted and DNA polymerase might put the wrong base into the new strand of DNA
What are two repair pathways we looked at?
Photolyase and nucleotide excision repair
What is photolyase? What organisms have it? How does it work?
An enzyme that cleaves photodimers. Plants, bacteria, fungi, and some animals (but not humans) have it. It requires light to work because it uses that energy to break the dimer
What is NER? What organisms have it? How does it work?
A set of enzymes (Uvr enzymes) that repair photodimers. All organisms have it. It cuts out a section of DNA around the photodimer and DNA polymerase fills in the gap
What are the two ways sunscreens work?
Either by using chemicals to absorb UV light or using minerals to scatter or reflect UV light
