Week 8 Lecture Content Flashcards
How frequent is a point mutation
Rare per replication but common in populations
1. Phenotype level: 10^-6 to 10^-8 / individual
2. DNA sequence level: 10^-9 per base per replication - consistent across all species due to intrinsic DNA replication process
Do beneficial mutations happen more often?
No it happens randomly
The Fluctuation Test of Luria and Delbruck
- Set up 3 different cultures and introduced T1 phage into them
- If random, the number of phage-resistant cells fluctuates substantially among populations as a result of random timing of mutation
- If adaptive, all populations will carry approximately the same proportion of phage resistant cells
- Found that proportions vary - random
In multicellular organisms, does the same mutation in different cell types have the same effects?
No, the same mutation in different cells can have very different effects
- Germ-line mutations: can be passed form one generation to the next
- Somatic mutations: not genetically impact the next generation
How are point mutations classified?
- Coding sequence mutations
- Regulatory mutations
Can also be classified as transition or transversion mutations
Coding sequence mutations
- Synonymous: no AA change
- Missense: changes on AA
- Nonsense: Creates a stop codon and terminates translation
- Frameshift: wrong sequence of AA
Regulatory Mutations
- Promoter: Changes timing or amount of transcription
- Polyadenylation: alters sequence of mRNA
- Splice site: Improperly retains an intron or excludes an exon
- DNA replication mutation: increases number of short repeats of DNA
Transition vs transversion mutations
Transition: A and G or T and C
Transversion: A to T or C and G to T or C
Forward mutation
A wild-type allele to a mutant allele
Reverse Mutation (reversion)
Mutant alleles to wild-type of near wildtype allele
- True reversion: another mutation restores wild-type DNA sequence
- Intragenic Reversion: second mutation elsewhere in the same gene restores gene function
-Second-site reversion (suppressor mutation): mutation in a different gene that compensates for the original mutation, restoring the organism to wild type
How are mutations generated?
- Changes in the chemical structure of a nucleotide base
- Errors in DNA replication: 1 x 10^-9 per base per replication
Strand slippage
The DNA polymerase temporarily dissociates and then reattaches to resume replication
- Leads to an altered number of repeat elements
Why is nucleotide repeat changes significant
many human disorders caused by repeated expansions
- Wild-type alleles have a certain number of DNA trinucleotide repeats
- Increases in the number of repeats beyond a certain threshold causes the disorders
What are the mechanisms of point mutations?
- Mispaired nucleotide during replication: Non-complementary base pairing can occur (incorporated error)
- Spontaneous nucleotide base changes
- Caused by chemical and or ionizing radiation
Depurination
The loss of a purine - apurinic site
- if not repaired, DNA polymerase will put an adenine into the site during replication
- type of spontaneous nucleotide base change
Base modification
Eg. Deamination
The loss of an amino group from nucleotide base
- Deamination of methylated cytosine produces thymine
- replication will produce mutant and wild-type sister chromatids
Mutagens
- Physical agents
- Chemicals agents
Modes of action of chemical mutagens
- Nucleotide base analogs
- Deaminating agents
- Alkylating agents
- Oxidizing agents
- Hydroxylating agents
- Intercalating agents
Radiation-induced DNA damage
- Higher energy radiation - more DNA damage (short wavelength)
- UV -> photoproducts: aberrant structures with additional bonds involving nucleotideWs
What kind of mutagenic event occurs as a result of an oxidizing agent
transversion mutation
How do we know if a chemical is a mutagen
The ames test
Ames test
- S9 extract is added to mutant strains of his- S. typhimurium
2.his-1 is a base substitution mutant, his-2 is a frameshift mutant - the S9-bacterial mixture from each strain is spread on one experimental plate and one control plate
- A paper disk is put on each plate. the test compound is added to the experimental plate disks
- The presence of a significant number of revertant colonies indicates the test compound induces base-substitution
- The control plates determine the rate of spontaneous his- to his+ reversion
- An insignificant number revertant colonies indicates the test compound does not induce frameshift mutations
Mutagenicity of Aflatoxin B determined by the Ames test
Used Ames test and found increased reversion with base-pair substitution bacteria
- means Aflatoxin B1 is a mutagen and causes base substitution, not insertion or deletion
What types of DNA damage repair systems are there?
Direct repair mechanisms
Photoreactive repair
Repair of UV-induced photoproducts catalyzed by photolyase activated by visible light
Base excisions repair
Removal of an incorrect or damage DNA base and repair by synthesis of a new strand segment
1. DNA N-glycosylase recognizes a base-pair mismatch
2. Removes the incorrect nucleotide creating an apyrimidinic site
3. AP endonuclease generates a single-stranded nick on 5’ side of the AP site
4. DNA polymerase removes and replaces several nucleotides of the nicked strand by nick translation
Nucleotide excision repair
removal of a strand segment containing DNA damage and replacement by new DNA synthesis
Mismatch repair
Removal of a DNA base-pair mismatch by excision of a segment of the newly synthesized strand followed by resynthesis of the excised segment
Hoe does the repair system know which base to remove in a mismatch base pair
Mismatch repair enzymes distinguish between the original, correct nucleotide and the new mismatch nucleotide through presence of methylation on original strand
1. MutH binds to hemimethylated DNA
2. MutS bings to a base-pair mismatch and attracts MutL and the complex contacts MutH
3. MutH cleaves the unmethylated DNA strand, generating a single-strand gap
4. The gap is filled by DNA polymerase activity to repair the mismated
How are large damages repaired
Translesion DNA synthesis by translesion DNA polymerase, which is error prone with no proofreading ability
How are double-strand breaks repaied
Nonhomologous end joining or synthesis-dependent strand annealing
Double strand bearks
- Chromosome instability - cell death
- Uncontrolled cell growth -cancer and chromosome structural mutations
Nonhomologous End joining
- X-ray or oxidative damage produces double-strand break in DNA
- Ku80-Ku70-PKcs protein complex binds DNA ends
- Ends are trimmed, resulting in a loss of nucleotides
- DNA ligase ligates blunt ends to reform an intact duplex
Synthesis-dependent Strand annealing
- One chromatid undergoes a double-stranded break
- Nuclease digest a portion of the broken strands. Rad51 binds the undamaged chromatid
- Strand invasion of the sister chromatid creates a D loop. a replication fork assembles on the D loop
- New strand synthesis takes place using the available intact strands as templates
- Partial strand excision occurs; duplexes reform, and strands are ligated
Key molecule involved in repair to prevent breast and ovarian cancer
BRCA1
Key molecule involved in repair to prevent tumor suppressor
p53
How do transposable genetic elements move and create mutations in genomes?
