define:
What is a mutation?
- a permanent change in genetic material
- doesn’t need to have a phenotypic effect
- can be spontanerous and induced
- cause of genetic varition in the sense that mutations are deletions
Classification of Mutations
Describe: Base Substitutions and Indels
Base Transition: of one base
- transition: purine to purine, pyrimidine to pyrimidine
- transervsion: purint to pyrimidine and vice versa
Indels: insertion or deletion of 3 bp or multiples of 3 nucelotides to cause frameshift
what is the likelyhood of a phenotypical effect due to a mutation
- mutations at the exon are more likely to have a functional consequence despite the intron and exon having the same probability of having a mutation occur
- the phenotype consequence of mutation is generally impossible predict
- it may have a phenotype
what are the different types of mutations
silent, missense, nonsense, frameshift
- silent mutation: the protein produced is the same as the wildtype
- missense: the protein produced is different from the wildtype
- nonsense: premature stop codon
- frameshift: caused by indels
Errors in replication
Explain Base Mismatch error by DNA polymerase
- can ocassionally add a wrong base pair
- 1/1000 BP error
- causes transitions; if it was a transversion there would be distorations with the helix and therefore is detected
- 1/10^6 effective mutation rate
- because of the 3’-> 5’ exonuclease activity, it is able to remove a mismatch and have a do over
Errors in replication
explain slippage
- DNA poly can dissociate and reattach, happens with long strands of the same seq
- if replicating strand slips, there is an insertion of an extra ntds
- indels
- fragile X order
- Huntingon’s Disease: mutant allele has greater than 40 repeats compared to WT with <28 repeats
- repeat of CAG
Spontaneous lesions
describe depurination
- loss of the purine base in the nucleotide
- in mammalian cell lose ~10,000 purines in a 20-hr cell cycle
- there are repair pathways to use other strand to fill up the AP site
Spontaneous lesion
Describe deamination
- very common: occurs in ~100 bases/cell/day
- requires replication to stabilize mutation
- loss of an amino group on cytosine and turns into Uracil
- causes a transition
Spontaneous lesion
Describe oxidative damage
- caused by reactive oxygen species (ROS)
- Guanine produces 8-oxoguanine: where the H bond becomes an O
- no longer is stable with Cytosine so it bonds with Adenine
- causes transversion
Spontaneous lesion
Describe tautomerization
- isomers: Keto and Enol form for purines; anime and imine form for pyrimidines
- causes to base pair with the wrong base
- ex. Gaunine KETO from is an H bond acceptor; ENOL form is an H bond donor
How do we identify a mutagen?
- Ames test: taking potential mutagen
1. activate mutagen
2. expose to His- bacterial strain
3. observe reversion rates
4. select for bacteria that are mutated again by mutagen
Types of Mutagens
What is 5-BU (base analogs)
- chemical that looks like a base but causes incorrect base pairing
- ex. 5-Bromo-uracil is thymine analog that base pairs with G instead
- causes transitions
Types of Mutagens
Describe Alkylating agents
EMS
- addition of Me or Et groups
- inferes with H bonding
- causes transtions
- ex. Gaunine to O6-methylguanine: cannot form the third H-bond with Cytosine so it base pairs with T
Types of Mutagens
Desrcibe Intercalating agents
Proflavin
- fit b/w stacked bases and cause distortions in double helix
- causes indels
Types of Mutagens
Describe Base Damage (UV, Aflatoxin B1)
Ex. UV Damage
- energy from UV light is absorbed by the rings of pyrmidines
- causes bonds to from b/w stacked bases: cyclopyrimidine dimer
- during DNA repl. lesion is filled by variable # of adenines
- can cause transitions and indels
- can be directly replaire by photolyase
How do we create mutants?
Describe Mutagensis screen
- looking for interesting phenotypes
1. start with known strain/ phenotype
2. choose a mutagen F1 will be heterozygous for induced mutations
3. score F2 and look for desired phenotypes
How do we create mutants?
Explain RNAi
- RNA interference
- introduction of a dsRNA triggers a trageted degradationg of all endogenous (natural) mRNAs
- RNAi mimics a knockout/ deletion mutation
How do we create mutants?
Explain CRISPR/cas 9
- “gene editing”
- uses naturally occuring enzymes from bacteria
- “search and replace function
- alter the genome in a specifi and targeted way
