Genetic Analysis Of Mutants Flashcards
Mutations in the promoter affect what?
Timing and abundance of gene product
Mutations in the open reading frame affect what?
Function and abundance of the gene product
Can you infer the effect on gene function by mutating the 5’ UTR?
No, you must know all regulatory mechanisms first
Mutation of a functional RNA can alter what?
Function
Structure
Stability
How conserved are tRNA?
Extremely. Every base change matters
Mutation of a protein coding gene can alter what?
Abundance
Structure
Stability
Function
What are SNPs?
Single Nucleotide Polymorphisms
Any base substitution between two related DNA sequences
Transitions
Purine to purine (AG)
Pyrimidine to pyrimidine (CT)
Transversions
Purine and pyrimidine switch
Are transitions or transversions more common?
Transitions are more common because they maintain H bonding
What’s the difference between SNPs and SNVs?
You can infer the original sequence with SNPs but not SNVs
Non-synonymous mutations are more likely to be caused where in the codon?
The first two sites of the codon
dN/dS <1
Negative selection
Mutation in housekeeping genes
dN/dS = 1
Neutral selection
Genetic drift
dN/dS > 1
Positive selection
Selective advantage
How can silent mutations be beneficial?
Can mutate to a more common codon
Quicker to find tRNA
Faster growth
Outcompetes WT
What’s the effect of indels on coding genes?
Mostly LoF
Frameshift if indel is not a multiple of 3
How do you use a transposon as a tool?
Insert a marker into transposon (such as antibiotic resistance)
Use helper plasmid which has the tnp
What does transposon mutagenesis allow you to do/tell?
Allows ID of mutated locus
Can make KO of genes
Can over-express a gene(s’
Use as a tag to find essential genes
How do you ID a mutated locus with transposon mutagenesis?
Integrate transposon with marker and helper plasmid
Digest genome and circularize it
Sequence piece containing the transposon to see where it integrated
How do you KO genes with a transposon?
Directly KO if integrated into a gene
Can KO downstream genes with promoter competition
Can over-express downstream genes if the tnp promoter is stronger than WT promoter
How do you confirm a KO from transposon integration into a gene?
Add WT gene copy
Move transposon
Target KOed gene specifically
How do you confirm a KO by transposon that did not integrate into that gene?
Use a merodiploid
If WT function restored then its a LoF mutation
How do you confirm over-expression of a gene downstream from the transposon?
If you over-express the gene in WT using a stronger promoter or a duplication it should have the same phenotype as the mutant
What are the pros of transposon mutagenesis?
ID of insertion site
Ability to titration amount of mutagen by controlling amount of transposons
What are the cons of transposon mutagenesis?
Only insertions
Sequence bias
Biased toward null mutations which can be lethal
Deciphering the mutational effect can be complex
How do you use a transposon as a tag?
ID essential genes under different conditions (genes with no integrations)
Can then select for mutants better or worse at a specific process
How could a silent mutation affect expression of a gene?
Change binding to tRNA or folding change
How could a nonsense mutation produce no phenotype change?
Close enough to the end to not matter or tRNA can decode STOP