- Cut and paste
- Copy and paste
Transposable genetic elements (TGE)
DNA sequences that can move within the genome through transposition
- Can vary in length, sequence composition and copy number
What are features of transposable elements
- Terminal inverted repeats on its ends
- The inserted transposable element is bracketed by flanking direct repeats
How do DNA elements transpose?
- Staggered cuts cleave the DNA strands of the target sequence
- Single-stranded ends result from staggered cuts of the target sequence
- The transposable element is inserted into the target sequence
- the gaps are filled by DNA polymerase
Categories of transposed elements
- DNA transposons: transpose as DNA sequence
- Retrotransposons: are composed of DNA but transpose through an RNA intermediate
Processes of DNA transposons
- Replicative: copy and paste
- Non-replicative: cut and paste
Process of retrotransposons
DNA to RNA to reversed transcribed into DNA then inserts into the new location
Insertional inactivation
If inserted into wild-type allele, can inactivate gene
Which type of transposon is more rare
DNA trasnposons
Transposable element Alu and cancers
- Insertion increases expression of gene
- Increases speed of cell cycle
- Leads to many cancer types
Forward genetics
Start with phenotypic difference to identify its genetic basis
Reverse genetics
Manipulate specific genetic change to identify its phenotypic effect
What is a common approach to identifying genes controlling phenotypic differences
Genetic cross and linkage mapping
Types of phenotypic variations
- Natural phenotypic variations
- Artificial random mutagenesis
Choosing an organism for fundamental questions
Select a model organism with features such as;
- Progress through its whole life cycle in a laboratory
- have a short generation time
- produce a reasonable number of progeny
- amenable to crossing and sexual reproduction
- amenable to genetic manipulations
Eg: E. coli, B. subtilis, baker’s yeast, fruit fly, zebra fish, mice
Choosing an organism for applied questions
Organism-specific and the organism is already selected for you
- Natural phenotypic variations
- Artificially introduced phenotypic variations through random mutagenesis
Choosing a mutagen
CHEMICAL
- Ethyl methanesulfonate
- mostly SNP
- usually loss-of-function, rarely hypermorphic
RADIATION
- Fast-neutron, X-ray, gamma-ray
- rearrangements (deletions, inversions, translocations)
- usually Loss-of-function, but can be gain-of-function
INSERTIONAL
- Transfer DNA, transposons
- Insertions
- Usually loss-of function
How to investigate the effect of mutagenesis in diploids
- Mutagenize sperm cells
- Mate with wild-type female
- Identify dominant mutations in F1 individuals
- If F1 progeny shows 1:1 phenotype ratio then dominant
- To check if recessive, intercross F1 individuals
What are the differences between mutations in haploid vs diploid organisms?
- Haploid: both recessive and dominant mutations can be identified directly
- mutations that cause lethality cannot be obtained in haploids but can be in diploids
How do you identify if a mutation is lethal in haploid
conditional mutant alleles
How to identify interacting genes
use a genetic modifier screen to identify a second gene that can modify the phenotype of the first mutation
Enhancer screen
Identifies second-site mutant allele that enhances the mutant phenotype
Suppressor screen
Identifies second-site mutation that suppress the effect of the first mutation
Synthetic Lethality
Combination of two viable mutations results in an inviable double mutant
Between-pathway gene interactions
Two pathways both preform the same essential function
- mutation of either alone may be inconsequential
- mutation of both will result in loss of the essential function
Within-pathway gene interactions
- Partial loss of function mutations alone reduce functions
- if both components are mutated the pathway may become nonfunctional
How to locate the mutated gene in the genome?
- Through genetic crosses and/or pedigree analyses
- Often involved multiple iterations
- can only identify to an approximate chromosomal location
- Genome-wide association analyses
How to identify the mutant genes?
- Construct DNA library
- Transformation and complementation
Genomic Library
Collections of cloned DNA fragments representing the ENTIRE genome of an organism
Complementary DNA libraries
Collections of cloned DNA fragments representing all mRNA produced by an organism
Complementation
Introduce a wild-type gene to the mutant and revert the mutant phenotype to wild type
What are 6 common reverse genetics technologies?
- Knockouts by homologous recombination
- CRISPR
- Random T-DNA
- Transposon insertions
- TILLING
- RNAi
Homologous recombination with circular DNA
- Single crossover results in integration of introduced DNA without replacement of target gene
- Double crossover results in replacement of target gene
Homologous recombination with linear DNA molecule
- Single crossover results in integration of introduced DNA and loss of chromosomes distal to integration site
- Double crossover results in replacement of target gene
