Mutations 1

Question 1

What would one need to do to regrow telomeres in a senescing cell culture?

Answer 1

1. understand what is limiting telomere length in the first place, could be a mutation, lack of transcription factor, wrong post-translational modifications
2. activate the three telomerase genes
3. check that your method to activate the gene resulted in longer telomeres

Question 2

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?

Answer 2

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

Question 3

what is a mutation

Answer 3

a stable change in DNA sequence

Question 4

the majority of coding region mutations are ______

Answer 4

detrimental

Question 5

name 4 types of point mutations

Answer 5

silent, sense, missense, nonsense

Question 6

what type of mutation is: a change in a DNA sequence that results in a new codon coding for the same amino acid

Answer 6

silent or sense

Question 7

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

Answer 7

missense

Question 8

what type of mutation is: a change in DNA that results in a premature stop codon

Answer 8

nonsense

Question 9

what type of mutation is caused by small insertion/deletions?

Answer 9

frameshift

Question 10

can large scale insertions/deletions/rearrangements that preserve reading frame still be disruptful?

Answer 10

yes (ex. huntingtons)

Question 11

how does a mutation in an intro disrupt gene expression?

Answer 11

introns contain transcription factor binding sites

Question 12

what does UTR mean

Answer 12

untranslated region

Question 13

5’ UTR mutations may interfere with _____

Answer 13

translation

Question 14

3’ UTR mutations may interfere with _____

Answer 14

polyadenylation

Question 15

mutations where would lead to abnormal/incomplete splicing?

Answer 15

splice sites

Question 16

Why do you think even in the same organism the rates of spontaneous mutations affecting different processes can be orders of magnitude different?

Answer 16

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

Question 17

how to identify auxotrophic mutants in bacteria?

Answer 17

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)

Question 18

name for a region of the chromosome more prone to mutations?

Answer 18

mutation hotspot

Question 19

what types of mutations can occur during DNA replication?

Answer 19

base mispairing due to change in H-bonding properties
syn vs anti base conformation
base analogs can be incorporated (manmade)

Question 20

what types of mutations are spontaneous?

Question 21

what types of mutations are induced by environmental of biological agents?

Question 22

explain tautomerization

Answer 22

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

Question 23

3 mechanisms by which changing H-bonding properties cause mispairing

Answer 23

1. tautomerization
2. syn vs anti conformation
3. H2O acting as a bridge to help pair 2 pyrimidines

Question 24

3 types of spontaneous DNA mutations?

Answer 24

1. deamination
2. depurination/depyrimidination
3. oxygen radical damage

Question 25

deamination involves transition of what?

Answer 25

NH2 to an O

Question 26

what happens if cytosine is deaminated?

Answer 26

it becomes uracil

Question 27

what is the “worst” type of deamination mutation and why?

Answer 27

conversion of methylcytosine to thymine

bad because there’s no repair systems that can recognize and remove it, a transition mutation will be retained

Question 28

what does spontaneous base loss create?

Answer 28

AP sites (apurinic/apyrimidinic or abasic)

Question 29

define an AP site

Answer 29

no base is attached to the sugar

Question 30

how does a cell respond to an AP site?

Answer 30

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

Question 31

ROS cause oxidative damage to ____ and _____ causing mutations

Answer 31

DNA and precursors to DNA (such as GTP)

Question 32

most important/damaging oxidation product after ROS exposure?

Answer 32

guanine –> 8-oxoguanine

mispairs with adenine and gives G-C to T-A transvertions

Question 33

there are many DNA base products that result from ROS, how to they cause mutations?

Answer 33

transcription stalling/replication fork stalling = DNA breaks

incorrect nucleotide is incorporated during repair leading to a mutation

Question 34

mechanism of nitrous acid mutagenesis?

Answer 34

oxidative demination (C to U, meC to T, A to hypoxanthine)

Question 35

what type of mutagenic agents add methyl or ethyl groups?

Answer 35

alkylating agents

Question 36

how do alkylating agent cause mutations?

Answer 36

addition of methyl or ethyl group changes base pairing propoerties

Question 37

what type of agent distorts the DNA helix, causing frameshift, insertions, or deletions

Answer 37

intercalating agents

ex. EtBr, acridine orange, actinomycin D

Question 38

3 mechanism that chemicals use to alter DNA structure?

Answer 38

bulky adducts

intra-inter strand crosslinks

DNA strand breaks

Question 39

chemical mutagens do not cause mutations directly but rather by _____

Answer 39

induction of mutagenic DNA repair processes

Question 40

explain Ames test

Answer 40

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

Question 41

how to make Ames test more sensitive?

Answer 41

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

Question 42

what is sometimes added to Ames test to account for metabolism?

Answer 42

liver extract containing metabolic enzymes

Question 43

most mutagenic fraction of UV radiation?

Answer 43

UVB

Question 44

how does UVA exert mutagenic effects?

Answer 44

production of ROS

Question 45

consequence of UVB exposure

Answer 45

CPDs and 6-4PPs

Question 46

how many bonds link adjacent bases in CPDs and 6-4PPs?

Answer 46

2 bonds in CPDs (5/6 carbons of base)

1 bond in 6-4PPs (6-4 carbons of base)

Question 47

how does ionizing radiation damage cells?

Answer 47

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

Question 48

what type of cells are more susceptible to mutations from ionizing radiation?

Answer 48

rapidly dividing cells

Question 49

name of mutation which arises from incorporation of foreign DNA in genome of another species?

Answer 49

insertional mutants

Question 50

if site of insertion is in coding or regulatory region function of gene will likely be ____

Answer 50

disrupted (insertional inactivation)

Question 51

how does insertion of foreign DNA activate a gene?

Answer 51

a foreign promoter is inserted in the vicinity of a certain gene