Module 1: Genes and Mutations

Question 1

What are the two levels of specificity required by tRNAs?

Answer 1

1. aminoacyl-tRNA synthase activity:
   - Identify elements in the acceptor step
2. Codon specificity

Question 2

What are the deviations from the standard genetic code?

Answer 2

1. Prokaryotes alternative start codons:
   - GUG and UUG instead of AUG
2. Candida species:
   - CUG encodes serine instead of leucine

Question 3

Start and stop codons

Answer 3

Start: AUG
Stop: UAA, UAG, UGA

Question 4

Wobble table

Answer 4

C --> G
A --> U
U --> A, G
G --> U, C
 I  --> U, C, A

Question 5

Forwards mutation

Answer 5

mutation from WT –> mutant

Question 6

Types of reverse mutations

Answer 6

overall: a second mutations that restores the original phenotype
1. Back mutation: a second mutation at the same site
2. Suppressor mutation: a second mutation at a different location

Question 7

Conditional lethal mutations

Answer 7

• lethal in the restrictive condition

- viable in the permissive condition

Question 8

What are the types of point mutations and their effects?

Answer 8

1. synonymous mutation:
   - new codon, no amino acid change
2. non-synonymous, conservative:
   - new amino acid but with similar physical properties to original
3. non-synonymous, non-conservative:
   - new amino acid with different physical properties

Question 9

Nonpolar, alipathic amino acids

Answer 9

1. valine (Val)
2. proline (Pro)
3. glycine (Gly)
4. leucine (Leu)
5. alanine (Ala)
6. methionine (Met)
7. isoleucine (Ile)

Question 10

Aromatic amino acids

Answer 10

1. Phenylalanine (Phe)
2. Tyrosine (Tyr)
3. Tryptophan (Trp)

Question 11

Polar, uncharged amino acids

Answer 11

1. Glutamine (Gln)
2. Serine (Ser)
3. Cysteine (Cys)
4. Asparagine (Asn)
5. Threonine (Thr)

Question 12

Positive amino acids

Answer 12

1. Arginine
2. Lysine
3. Histidine

Question 13

Negatively charged amino acids

Answer 13

1. Aspartate (Asp)

2. Glutamate (Glu)

Question 14

Missense mutations

Answer 14

• amino acid substitution leads to protein being defective

Question 15

Nonsense mutation

Answer 15

• creates a stop codon, prematurely ending the codon –> loss of function mutation

Question 16

Tautomeric Shifts

Answer 16

Isomerisation caused by a reversible change in the location of a hydrogen atom in a base
Can cause substitution mutations by modifying base pairing

Question 17

Types of base substitutions

Answer 17

Transitions: pyrimidine -> pyrimidine; puring -> purine
Transversions: pyrimidine -> purine; purine -> pyrimidine

Question 18

Mutagenic effects of 5-bromouracil

Answer 18

1. C:G –> T:A substitution and visa versa

cytosine –> 5BU
need to finish this

Question 19

Mutagenic affects of nitrous acid

Answer 19

Causes oxidative deamination of bases

1. Cytosine –> uracil (pairs with A instead of G)
2. adenine –> hypoxanthine (pairs with C instead of T)

Question 20

Mutagenesis by UV radiation

Answer 20

1. hydrolysis of cytosine causes mispairing during replication
2. Cross liking of adjacent thymines –> thymine dimers block DNA replaication and activate error-prone DNA repair

Question 21

Mutagenesis by acridine dye

Answer 21

Intercalation of acridine dye causes frameshift mutations

• IN/DEL of base pairs not divisible by three alters reading frame
• usually positive and inserts into DNA

Question 22

How do IN/DELs affect phenotype

Answer 22

• in ORF: cause frameshift mutations
• usually result in frameshift mutations (premature stop)
• usually encode nonsense proteins

Question 23

How were suppressor mutations discovered?

Answer 23

1. mutate bacteria, select and auxotroph (e.g. leu-)
2. grow up leu- mutant on medium containing leucine
3. mutate the leu- cells
4. select second mutant that is now prototrophic
5. sequence the gene encoding leu biosynthesis enzyme in:
   - WT
   - first mutant (Leu-)
   - second mutant (revertant to Leu+)
6. compare sequences

Question 24

Intragenic vs Intergenic suppressor mutations

Answer 24

1. Intragenic:
   - second mutation within the same gene, but different codon
2. Intergenic:
   - second mutation in a different gene

Question 25

Amber mutations and their suppression

Answer 25

Amber: TAG stop codon

Question 26

Describe photreactivation in E.coli

Answer 26

- thymine dimers are caused by UV radiation - fixed using light dependent repair - photolyase absorbs blue light for energy - cleaves thymine dimers, restoring original state

Question 27

Base excision repair

Answer 27

- deamination of cytosine to uracil - excision of uracil - sugar-phosphate removed by AP endonuclease and phosphodiesterase - DNA polymerase adds cytosine base back -> uses the undamaged complementary strand and the template - DNA ligase seals the break left by polmerase

Question 28

Nucleotide excision repair

Answer 28

- Thymine dimer in DNA strand - Polypeptide trimer (containing 2 x UvrA and 1 x UvrB) recognises and binds to damaged DNA - Energy from ATP is used to bend DNA and change conformation of UvrB --> UvrA released - UvrC binds to UvrB and DNA and cleaves DNA around UvrB protein - UvrD helicase releases excised oligodimer - DNA pol1 replaces UvrB and fills in gaps using complementary strand as template - DNA ligase seals the nick

Question 29

The Ames Test

Answer 29

??

Question 30

Mismatch Repair in E.coli

Answer 30

1. MutS recognises mismatches and binds to them to initiate the repair process 2. MutH and MutL join the complex 3. MutH (endonuclease) cleaves the unmethylated strand at hemimethylated GATC sequences on eitther side of the mismatch 4. Excision requires MutS, MutL, MutU and an exonuclease 5. DNA pol III fills in the gap and ligase seals the nick

Question 31

How are thymine dimers dealt with post replication?

Answer 31

1. a thymine dimer in the template strand blocks replication 2. DNA pol III restarts DNA synthesis past the dimer, leaving a gap in the nascent strand 3. RecA binds to the single strand of DNA at the gap and mediates base pairing with the homologous segment of the sister double helix to fill the gap 4. DNA pol fills the gap using information from the other strand 5. ligase seals the nick

Question 32

What happens in the SOS response in E.coli

Answer 32

1. if DNA is heavily damaged by mutagens, the SOS response is activated 2. DNA pol V replicates the damaged regions, but damaged sequences are not replicated accurately 3. When extensive DNA damage occurs, RecA binds to single-stranded regions of DNA in damaged regions 4. This activates RecA, which stimulates LexA to inactivate itself, turing on SOS 5. LexA usually represses that regulate SOS response

Question 33

Describe the Holliday Model of recombination and crossing over

Answer 33

1. Pairing of homologous chromosomes 2. Formation of single-strand breaks --> endonucleases nick chromsomes 3. Helicase and DNA single stranded binding proteins unwinds DNA 4. Strand exchange occurs by RecA type protein 5. Formation of a covalent, single-stranded bridge