Topic 5: Mutations Flashcards
Mutations are the source of…
Genetic variation, providing the raw material for evolution
Diseases and disorders
Mutations are useful for…
Probing fundamental biological processes
What is a mutation
A permanent change in the genetic material ie DNA seq
does not need to result in phenotypic change
Spontaneous vs induced mutation
Spont = in nature (UV)
Induced = in lab
Genetic variation is the result of…
Mutations combined with processes of recombination, independent segregation, fertilization of gametes
What are somatic mutations
Arise in somatic tissues (non-heritable mutations)
What are germ-line mutations
Arise in germline tissues/cells (sexual tissues; make gametes (eggs/sperm) -> are heritable
Mutation affects one allele or one homolog
Gene vs chromosomal mutations
Gene = alter genes - relatively small
Chromosomal = altering order or presence of genes on a chromosome - relatively large
Slide 7
Somatic vs germ-line mutations
Mutations that usually cause phenotypic changes
Mutations in protein coding genes, non-coding genes (fxnal RNA), regulatory elements
Types of mutations (based on molecular nature)
Base substitutions
Insertions and deletions
Types of base substitution mutations
Transitions
Transversions
Insertions and deletions can cause.. Why wouldn’t they
frame-shift mutations
Unless the indel occurs in a unit of 3
What is a transition
Base substitution
Substitution of a purine for a purine or of a pyrimidine for a pyrimidine
What is a transversion
Base substitution
Substitution of a pyrimidine for a purine or a purine for a pyrimidine
What are expanding nucleotide repeats
Type of in-frame indel mutation
Increases the number of copies of a set of repeated nucleotides
What is the fragile site
Associated with characteristic constriction on X chromosome
Forward vs reverse mutation
Forward = wild type -> mutant type
Reverse = mutant type -> WT
What are missense, nonsense, silent and neutral mutations
Missense = aa -> diff aa
Nonsense = sense codon -> nonsense codon (stop)
Silent = codon -> synonymous codon
Neutral = no change in fxn
Slide 13
Missense, nonsense, silent mutations
Two phenotypic effects of mutations
Loss-of-function mutation
Gain-of-function mutation
What is a loss-of-function mutation
DNA that is mutated stops its normal fxn
Straight forward relationship between genotype and phenotype
What is gain-of-function mutation
More of the same fxn or totally new fxn
Not straight forward
What can mutations be caused by
- spontaneous replication errors
- spontaneous chemical changes
- chemically induced mutations
- radiation
Types of spontaneous replication errors
- tautomeric shifts
- non-standard base pairing
- causes of deletions and insertions (strand slippage, unequal crossing over)
What are tautomeric shifts
H moves to different N on base, causes it to bond to diff base
Examples of non-standard base pairing
Wobble pairing
What is wobble pairing
Non-standard base pairing where thymine binds with guanine or cytosine binds with adenine
How are DNA lesions usually repaired
DNA polymerase proofreading
mismatch repair
direct and excision repair mechanisms
What happens if lesions are not reparied
become replicated errors in subsequent round of DNA synthesis
Slides 18-22***
Wobble, strand slippage, unequal crossing over
Examples of spontaneous chemical changes
Depurination: loss of purine
Deamination: loss of amino group
Slide 24, 25**
Depurination, deamination
Mutagens of chemically induced mutations
Base analogs
Alkylating agents
Oxidative reaction
E.g. of base analog, what does it do
5-bromouracil is an analog of thymine
If present, can be incorporated into DNA during replication in place of T
Slide 28**
Base analog 5-bromouracil
What is an intercalating agent? Causes…
Inserts themselves between adjacent bases in DNA, distorting the 3D structure of the helix
Causes single-nucleotide indels during DNA replication
Examples of ionizing radiation, what does it do?
X-rays, gamma rays and cosmic rays
Penetrate tissue and damage DNA
- dislodge electrons
- break phosphodiester backbone (DS break-> if error in repair = mutation)
Energy level of UV radiation vs ionizing
UV has less energy
UV radiation can cause…
Pyrimidine dimer; two thymines block replication
Pyrimidine dimers block…
Cell division
- block DNA polymerase from continuing replication
- cells get stuck in G2 = death
How do pyrimidine dimers cause mutations
Specific repair mechanisms deal with them, but if mistake is made = mutation
Slide 32***
The Ames Test
What is forward genetics
Begins with a phenotype we try to find the genes encoding that phenotype
What is reverse genetics
Begins with a gene of unknown function, induce mutations, check the effect of the mutation on the phenotype
Slide 34
Forward and reverse genetics
Steps of creating random mutations
- Apply mutagens (e.g. radiation) to model species
- Screen for interesting phenotypes in the F2 generation
Slide 36
P0, F1 and F2 generations
Forms of targeted mutagenesis
Insertion mutants
RNAi mutants
CRISPR-Cas9 system
What is transgenic insertion mutant
Piece of DNA is inserted into your gene of interest using transgenic technology
Insertion acts like large insertion mutation (frameshifts if in coding region = “kockout” mutant = LOF)
Transgenic insertion mutants mice
neo/tk system is often used to introduce insertion into a target gene
Slides 39, 40**
Transgenic insertion mutants in arabidopsis
What is RNAi (functions)
RNA interference, has two functions:
1. controls gene expr at post-transcriptional level by destroying target mRNAs
2. Protect against invading viruses by destroying viral RNAs
Two proteins in RNAi
Dicer: finds and cuts dsRNA into small interfering RNAs or micro RNAs
RISC: RNA-induced silencing complex: uses RNAs as template for search and destroy (of other ssRNAs that complement with it)
How do we silence genes with RNAi
Engineer transgenic organisms that produce siRNA (small interfering RNA) that will complement our gene of interest, triggers RNAi process to destroy all mRNA produced from our gene of interest
What are knock-downs
In RNAi, mutant b/c some mRNAs will always escape the RISC
What does the CRISPR/Cas9 system do
Combines single guide RNA with a nuclease, together they attach to specific DNA sequences and make double-stranded cuts
Repair these cuts by nonhomologous end joining or homology-directed repair, providing the means to introduce alterations to the genome
What does the single guide RNA do in CRISPR/Cas9
Provides specificity through complementation
