How do you determine if a compound is mutagenic? - AMES Test
Use 4 strains of the bacteria strain salmonella typherium (all oxytrophic which means they can't grow on minimal media)
One strain is especially susceptable to base pair substitution. The other 3 are susceptible to frameshift mutation
(3:10) Can this compound cause mutation and result in a significant appearence of prototrophs.
Auxotrophic strain + liver enzymes. Many mutagenic compounds only become mutagenic after exposure to liver enzymes.
Plate the strain on two minimal media plates
Mix your potential mutagen with liver enzyes - add to a piece of filter paper
filter paper is placed on one of two minimal media plates
Incubate overnight and determin number of prototrophs on both minimal media plates.
if experimental plate has a substantially larger number of prototrophs then the control then compound is a mutagen
If your experimental and control plates have similar numbers of prototrophs then it is most likely not a mutagen. (test was developed in 1970's and was used on carcinogens, 80% resulted in mutation)
(13:15) DNA Repair
- Photoreactivation repair (involves prokaryotes) - requires activation from UV light
- How it was discovered:
- What they knew: If you exposed DNA to light from the blue spectrum shortly after exposing DNA o UV light you can reverse some damage done to the DNA. Relied on light-dependent enzyme which was also temperature sensitive.
- What they discovered: Found a protein called photoreactivation enzyme (originally isolated from E. coli) and discovered that this eznyme absorbed 1 photon of light and thus became active. As it became active the enzyme gains the ability to cleave the bonds in a thymine dimer.
- Nucleotide excision repair (found in the vast majority of organisms) 3 step process.
- - Begin with damaged piece of DNA -
- Nucleases search for and identify these lesions. When they find it they cut out the damaged DNA along with a number of bases on either side. This results in a gap in the DNA.
- (23:40) Form of DNA polymerase which fills in the gap with new nucleotides and in the process corrects the mutation.
- (25:05) DNA ligase functions to seal gaps in the DNA strand.
- (26:45) Base excision repair: Corrects minor alteration to DNA molecules (a bit more focused then nucleotide excision repair)
- DNA glycosylase (an enzyme) functions to cleave a base from the sugar phosphate backbone
- AP endonuclease recognizes a sugar without a base (whatever glycosylase leaves behind). When it does this is leaves a nick in the DNA strand upsteram of the sugar where there is no base.
- DNA polymerase comes in and removes the sugar and replaces it with a new nucleotide.
- DNA ligase comes and seals the gap in the DNA strand.
- (32:20) Proofreading during DNA replication
- During DNA replication, DNA polymerase functions to add nucleotides to a growing DNA chain.
- After DNA polymerase adds a base it pauses to check that base looking specifically for "correctness". Did the polymerase add the right base. TWO things can happen: if the base is correct DNA polymerase moves on. If the base is not correct DNA polymerase (with its 3' to 5' exonuclease activity) will function to cleave and remove an incorrect base. After removal DNA polymerase replaces the base and moves on.
- (37:10) SOS repair system: responds to gaps in DNA strands
- Use an alternative DNA polymerase (DNA POL V).
- Typically gaps cause DNA replication to stall out. DNA POL V comes to a gap like this and can fill, it solves the problem. However, in filling the gap, DNA POL V uses less stringent base pairing rules. By-product is increased mismatched base pairs.
No comments:
Post a Comment