Mutations Flashcards
mutant
genetic state of an organism, different from the wild type
mutation
structural alteration of DNA in a mutant, always change in base sequence of DNA
mutagen
physical or chemical agent that causes mutations
mutagenesis
process of producing a mutation
spontaneous vs. induced mutagenesis (background level of mutation, about 1 per 107-109 divisions)
TYPES OF MUTATIONS 5
- wildtype - normal state of the MO
- base substitution
- base addition or insertion
- base rearrangement (recombination)
- base deletion
Point mutation
single base pair changed
Multiple mutation
2 or more bp change
_____ sequence> __ ___ sequence >___ ___ ____
base sequence, AA sequence, 3D protein structure
Silent mutation
does not alter AA sequence
Neutral mutation
AA substitution - no effect- does not change structure of protein
Leaky mutation
reduction but not loss of activity
not in active site
Conditional Mutations
affected by temperature - exhibit mutant phenotype under certain conditions.
Missense mutation
change in AA - substitution
Nonsense mutation (chain termination)
(amber, ochre, opal - stop codons) - codon stops protein synthesis
Detection of Mutants - Screening
- plate a large number of bacteria on agar with antibiotic or any compound in agar
- sugar-utilization mutants
color indicator plates
EMB agar (pH colonies) incorporate dyes or not (Lac+ black or Lac- white)
MacConkey, tetrazolium agars - leucine requiring - mutant (Leu-)
Replica plating for Ts mutants
- grow at different temperatures.
ENRICHMENT FOR MUTANTS
Selection
- only mutant cells will grow
1. antibiotic resistance mutants
2. grown on substrates that normally cannot be used
ENRICHMENT FOR MUTANTS
- spontaneous mutation - Leu- 1 per 107 cells, 5x104 plates
- induced mutation rate - 1x105, 500 plates
- need to favor growth of mutant over wildtype
Example: Penicillin Selection Techinque
cells in Leu- media, synthesize Leu+ grow & explode
99% die Leu-, now 1/104, need only 50 plates
2nd cycle repeat pen enrichment 1 leu-/102
Origin of Spontaneous Mutations
Sources
- errors during replication
corrected by Pol III proofreading
corrected by recombination repair - spontaneous alteration of a nucleotide - deamination
spontaneously deaminated MeC->T - depurination - N glycosylic bond for purine breaks
errors during replication
corrected by Pol III proofreading
corrected by recombination repair
can happen if:
-tautomerision of nucleotides pg. 197
boat & chair forms
Types of Mutagens
- Base-analogue mutagens
- Chemical mutagens
- UV light
- INtercalating agents
- Transposable Elements
- Base-analogue mutagens
tautomerize, two modes of H bonding
a. 5-bromouracil (BU) analogue of thymine
usually pairs A, keto form pairs with A ,*rare enol form pairs with G AT -> GC
- pair with G –> G:C pair
- occassionally BU analogue for C can pair the other way rarely to cause a mutation GC -> AT
b. 2-aminopurine (AP) substitute for A base pair w/T or C
Chemical Mutagens
substance alters a base already in DNA helix, change H-bond properties
oxidative
a. nitrous acid - amino groups ——————-> keto groups
deamination
cytosine & adenine are deaminated
cytosine -> uracil -> UA - next round of replication
adenine -> hypoxanthine -> HC (in vitro reaction)
b. hydroxylamine - reacts with C –> converts to a base that pairs with A
c. EMS (ethyl methane sulfonate) (used w/eukaryotes)
alkylating agent
react with G, sometimes T
- add alkyl group (alkane minus one hydrogen CH3CH2) to N-7 position of the purine ring
1. G pairs with T in ionized form
2. effects SOS repair system
2. depurination occurs, another base inserted before repair mechanism can fix original loss
- UV light
thymine dimers - SOS repair system
- Intercalating agents
x. acridine orange, proflavine, acriflavine, EB (dyes)
-planar, 3 rings, same size as base pair - intercalation causes frameshift mutations
class=Section3>
used in very low concentrations
also prevent plasmids from replicating/partitioning (plasmid curing)
Transposable Elements (mobile DNA segments)
100’s-1000’s bp in length
-copies of Tn can be inserted at other locations on the genome
one copy at original site with a new copy in another site.
