1 - genes & mutation

Question 1

to which end of the tRNA is the amino acid attached?

how are they connected?

Answer 1

aa connects to 3’ end of tRNA

attached via covalent bond

Question 2

describe the structure of tRNA

Answer 2

secondary structure = “cloverleaf”

some ss areas and some ds areas

Question 3

what are the levels of specificity expressed by tRNAs

Answer 3

1) aminoacyl-tRNA synthetase specificity:
- each tRNA only has ONE specific amino acid that can be added

2) codon specificity:
- codon/anticodon interaction specificity what tRNA is used

Question 4

in what way to codons and anticodons interact

Answer 4

hydrogen bonds

Question 5

start codon

Answer 5

eukaryotes:
AUG = Met

prokaryotes:
AUG, GUG, UUG (only at start)

Question 6

stop codons

Answer 6

eukaryotes:

UAA, UAG, UGA

Question 7

deviations from standard genetic cose found in Candida species

Answer 7

CUG encodes serine instead of leucine (Candida albicans)

Question 8

wobble hypothesis

Answer 8

tRNA base 1st position in anticodon —–(recognises)—–> mRNA base 3rd position in codon

C -----> G
A -----> U
U -----> A or G
G -----> U or C
I  -----> U, C or A (=H)

Question 9

types of mutations: spontaneous, induced, germinal and somatic, forward, reverse (back and suppressor)

Answer 9

spontaneous: random mutations to do metabolic errors or unknown agents
induced: mutations induced deliberately by mutagens
germinal: occur in germ-line cells –> hereditary
somatic: occur in soma –> not hereditary
forward: mutation of a wild-type allele to a mutant allele

reverse: a second mutation that restores the original phenotype
- –> back: a second mutation at the same site
- –> suppressor: a second mutation at a different location in the genome

Question 10

in bacteria and fungi, at what point in mutagenesis does mutation most commonly occur?

Answer 10

stationary phase
this stage occurs under environmental stress
population is not advancing, therefore random mutations occur

Question 11

conditional lethal mutations

Answer 11

lethal in restrictive condition

viable in permissive condition

Question 12

auxotroph

Answer 12

auxotrophs are unable to synthesise an essential metabolite that is synthesised by prototrophs, and can grow only when it is supplied in the medium

Question 13

describe three types of point mutations and how they effect amino acids

Answer 13

1) synonymous mutation:
- new codon but no amino acid change
- e.g. GCC (Ala) —> GCA (Ala)

2) non-synonymous mutation, conservative substitution:
- new amino acid that has similar properties
- no functional change
- e.g. GCC (Ala) —> GTC (Val)

3) non-synonymous mutation, non-conservative substitution:
- new amino acid that has very different properties
- functional change
- e.g. GCC (Ala) —> GAC (Asp)

Question 14

list the non-polar amino acids

Answer 14

1. glycine (gly)
2. alanine (ala)
3. proline (pro)
4. valine (val)
5. leucine (leu)
6. isoleucine (ile)
7. methionine (met)

GAPVLIM

Question 15

list the polar, uncharged amino acids

Answer 15

1. glutamine (gln)
2. asparagine (asn)
3. cysteine (cys)
4. threonine (thr)
5. serine (ser)

GACTS

Question 16

list the aromatic amino acids

Answer 16

1. phenylalanine (phe)
2. tyrosine (tyr)
3. tryptophan (trp)

Question 17

list the positively charged amino acids

Answer 17

1. lysine (lys)
2. arginine (arg)
3. histidine (his)

Question 18

list the negatively charged amino acids

Answer 18

1. aspartate (asp)

2. glutamate (glu)

Question 19

describe tautomeric shifts

Answer 19

spontaneous isomerization of a nitrogen base to an alternative hydrogen-bonding form, possibly resulting in a mutation

for example:

• G turns into rare enol form —> binds with T not C
• hopefully the mutant is repaired
• if not repair, mutant goes through replication
• two replicated DNA strands have different code —> GC bp and AT bp

Question 20

describe how UV radiation can cause mutagenesis

Answer 20

• UV radiation facilitates hydrolysis of cytosine to cytosine hydrate
• this may cause mispairing during replication
• UV radiation facilitates cross-linking of adjacent thymine forms thymine dimers
• dimers cannot be recognised by DNA pol. –> leaves a gap on strand with no genome –> results in single stranded domain –> highly fragile –> activate error-prone DNA repair mechanisms (e.g. SOS response)

Question 21

describe the effect nitrous acid has on base pairs

Answer 21

nitrous acid causes oxidative deamination of bases (amine functional group removed –> keto form)

adenine —> hypoxanthine
cytosine —> uracil
guanine —> xanthene

these rare bases are not meant to be in DNA

when identified, these bases are removed

this increases the chance of DNA mutation

Question 22

describe the effect of indels

Answer 22

in an open reading frame, indels can cause frameshift mutations

the closer the frameshift to the start codon, the higher the chance it will be deleterious
—> commonly results in nonsense mutations and nonsense proteins

Question 23

describe the effect of ionising radiation

Answer 23

ionising radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations

Question 24

describe the effect of hydroxylamine

Answer 24

hydroxylamine hydroxylates the amino group of cytosine and leads to G —> T transitions

Question 25

describe the effect of alkylating agents

Answer 25

alkylating agents are chemicals that donate alkyl groups to other molecules, inducing transitions, transversions, frameshifts, and chromosome aberrations

Question 26

describe suppressor mutations and how they were discovered

Answer 26

suppressor mutation: when a second mutation overcomes the deleterious effects of a first mutation

discovered via studies into bacterial genetics:
1. mutate bacteria, select an 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:
− wild-type
− first mutant (Leu-)
− second mutant (revertant to Leu+)
6. compare sequences

can revert to original amino acid or amino acid with similar properties

Question 27

describe intragenic suppressor mutations and how they can revert frameshift mutations

