week 6 (gene mutations + epigenetics) Flashcards
define: point mutation
- substitution, insertion, or deletion of single base pair in gene
question: germ line mut. vs somatic mut.?
- germ line = generated w/in gametes
⤷ can be passed to next gen. - somatic = generated during mitosis
⤷ not passed on but affects indiv.
name + define: types of coding seq. point mutations (6)
-
synonymous
- no AA seq. change
- change one base pair but no change to AA -
missense
- changes 1 AA
- changing one base pair changes the AA -
nonsense
- creates stop codon and stops translation -
frameshift
- wrong seq. of AA
- insertion or deletion shifts sequences
- if insert/delete in multiples of 3 = no change -
transition
- changing a purine -> purine or pyrimidine -> pyrimidine
- A and G = purines
- T and C = pyrimidines -
transversion
- purine -> pyrimidine (vv)
question: forward mutations vs reverse mutations?
- forward = wild type -> mut.
- reverse = mut. -> wild type
define: true, intragenic, and second site reversions
- true = mut. restores exact wild type DNA seq.
- intragenic = mut. elsewhere in same gene restores wild type gene function (not same sequence as WT, but same pheno.)
- second site = mut. in different gene compensates for OG mut.
⤷ suppresses mut.
name: example of second site reversion
- colour
- 2 genes encoding pigment transportation
- mut. in one gene -> less pigment transported
- mut. in second gene -> upregulates pigment transportation
- if mut. in both, gene 1 transports less pig. but gene 2 compensates but transporting more -> wild type pheno.
name: mechanisms of point mutations (3)
- mispaired nucleotides during replication
- spontaneous nucleotide base change
- mutagens
explain: effect of mispaired nucleotides
- happens in replication
- causes point mutation
- base pairs are non-complementary
⤷ G with T
⤷ C with A - left w/out repair -> mut.
explain: effects of spontaneous nucleotide base changes
- depurination -> losing a purine
- deamination -> losing an amino group
- depurination
⤷ DNA polymerase puts adenine
⤷ often causes G to become A - deamination
⤷ methylated cytosines often become thymines
⤷ causes mismatch but can be repaired (bc using the separate mismatched strands as templates -> leads to either wild type or transition mutation)
explain: effect of chemical mutagens (6)
-
nucleotide base analogs
- chem. w/ similar structure of DNA incorporates into DNA during rep. and induces point mut. -
deaminating agents
- removes amino groups
⤷ often CG pairs become TA (spontaneous nucleotide base change) -
alkylating agents
- adding methyl or ethyl group
- distorts helix -> mut. -
oxidizing agent
- oxidizes base
⤷ often -> transversion -
hydroxylating agents
- adding hydroxyl group
⤷ often pairs C and A -
intercalating agents
- molecules that fit between base pairs
- distorts DNA
⤷ often causes frameshift mutations
define: ames test
- to test if a chemical is a mutagen
- exposing bac. to a chem. in the presence of enz. from a mammal’s liver
explain: example ames test
- tested genes that prevent histidine synthesis
- grow bac. on media without histidine
- if bac. mutants grow = mutations occurred to allow bac. to synthesize. histidine
⤷ means chemical had mutagenic properties
question: how to test how mutagenic a chemical is?
- ames test
- count number of colonies of bac. to compare
question: what does high energy radiation do?
- all high energy radiation is mutagenic
- UV, x-rays, gamma, cosmic
- induces mutations in germ line
⤷ passes to next gen.
question: what does UV radiation do?
- can form thymine dimers
- covalent bonds between C5-C6 or C4-C6 of adjacent thymines
- if not repaired -> disrupts rep. and causes mutations
- strong assoc. with causing skin cancer
question: how are damaged and misrepaired DNA repaired?
- precision/direct repairs (BER, NES, mismatch repairs)
- error-prone translesion
question: how are double-stranded breaks repaired? (name types of repair mechanisms)
- nonhomologous end joining
- synthesis dependent strand annealing (homologous recomb.)
define: base excision repair
- BER = removing incorrect/damaged DNA base and repaired by synthesis of new segment
- nick translation
- replaces several nucleotides around the nick
- N-glycosylase starts removing base -> AP site
- AP endonuclease generates nick
define: nucleotide excision repair
- NER = removing strand segment of damage and replace by new DNA synthesis
- specialized for thymine dimers
⤷ often helps repair UV damage - UVR AB complex binds to the thymine dimer
- UVR B denatures and UVR C catalyzes the cuts
- UVR D helps release the damaged strand
define: mismatch repair
- removing base-pair mismatch by excision of segment of the newly synthesized strand + resynthesis
- MutHbinds to unmethyulated daughter
- MutS binds to mismatch between parent and daughter strands
- MutL connects H and S
- H cleaves the daughter strand
define: translesion DNA synthesis
- error-prone translesion
- unrepaired damage can block DNA poly II
- activating translesion DNA polymerase bypasses lesions and synthesizes short DNA segments
- used as last resorts bc no proof reading so more prone to errors
- SOS repair = system in E.coli to repair massive DNA damage
define: double stranded break repairs
- lack templates for DNA repair
- causes chromo. instability, cell death, cancer
- NHEJ
- SDSA
explain: NHEJ
- non homologous end joining
- can help repair after error prone mech.
