Genetic Analysis Of Mutants

Question 1

Mutations in the promoter affect what?

Answer 1

Timing and abundance of gene product

Question 2

Mutations in the open reading frame affect what?

Answer 2

Function and abundance of the gene product

Question 3

Can you infer the effect on gene function by mutating the 5’ UTR?

Answer 3

No, you must know all regulatory mechanisms first

Question 4

Mutation of a functional RNA can alter what?

Answer 4

Function
Structure
Stability

Question 5

How conserved are tRNA?

Answer 5

Extremely. Every base change matters

Question 6

Mutation of a protein coding gene can alter what?

Answer 6

Abundance
Structure
Stability
Function

Question 7

What are SNPs?

Answer 7

Single Nucleotide Polymorphisms
Any base substitution between two related DNA sequences

Question 8

Transitions

Answer 8

Purine to purine (AG)
Pyrimidine to pyrimidine (CT)

Question 9

Transversions

Answer 9

Purine and pyrimidine switch

Question 10

Are transitions or transversions more common?

Answer 10

Transitions are more common because they maintain H bonding

Question 11

What’s the difference between SNPs and SNVs?

Answer 11

You can infer the original sequence with SNPs but not SNVs

Question 12

Non-synonymous mutations are more likely to be caused where in the codon?

Answer 12

The first two sites of the codon

Question 13

dN/dS <1

Answer 13

Negative selection
Mutation in housekeeping genes

Question 14

dN/dS = 1

Answer 14

Neutral selection
Genetic drift

Question 15

dN/dS > 1

Answer 15

Positive selection
Selective advantage

Question 16

How can silent mutations be beneficial?

Answer 16

Can mutate to a more common codon
Quicker to find tRNA
Faster growth
Outcompetes WT

Question 17

What’s the effect of indels on coding genes?

Answer 17

Mostly LoF
Frameshift if indel is not a multiple of 3

Question 18

How do you use a transposon as a tool?

Answer 18

Insert a marker into transposon (such as antibiotic resistance)
Use helper plasmid which has the tnp

Question 19

What does transposon mutagenesis allow you to do/tell?

Answer 19

Allows ID of mutated locus
Can make KO of genes
Can over-express a gene(s’
Use as a tag to find essential genes

Question 20

How do you ID a mutated locus with transposon mutagenesis?

Answer 20

Integrate transposon with marker and helper plasmid
Digest genome and circularize it
Sequence piece containing the transposon to see where it integrated

Question 21

How do you KO genes with a transposon?

Answer 21

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

Question 22

How do you confirm a KO from transposon integration into a gene?

Answer 22

Add WT gene copy
Move transposon
Target KOed gene specifically

Question 23

How do you confirm a KO by transposon that did not integrate into that gene?

Answer 23

Use a merodiploid
If WT function restored then its a LoF mutation

Question 24

How do you confirm over-expression of a gene downstream from the transposon?

Answer 24

If you over-express the gene in WT using a stronger promoter or a duplication it should have the same phenotype as the mutant

Question 25

What are the pros of transposon mutagenesis?

Answer 25

ID of insertion site
Ability to titration amount of mutagen by controlling amount of transposons

Question 26

What are the cons of transposon mutagenesis?

Answer 26

Only insertions
Sequence bias
Biased toward null mutations which can be lethal
Deciphering the mutational effect can be complex

Question 27

How do you use a transposon as a tag?

Answer 27

ID essential genes under different conditions (genes with no integrations)
Can then select for mutants better or worse at a specific process

Question 28

How could a silent mutation affect expression of a gene?

Answer 28

Change binding to tRNA or folding change

Question 29

How could a nonsense mutation produce no phenotype change?

Answer 29

Close enough to the end to not matter or tRNA can decode STOP