Answer 27

intragenic suppressor mutation = second mutation in same gene but different codon

upon first indel = frameshift mutation occurs as aa sequence is disrupted –> nonsense protein

second indel a few base pairs downstream

reading frame is restored from site of second mutation

to work, total change between two indels must be 0 or +/-3 nucleotides

Question 28

describe amber mutations and how they can be suppressed

Answer 28

amber mutation = tyr codon (UAC/UAU) converted to stop codon (UAG), resulting in premature termination

SUPPRESSION:
1) tyrosine codon (UAC) recognised by tRNA tyr anticodon (AUG)

2) amber mutation occurs, tyrosine codon UAC mutated to UAG i.e. a stop codon
3) stop codon recognised by release factors during translation –> premature termination. tRNA tyr anticodon not recognised

4) a) suppress via reverse mutation in tyr codon i.e. UAG back to UAC or UAU
4) b) mutation in anticodon from AUG to AUC –> tyrosine bought in when stop codon is present. this is not effective. one third of OFR in genome will contain UAG stop codon. there is a chance that the mutated tyrosine anticodon will bind to UAG stop codon, preventing termination of translation. this results in C-terminus extensions, leading to defective function

Question 29

describe how the ames test detects mutagens

Answer 29

mutagens cause reversions, i.e. autotrophs –> prototrophs

1) isolate enzymes from rat liver
2) prepare solution of potential mutagen
3) grow auxotrophs carrying a frameshift mutation
4) mix enzymes and auxotrophs and spread on agar plate = control plate
5) mix enzymes and potential mutagen on filter disk and place on plate spread with auxotroph (with tiny amount of aa to enable growth initiation) = experimental plate
6) incubate plates and compare results

control:
- few spontaneous reversions to prototroph observed

experimental:

• many reversions observed
• confirmed mutagen

Question 30

describe the process of photoreactivation

Answer 30

UV radiation induces formation of thymine dimers

can be fixed via photoreactivation = light-dependent repair

photolyase absorbs blue light for energy

cleaves thymine dimers, restoring original state

Question 31

describe the process of base excision repair

Answer 31

1) deamination of a base –> resulting in abnormal base
2) identified by glycosylase (specified for specific base) –> mutant neatly clipped off, leaving phosphate backbone in place
3) sugar phosphate removed by AP endonuclease and phosphodiesterase
4) DNA polymerase fills the single stranded break
5) DNA ligase seals the nick left by DNA polymerase

Question 32

describe the process of nucleotide excision repair

Answer 32

for bulkier defects, e.g. thymine dimer

1) UV photon creates thymine dimer
2) UVRB and two UVRA molecules bind to damaged DNA
3) molecules use energy from ATP to bend DNA, releasing UVRA molecules and recruiting UVRC
4) using nuclease activity, incision made on strand containing mutation (8bp in one direction and 4 in other)
5) helicase unwinds DNA and removed mutated region of DNA (to be recycled)
6) DNA polymerase fills the single stranded break
7) DNA ligase seals the nick left by DNA polymerase

Question 33

explain how bacteria use methylation to repair mismatched bases
describe the mechanism of mis-match repair

Answer 33

the A in GATC sequences is methylated subsequent
to DNA replication

in newly replicated DNA, the parental strand is methylated, but the new strand is not —> this difference allows the mismatch repair system to distinguish the new strand from the old strand

if the wrong base is incorporated into a newly synthesised DNA strand, the mismatched nucleotide is excised from the new strand and replaced with the correct nucleotide, using the methylated parental strand as a template

MEHCANISM:

1) mutS recognises mismatches and binds to them –> initiates repair process
2) mutH and mutL join the complex
3) mutH (an endonuclease) cleaves the unmethylated strand at GATC sequences on one side of the mismatch
4) helicase and an exonuclease excise the mismatched base on the unmethylated strand
5) DNA polymerase III fills in the gap, and DNA ligase seals the nick

Question 34

describe how bacteria repairs heavily damaged DNA

Answer 34

bacteria use the SOS response to fix heavily damaged DNA

1) recA recognises damaged ss regions of DNA and stimulates lexA to inactivate it’s self

2) lexA represses SOS response by binding to promoter of genes controlling DNA pol. 5
lexA iniactivated = DNA pol. 5 activated

3) DNA pol. 5 not disrupted by damaged DNA when replicating, but is very error prone

Question 35

describe how recombination occurs using the Holliday Model

Answer 35

1) two chromosomes come together
2) endonuclease and nicks one strand of each
3) helicase unwraps each strand
4) single stranded binding protein (SSBP) binds to protect strands
5) RecA facilitates strand exchange
6) DNA ligase repairs nick
7) DNA strands change conformation into Chi structure
8) nuclease separates two chromosomes
9) results in mix of DNA material between two homologs

Question 36

describe the process of complementation testing

Answer 36

only works with recessive mutations

draw diagram

cis-heterozygote: mutations in one gene
= cis-heterozygote will have WT phenotype

cis-heterozygote: mutations in different genes
= cis-heterozygote will have WT phenotype

trans-heterozygote: mutations in one gene
= no functional products –> trans-heterozygote will have mutant phenotype

trans-heterozygote: mutations in different genes
= functional products of both genes–> trans-heterozygote will have WT phenotype

Question 37

what is an opal codon?

Answer 37

UGA stop codon

Question 38

what is an amber codon?

Answer 38

UAG stop codon

Question 39

what is a ochre codon?

Answer 39

UAA sop codon