Lecture 14: Mutations, Genetic Screens, & Selections

Question 1

Mutation Rate

Answer 1

The chance of mutation to a particular phenotype over a certain time interval. Can differ for phenotypes depending on how many mutations can lead to the phenotypic change.

Question 2

What does high mutation rate suggest

Answer 2

A specific event like loss of a plasmid or programmed recombination event occurs

Question 3

What does unusually low mutation rate suggest

Answer 3

2 independent mutations required for phenotypic change

Question 4

Single Nucleotide Polymorphisms (SNPs)

Answer 4

-Most common mutations
-Single nucleotide change that occurs in at least 1% of population

Question 5

Transition Mutations

Answer 5

-Purine is swapped for a purine
-Pyrimidine swapped for a pyrimidine

Question 6

Transversion mutation

Answer 6

-Purine swapped for pyrimidine
-Pyrimidine swapped for purine

Question 7

Common causes of mutations

Answer 7

-Chemical mutagens
-UV light
-DNA replication
-Oxidative damage
-Homologous recombination

Question 8

Most common cause of SNPs

Answer 8

DNA replication

Question 9

Speed of DNA replication in E. Coli

Answer 9

-4 million BP per 80 minutes
OR
-50,000 BP per minute
OR
-833 BP per second

Question 10

Why are there so many wrong base incorporations during DNA synthesis

Answer 10

Nucleotides possess tautomeric forms

Question 11

Tautomers

Answer 11

-Aromatic bases are composite of all possible electron distributions
-Tautomers are representation of different isomeric forms of bases
-Leads to sequence independent DNA replication errors
-At physiological pH and conditions, the tautomeric forms represent 1 in a million nucleotides (similar to error rate)

Question 12

If error rate is 1 in a million, why is the OBSERVED error rate 1 in a billion

Answer 12

Repair mechanisms in the body

Question 13

Outcome of SNP mutations

Answer 13

-Change in gene sequence, causing change of protein sequence leading to change in structure and function of proteins
-A random mutation will almost always result in protein loss of function
-Gain of function mutation will VERY RARELY arise which can increase or change protein activity

Question 14

Missense mutation

Answer 14

Change in amino acid sequence of protein

Question 15

Silent mutation

Answer 15

Codon sequence is changed but amino acid sequence remains the same

Question 16

Nonsense mutation

Answer 16

Change in amino acid codon to stop codon

Question 17

Slipped strand mispairing

Answer 17

DNA polymerase tracks over a repetitive sequence it can “Slip” forward or reverse causing insertions (Omega) or deletions (Delta)

Question 18

Frameshift mutation

Answer 18

Insertion or deletion of BP in amount not divisible by 3, completely altering the amino acid sequence.

Question 19

UV mutations

Answer 19

Thymine and pyrimidine dimers can form which will alter the shape of the double helix which does not introduce change in the DNA sequence

Question 20

X-ray exposure mutations

Answer 20

Ionizing radiation can break bonds and affects DNA in several ways

Question 21

Alkylating Agents

Answer 21

Some chemicals react with the nucleobases and/or phosphates and attach alkyl groups

Question 22

Oxidizing agents

Answer 22

Metabolism can generate ROS which can cause numerous problems in DNA pairing

Question 23

Deamination

Answer 23

At low frequency, nucleobases (Mostly cytosine) can spontaneously be deaminated.

Question 24

Selection vs Screen

Answer 24

-Selection: Experiments where wildtype dies and mutant lives. You select for desired mutants.
-Screen: Approach to identify mutations that result in a difference in phenotype not associated with wild-type lethality

Question 25

Lac- screenings

Answer 25

-Using metabolism to screen for changes in transcriptional regulation -MacConkey agar can isolate lac- by using the pH change from lactose fermentation to induce color change on the agar. Lac- will not turn red

Question 26

Replica plating

Answer 26

Trasnfer a sample of colonies onto a sterile cloth and then imprint it onto successive agar plates containing different media

Question 27

MacConkey agar

Answer 27

Inhibits gram positive bacteria due to bile salts. Contains a neutral red dye thatll turn pink if bacteria ferment lactose

Question 28

What do lac screenings identify

Answer 28

-Mutations compromising LacZ -Mutations that compromise LacY -LacI mutants blind to lactose -LacI mutants that bind tighter to DNA -DNA operator mutations that bind tighter to LacI -Mutations in CAP -Mutations in DNA at CAP activation site

Question 29

How can you use growth to screen for changes in transcriptional regulation

Answer 29

Plate mutagenized cells onto LB agar and replica plate them onto minimal medium with and without lactose. Search for mutants that can frow on lactose as sole carbon source

Question 30

How could you identify bacteria that lost the ability to be resistant to a particular antibiotic

Answer 30

Plate mutagenized cells onto LB agar and replica plate them onto medium with and without antibiotic. Search for mutants that can grow in the presence of antibiotics

Question 31

What phenotypes can you select for

Answer 31

-Resistance to death: Phage or other predators, antibiotics, or environmental stresses -Metabolism: Ability to grow without certain nutrients (Gain of function experiments)

Question 32

Analysis of temperature sensitive mutants

Answer 32

Identify temp-sensitive mutants in the essential genes. Can be combined with other selective pressures to identify temperature-sensitive mutants that target a particular phenotype

Question 33

Nonsense suppressors

Answer 33

-Suppressor tRNA that has an anticodon to a stop codon but it charged with an amino acid -Stop codons in a target open reading frame will be translated with the suppressor tRNA