1 - genes & mutation Flashcards
to which end of the tRNA is the amino acid attached?
how are they connected?
aa connects to 3’ end of tRNA
attached via covalent bond
describe the structure of tRNA
secondary structure = “cloverleaf”
some ss areas and some ds areas
what are the levels of specificity expressed by tRNAs
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
in what way to codons and anticodons interact
hydrogen bonds
start codon
eukaryotes:
AUG = Met
prokaryotes:
AUG, GUG, UUG (only at start)
stop codons
eukaryotes:
UAA, UAG, UGA
deviations from standard genetic cose found in Candida species
CUG encodes serine instead of leucine (Candida albicans)
wobble hypothesis
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)
types of mutations: spontaneous, induced, germinal and somatic, forward, reverse (back and suppressor)
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
in bacteria and fungi, at what point in mutagenesis does mutation most commonly occur?
stationary phase
this stage occurs under environmental stress
population is not advancing, therefore random mutations occur
conditional lethal mutations
lethal in restrictive condition
viable in permissive condition
auxotroph
auxotrophs are unable to synthesise an essential metabolite that is synthesised by prototrophs, and can grow only when it is supplied in the medium
describe three types of point mutations and how they effect amino acids
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)
list the non-polar amino acids
- glycine (gly)
- alanine (ala)
- proline (pro)
- valine (val)
- leucine (leu)
- isoleucine (ile)
- methionine (met)
GAPVLIM
list the polar, uncharged amino acids
- glutamine (gln)
- asparagine (asn)
- cysteine (cys)
- threonine (thr)
- serine (ser)
GACTS
list the aromatic amino acids
- phenylalanine (phe)
- tyrosine (tyr)
- tryptophan (trp)
list the positively charged amino acids
- lysine (lys)
- arginine (arg)
- histidine (his)
list the negatively charged amino acids
- aspartate (asp)
2. glutamate (glu)
describe tautomeric shifts
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
describe how UV radiation can cause mutagenesis
- 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)
describe the effect nitrous acid has on base pairs
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
describe the effect of indels
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
describe the effect of ionising radiation
ionising radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations
describe the effect of hydroxylamine
hydroxylamine hydroxylates the amino group of cytosine and leads to G —> T transitions
describe the effect of alkylating agents
alkylating agents are chemicals that donate alkyl groups to other molecules, inducing transitions, transversions, frameshifts, and chromosome aberrations
describe suppressor mutations and how they were discovered
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
describe intragenic suppressor mutations and how they can revert frameshift mutations
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
describe amber mutations and how they can be suppressed
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
describe how the ames test detects mutagens
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
describe the process of photoreactivation
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
describe the process of base excision repair
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
describe the process of nucleotide excision repair
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
explain how bacteria use methylation to repair mismatched bases
describe the mechanism of mis-match repair
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
describe how bacteria repairs heavily damaged DNA
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
describe how recombination occurs using the Holliday Model
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
describe the process of complementation testing
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
what is an opal codon?
UGA stop codon
what is an amber codon?
UAG stop codon
what is a ochre codon?
UAA sop codon
