Mutation Flashcards
mutant (def.)
an organism in which the base sequence of DNA has been changed
mutation (def.)
any heritable change in the DNA sequence
phenotype (def.)
- observable properties of an organism
- mutant vs. wildtype
genotype (def.)
actual sequence of the DNA of an organism
allele (def.)
- different forms of the same gene
- mutant vs. wildtype
Reversion (def.)
-a restoration of the original phenotype to a mutant
-due to restoration of the original sequence of the gene
Consequences of Mutation: - mutations that alter phenotype disrupt _____
protein function
Alteration of DNA/RNA/protein function - must disrupt DNA/RNA/protein sequence (ways):
1) _______
2) _________
-change sequence of one or more codons
-change reading frame (ORF = open reading frame)
silent MUTATIONS vs leaky mutations
silent: amino acid substitution that does not affect protein structure/activity
leaky: partial disruption in a protein’s activity
Conditional vs Non-conditional mutations
non- conditional: mutant phenotype expressed under all conditions
conditional: mutant phenotype only expressed under
certain conditions (ex. Temperature sensitive, Suppressor sensitive, Auxotrophic)
point mutation change? characteristics?
-alteration single base pair (transitions/reversions, single/double/triple), missense/nonsense
-can be leaky, revert
deletion mutation change? characteristics?
-removal of DNA
-not leaky, never revert
insertion mutation change? characteristics?
-addition new DNA
-not leaky, can revert
inversion mutation change? characteristics?
-inversion existing DNA
-not leaky, can revert
frameshift mutation change? characteristics?
-addition or deletion not equal to multiple of 3nt
-not leaky
-can revert
point mutation: transitions
Pu:Py pair -> Pu:Py pair
Py:Pu pair-> Py:Pu pair
point mutation: transversions
Pu:Py pair -> Py:Pu pair
Py:Pu pair -> Pu:Py pair
1952 Lederberg & Lederberg demonstrated direct evidence that phage T1r mutations occurred _________
spontaneously in E. coli before exposure to phage
1952 Lederberg & Lederberg lab technique
-used replica plating
- grew colonies on non-selective medium (no T1)
- replica plate to multiple plates spread with T1 phage
- T1r colonies appeared in same positions on each of replica plates
mutation rates (def.)
probability that a gene will be mutated in a single generation- also referred to as chance of mutation to a particular phenotype
mutations rate widely variable amongst different genes: dependent upon: _______ (3)
- gene size
- nucleotide sequence
- number of genes that dictate phenotype
Generation of Spont. mutations (3)
1) Replication errors
2) alteration of nucleotides
3) recombination
2 types of replication errors
i) errors in nucleotide incorporation: incorporation of tautomeric bases during replications; causing transitions
ii) slipped strand mispairing (one strand of DNA slips during replication)
Tautomer (def.)
alternative forms of bases with altered base-pairing properties
adenine (imino form) bind ____; thymine (enol form) bind _____
-cytosine
-guanine
tautomeric transitions may escape repair mechanisms because: ________ (2) p
1) proofreading -> tautomers are correctly hydrogen-bonded
2) mismatch repair -> tautomer may later assume normal structure, could be removed by the “mismatch repair”, but only if the tautomer is found in unmethylated daughter strand
extra base is either added (slippage of ____ strand)
or deleted (slippage of _____ strand). slipping occurs in ______ sequences; causes _____ mutations
-daughter
-template
-short, repeated
-frameshift
2 Alteration of Nucleotides
1) deamination: about 5% of cytosines in bacterial DNA are methylated (MeC); both MeC and C are deaminated occasionally
2) oxidation: reactive forms of oxygen from metabolism(peroxide/free radicals), react with and alter DNA bases (eg. guanine -> 8-oxo-guanine pairs with adenine instead of cytosine causing GC-> TA transversions)
deamination C-> U
-U can pair with A
-GC -> AT transition
-repaired by uracil-N-glycosylase so that this rarely happens
deamination MeC-> T
-T can pair with A
-GC -> AT transition
-MeC:G base pairs can be hotspots for mutation
recombination between direct repeats leads to ____
deletions
recombination between inverted repeats leads to ____
inversions
mutagenesis -> treatments that alter the DNA; exposure of the whole organisms or just DNA to ______
-mutagens
Base-Analog Mutagens: compound similar to ______ that can be incorporated into DNA during replication; H-bonds correctly, so escapes ____. to be mutagenic, must have _____
- eg. 5-bromouracil (BU); analog of _____ (tautomeric enol form predominant that frequently pairs with ___)
-causes _____ MUTATIONS
-if replaces a T in keto form, can tautomerize into enol form to yield _____
-can be misincorporated in enol form to yield ______
Base analog mutagens ALWAYS induce _____
-one of 4 DNA bases
-editing
- 2 modes of H-bonding
-thymine
-G
-TRANSITION
-AT -> GC
-GC -> AT
-transitions
Chemical Mutagens -alter bases already incorporated into DNA to change _____
-H-bonding specificity
types of chemical mutagens
1) nitrous acid (C-> U)
2) hydroxylamine
3) ethylmethane sulfonate (EMS)
Intercalating Agents (def.)
- planar, heterocyclic molecules with same dimensions as a Pu:Py base pair
Intercalating Agents intercalates between _____ . During replication, _______ are inserted into DNA containing intercalating agents and generates _____ mutations. this may work by stabilizing looped out bases in _______
- base pairs
-extra base pairs - frameshift
- slipped strand mispairing during replication
mutagenesis by mutator genes: mutations in specific genes (_____) whose products are involved in _____; leads to _________ at other genes throughout the chr. (aka _____ phenotype)
-mut genes
-DNA repair
-a high mutation frequency
-mutator phenotype
mutatator gene: mutD/dnaQ dam function
proofreading functionof DNA pol II; methylates DNA; directs mismatch repair to correct stand
mutatator gene: mutH, mutL, mutS
mismatch repair
isolation of mutations: screens
- Mutants grow on media where phenotype of the mutants can be distinguished
from parent - eg. COLOR INDICATOR PLATES
- eg. identification of auxotrophic mutants based on inability to grow in the absence of certain metabolites (replica plating)
isolation of mutations: selections
- Mutants are identified based on their ability to grow under conditions that the
parent cannot (Very Powerful) - can identify a mutant on single plate of 10^10 bacteria/phage
- eg. resistance mutants (antibiotic, phage resistance)
- consider a rare mutation (1/10^6)
screens vs . selections
screen: 100 colonies/plaques per plate; must screen 10 000 plates
selections: 10^10 bacteria/phage; select mutant off 1 plate
isolation of mutations: enrichments
- procedures that enrich for mutant organisms
- living, multiplying cells are killed by treatment, while non-growing
mutant cells are not (eg. penicillin enrichment)
isolation of mutations: Isolation of Conditionally Lethal Mutations
REPLICA PLATING
- mutants plated under permissive conditions –> selected under nonpermissive conditions (e.g. Host mutants)