- both ends of DNA are trimmed and rejoined w/ DNA ligase
- trimming causes loss of nucleotides (can’t eb replaced)
⤷ can lead to frameshifts
explain: SDSA
- synthesis dependent strand annealing
- error-free
- use the intact sister chromatid to help repair the other damaged strands
- use strand invasion to offer a new template to repair
⤷ broken strand invades other sister -> D loop
explain: effect of CRISPR w/ NHEJ and SDSA
- SDSA doesn’t happen without a template
- NHEJ repairs as well as it can
- CRIPR can mutate the gene bc there are deleted areas but not replacements
- so if you want to control a replacement area, you need a template
- ex. NHEJ used -> deleting gene (knock out)
⤷ only with a template can you knock in (reinsert modified gene)
define: transposable genetic elements
- TGE
- DNA seq. that move w/in genome through transposition
- can vary in length, seq, copy numbers
- 2 ways of mvt
- non replicative transposition
- replicative transposition
define: ways TGE can move (2)
-
non replicative transposition
- excision of element from OG spot and inserting it in a new spot
- cut and paste -
replicative transposition
- duplication of element and inserting it in a new spot too
- copy and paste
question: what’s the structure TGE?
- terminal inverted repeats on the ends
- surrounded by flanking direct repeats
⤷ not considered part of TGE
FDR - TIR - middle region - TIR - FDR
question: how does transposition work?
- staggered cuts cleave the DNA strands
- have single stranded overhanging ends
⤷ sticky ends - transposable element is inserted into the seq.
- gaps filled by DNA poly
name + define: categories of transposable elements (2)
-
DNA transposons
- transpose as DNA seq.
- replicative or non-replicative -
retrotransposons
- made of DNA but transposed through RNA intermediate
- DNA -> RNA -> reverse transcribed into DNA and inserted
question: how can transposition be mutagenic?
- when TGEs insert themselves into crucial genetic regions -> mut.
⤷ ex. coding regions, promotor regions
explain: P-elements and transposition
- P-elements = transposable elements in drosophila
- used int eh past to generate transgenic flies (before CRISPR)
- process:
⤷ clone gene of interest into a plasmid w/ inverted repeats (like a TGE)
⤷ inject embryo with plasmid
⤷ gene of interest will randomly insert itself into embyro’s genome
define: epigenetics
- studying traits above inheritance
- how envrt. and behaviour shape genes
name: 5 features of epigenetic modifications
- epigenetic mod. patterns alter chromatin struc.
- transmissible during cell div.
- reversible
- directly assoc. w/ gene transcription
- do not alter DNA seq.
question: euchromatin vs hetero chromatin?
- euchromatin = loose, more transcriptionally active
⤷ less histone prot. - hetero = dense, less transcriptionally active
⤷ more histone prot.
question: constitutive heterochromatin vs facultative?
- constitutive = always hetero
- facultative = switch back and forth between eu and hetero
explain: structure of a nucleosome
- DNA wound around 8 histone prot.
- histone port. allow DNA to coil around -> condenses DNA into chromatin
name + explain: chromatin modifiers (2)
-
acetylation
- relaxes histone DNA interactions making areas more transciptionally active (more euchromatic)
- histone acetyl transferases HATs
⤷ add acetyl groups
⤷ leads to euchromatin - histone deacetylases HDACs
⤷ remove acetyl groups
⤷ leads to heterochromatin
-
methylation
- assoc. w/ heterochromatin but can also lead to euchromatin
- histone methyltransferases HMTs
⤷ add methyls
- histone demethylases HDMTs
⤷ remove methyls
explain: PEV
- position effect variegation
- heterochromatic areas spread into euchromatic areas
⤷ silences transcription of genes - seen in drosophila eyes
⤷ inversion of X chromo. placed white gene near centromere in heterochromatic region
⤷ caused mosaic red and white eyes
⤷ pheno = mut but geno = still wildtype
question: E(var) vs Sur(var)?
E(var)
- enhancers of PEV
- enhances mutant pheno. by encouraging spread of heterochromatin
Sur(var)
- suppressors of PEV
- restricts heterochromatin spread
⤷ encourages wild-type pheno.
explain: x-inactivation in female mammals
- any given cell inactivates either maternal X or paternal X randomly
- every female is a mosaic of 2 cell types
occurs early in embryonic dev.
question: how does x-inactivation affect a female and colour blindness?
- if majority inactivated wildtype -> more are expressing colour blind
⤷ will be RG colour blind - if 50/50 x-inactivation -> normal vision
define: long coding RNA
- long RNA lacking open reading frames
- acts as scaffolds that link regulatory prot.
- involved in x-inactivation
⤷ ex. x-inactivation-specific-transcript (Xist)
explain: Xist
- the x-inactivation center on an x chromo.
⤷ active in heterochromatic X, inactive in euchromatic X - ex. of long coding RNA
- Xist RNA on chromo. to be inactivated
⤷ spreads along chromo. and inactivates all the genes -> silences the chromo.
define: genomic imprinting
- a heritable epigenetic phenomenon
- genes expression in offspring dep. on the parent that passed it on
⤷ maternal imprinting = allele from mother is inactivated -> expresses father allele
⤷ paternal imprinting (vv) - can be x-linked or autosomal
compare: maternal vs paternal imprinting
MATERNAL
- only females switch allele off when passing it on
- look for mothers with affected mother with all unaffected children
- only affected males or carrier males can have affected children
PATERNAL
only males switch allele off when passign it on
- look for affected fathers with all unaffected children
⤷ can be carrier children or unaffected children
explain: IGF2 and H19 example
- both genes on chromo. 11
- H19 only expressed on maternally inherited chromo.
- IGF2 only expressed on paternally inherited chromo.
- Russell-Silver syndrome = both chromo. express maternal
⤷ born underweight - Beckwith-Wiedemann syndrome = both chromo. express paternal
⤷ overgrowth of tissue
question: how is expression decided for IGF2 and H19?
MATERNAL
- enhancer drives expression of H19
- insulator blocks OGF2
PATERNAL
- methylation inactivates insulator (ICR) and blocks H19 expression
- IGF2 gets enhanced
