Mutations 1 Flashcards
What would one need to do to regrow telomeres in a senescing cell culture?
- understand what is limiting telomere length in the first place, could be a mutation, lack of transcription factor, wrong post-translational modifications
- activate the three telomerase genes
- check that your method to activate the gene resulted in longer telomeres
Can you think of a way to compare the lengths of the telomeres of two cell culture samples (e.g., a cell line before and after telomere regrowth) using qPCR?
in repetitive regions the primer will anneal many times meaning you will have a lower ct value, compare the ct values of your telomere to that of a housekeeping gene, the ratio between the two is what you will compare
what is a mutation
a stable change in DNA sequence
the majority of coding region mutations are ______
detrimental
name 4 types of point mutations
silent, sense, missense, nonsense
what type of mutation is: a change in a DNA sequence that results in a new codon coding for the same amino acid
silent or sense
what type of mutation is: a change in DNA that results in a different codon, a different amino acid is substituted in the corresponding protein
missense
what type of mutation is: a change in DNA that results in a premature stop codon
nonsense
what type of mutation is caused by small insertion/deletions?
frameshift
can large scale insertions/deletions/rearrangements that preserve reading frame still be disruptful?
yes (ex. huntingtons)
how does a mutation in an intro disrupt gene expression?
introns contain transcription factor binding sites
what does UTR mean
untranslated region
5’ UTR mutations may interfere with _____
translation
3’ UTR mutations may interfere with _____
polyadenylation
mutations where would lead to abnormal/incomplete splicing?
splice sites
Why do you think even in the same organism the rates of spontaneous mutations affecting different processes can be orders of magnitude different?
because the “genetic footprint” of genes controlling a certain function varies, the more genes controlling one function the more likely they are to be mutated
how to identify auxotrophic mutants in bacteria?
replica plates are made from a master plate, the replicas are grown with/without histidine, growth on the plates is compared, a colony that grows on media with histidine but couldn’t grow on media without histidine is auxotrophic (histidine requiring mutant)
name for a region of the chromosome more prone to mutations?
mutation hotspot
what types of mutations can occur during DNA replication?
base mispairing due to change in H-bonding properties
syn vs anti base conformation
base analogs can be incorporated (manmade)
what types of mutations are spontaneous?
what types of mutations are induced by environmental of biological agents?
explain tautomerization
a change in the H-bonding properties of the base, “the addition of a H-atom (proton) at one molecular site and its removal from another”
ex: adenine usually pairs with thymine, when a double bond is shifted within adenine is shifted it can no longer bond with thymine but will bond with cytosine
3 mechanisms by which changing H-bonding properties cause mispairing
- tautomerization
- syn vs anti conformation
- H2O acting as a bridge to help pair 2 pyrimidines
3 types of spontaneous DNA mutations?
- deamination
- depurination/depyrimidination
- oxygen radical damage
deamination involves transition of what?
NH2 to an O
what happens if cytosine is deaminated?
it becomes uracil
what is the “worst” type of deamination mutation and why?
conversion of methylcytosine to thymine
bad because there’s no repair systems that can recognize and remove it, a transition mutation will be retained
what does spontaneous base loss create?
AP sites (apurinic/apyrimidinic or abasic)
define an AP site
no base is attached to the sugar
how does a cell respond to an AP site?
a random base will be inserted across from an abasic site resulting in a mutation, only 25% of the time will the right base be incorporated
ROS cause oxidative damage to ____ and _____ causing mutations
DNA and precursors to DNA (such as GTP)
most important/damaging oxidation product after ROS exposure?
guanine –> 8-oxoguanine
mispairs with adenine and gives G-C to T-A transvertions
there are many DNA base products that result from ROS, how to they cause mutations?
transcription stalling/replication fork stalling = DNA breaks
incorrect nucleotide is incorporated during repair leading to a mutation
mechanism of nitrous acid mutagenesis?
oxidative demination (C to U, meC to T, A to hypoxanthine)
what type of mutagenic agents add methyl or ethyl groups?
alkylating agents
how do alkylating agent cause mutations?
addition of methyl or ethyl group changes base pairing propoerties
what type of agent distorts the DNA helix, causing frameshift, insertions, or deletions
intercalating agents
ex. EtBr, acridine orange, actinomycin D
3 mechanism that chemicals use to alter DNA structure?
bulky adducts
intra-inter strand crosslinks
DNA strand breaks
chemical mutagens do not cause mutations directly but rather by _____
induction of mutagenic DNA repair processes
explain Ames test
tests the mutagenicity of a chemical
tester strains of bacteria cannot synthesis histidine due to specific frameshift or point mutations
bacteria are exposed to chemical, seen if the are able to grow in absense of histidine
if yes than a mutation of the specified type occured
how to make Ames test more sensitive?
also mutate DNA excision repair machinery and LPS synthesis, will make the walls of bacteria more permeable (chemical can get inside) and will ensure DNA damage isn’t repaired if chemical is mutagenic
what is sometimes added to Ames test to account for metabolism?
liver extract containing metabolic enzymes
most mutagenic fraction of UV radiation?
UVB
how does UVA exert mutagenic effects?
production of ROS
consequence of UVB exposure
CPDs and 6-4PPs
how many bonds link adjacent bases in CPDs and 6-4PPs?
2 bonds in CPDs (5/6 carbons of base)
1 bond in 6-4PPs (6-4 carbons of base)
how does ionizing radiation damage cells?
free radicals a formed which cause:
Breaks in one or both strands (can lead to rearrangements, deletions, chromosome loss, death if unrepaired)
Damage or loss of bases
Crosslinking of DNA to itself or proteins
organelle damage, membrane damage
what type of cells are more susceptible to mutations from ionizing radiation?
rapidly dividing cells
name of mutation which arises from incorporation of foreign DNA in genome of another species?
insertional mutants
if site of insertion is in coding or regulatory region function of gene will likely be ____
disrupted (insertional inactivation)
how does insertion of foreign DNA activate a gene?
a foreign promoter is inserted in the vicinity of a certain gene