-some contain sequences that terminate transcription
big problem in polycistronic MRNA
-can find Tn when you induce mutation
SITE SPECFIC MUTAGENESIS
- Production of deletions with endonucleases (restriction)
2 cuts, remove piece of DNA and then reanneal - Point mutations at random sites in a particular region
remove fragment as in #1
treat fragment with mutagenic agents
reinsert into DNA - Point mutations at a particular base sequence
a. synthetic piece of DNA is made - M13 method pg. 365, Fig. 15.9
b. dut ung method
MUTATOR GENES
genes that in a mutant state cause mutations to occur in other genes
actually gene function is to keep mutation rate low; defective product of mutator gene, then accompanying mutations occur
MUTATOR GENES- mechanism
- polymerase is defective - (altered), stops the editing function
- dam gene for methylation of DNA, 3’–>5” editing function for mismatch repair if no methylation doesn’t know which base to repair in which strand, daughter or parent.
- enzyme altered that would carry out excision step (mismatch repair)
- proteins changed that keep SOS turned off.
Mutational Hot Spots
map location of mutants on the genome - certain sites altered with high frequency explanations (20-500 times more often)
Reasons for Hot Spots
- area that breathes more often
- area accept base analogue or SOS repair
- average in DNA - 1% prok., 5% euk. cytosines are methylated (MeC), when these are deaminated (usually spontaneously) –> 5-methyluracil or thymine
____ doesn’t occur w/ high frequency in areas where it affects a ___ _____
MeC, gene product
REVERSION
(back mutation, reverse mutation)
result of spontaneous mutagenesis - random
some mutants that don’t revert at a detectable frequency
reasons:
- double mutant (2 base sequences changed)
2. deletion of many consecutive bases - replacement is very rare
Intragenic Reversion
-inside the gene
1. Same site reversion - rare - true reversion
2. Second site or suppressor mutation
reversion not at the 1st mutation site but by a mutation at a second site (reversion doesn’t return to genotype, but does exhibit the same phenotype)
Types of Reversion
a. Charge of AA
b. Size of AA
Types of Intragenic Revertants:
- wildtype - return to wt genotype
- same site revertant
- second site revertant
Second site - frameshift reversion (shift back to right reading pattern)
Two requirements for this reversion:
- reverting event - near mutation site
- polypeptide chain area can be withstand the alterations (outside active site or regulatory sites)
Suppression or Intergenic Reversion
change in second gene that eliminates or suppress first mutation…
Information suppressors
- change cell’s translational machinery
- read mutation as “normal”
a. mutations in tRNA genes
b. mutations in ribosomal genes - usually these are unhealthy mutations
Interaction suppressors
- two proteins that interact
- mutation in second gene restores overall protein function
- used to study protein interactions
Overproduction suppressors
- mutant protein has lower activity (leaky mutation)
- mutation in regulatory gene or regulatory site on protein -> overproduction of mutant protein
Bypass suppressors
-turn on a new pathway that eliminates need for mutant gene
Physiological suppressors
-suppression by general changes in cell metabolism, mutations that increase conc of “chaperone” proteins (shock proteins)
tRNA Suppressors
suppress original mutation & others as well.
-“suppressor-sensitive mutations”
Nonsense or chain termination mutations
presence of a stop codon is not enough to stop protein synthesis
a. altered tRNA - reads a stop codon as an AA codon
Reasons tRNA terminator suppressors cells are viable
a. Protein factors are also involved in chain termination and will terminate even if tRNA is bound to stop codon - much of the time - get less of the protein.
b. Often chain termination codons are in pairs - second terminator works.
Missense Suppressors
(exchange one AA or another)
a. mutant tRNA that can recognize 2 codons
b. mutant tRNA incorrectly recognized by an aminoacyl synthetase
c. mutant animoacyl synthetase that puts wrong AA on the tRNA
Ames Test
test for mutagenesis/carcinogenic
- His- reverting to His+
- microsomal fraction (isolated from a rat liver) converts some chemical to carcinogenic form
