Mutation terms Flashcards
(27 cards)
What are informational suppressors?
Changes to tRNAs or ribosomes
Subtypes: nonsense, missense, frameshift suppressors
What are interaction suppressors?
They are binding partners in a protein complex that makes a compensating change.
What do dosage compensation suppressors do?
Increase activity of a protein (e.g. downstream in pathway) that compensated for a partly inactive protein
What do bypass suppressors do?
New cellular activity compensated for a missing function (e.g. de-repress a silent gene)
What do physiological suppressors do?
They restore activity of the mutant protein by changing cell physiology (e.g. protein chaperone, ion requirement)
What do nonsense suppressors do?
They translate stop codons into aa
Inefficient, widely tolerated
What do missense suppressors do?
Substitute one aa for another
Inefficient, often lethal
What do frameshift suppressors do?
Translate 4 bases into aa
Inefficient, often lethal
What are examples of reversion in the same gene?
- Direct reversion of the original base change
- Reversion of the mutant aa to a functional but different aa
- Intragenic suppression where there is compensating change elsewhere in gene
Forward genetics is
Selection of a mutation with a known phenotype and find out which gene is effected
Reverse genetics is
The making of a defined mutation in a gene and then discovering what phenotype is effected
What is a transition?
Replacement of like nucleotides
What is a transversion?
Replacement of unlike nucleotides
What is an Ames test?
Tests for mutagens
Uses His- mutants as the reversion patterns are well defined
His- plated as a control for natural revertants (on plat that requires His+)
His- with possible mutagen added is plated and number of revertants counted - if significantly different from control it indicates a mutagenic substance
What are three typed of mutation studies?
- Random mutagenesis of the whole genome
- Random mutagenesis of a defined target region
- Defines (controlled) nucleotide changes
What are some ways to conduct random mutagenesis of a whole genome?
EMS/NTG - mostly point mutations
Fast Neutron radiation - mostly deletion mutations
Transposons - nulls
What are some ways to conduct random mutagenesis of a defined target region?
-Error prone PCR - use of Mn instead of Mg, use of error-prone polymerase
What is a way to conduct defined nucleotide changes?
-Oligos
What is saturation?
Well over 4.6x hits per gene
All possible mutations gained
What are the benefits of EMS/NTG chemical mutagenesis?
- Simple
- Easy to perform
- Very high hit rate
- Multiple mutants per genome (25-50)
- Wide range of mutation types (nulls and partially active included)
- MOSTLY G to A TRANSITIONS
What are the disadvantages of EMS/NTG chemical mutagenesis?
- Often multiple hits clustered in certain regions, so hard to identify the causal mutation
- No simple way to identify the mutation change => sequencing now feasable for bacteria
- Difficult to clone gene of interest and sequence it
What are the advantages of transposon mutagenesis?
- Makes nulls/knockouts
- Known DNA sequence tag so possible to sequence out from this to the gene of interest. This also simplifies cloning process (probe usage for tag)
What do you use to sequence from a known region?
Inverse PCR - primers to known region, ligate DNA to circles in low concentrations, Use PCR to amplify in each direction
Adaptor-mediated PCR - prime known region, ligate an adaptor to side of unknown region
What are the steps in Adaptor-mediated PCR?
- Digest DNA with restriction enzymes
- Ligate adapter to all termini - this is modified with one strand unphosphorylated so it will not copy during first PCR round (sometimes one strand has N group instead)
- PCR with primers to TE and adapter
