Complementation and New Advances in Genetics Flashcards
1
Q
Complementation
A
- characterizing mutants is a way to identify gene function
- complementation restores a genotype by replacing the mutated gene with a functional one
- important in bacteria because of operons
- some mutants cannot be complemented
2
Q
Complementation Studies with Phage
A
- viruses of two or more different genotypes can simultaneously infect a bacterium
- the DNA molecules of one of the infecting viruses can recombine with that of another forming recombinant molecules
- the huge humber of viruses released from a huge number of hosts enables rare recombination events to be detected
- Seymour Benzer exploited these properties of phage T4 to show that different genes were responsible for rapid lysis (rll) phenotype
3
Q
Lytic Cycle of Phage (5)
A
- Injection of phage DNA
- replication of DNA
- synthesis of new phage particles
- packaging of DNA into head
- lysis releasing progeny
4
Q
Mapping the rll phenotype
A
- to test if mutants could recombine Benzer infected a permissive strain of E.coli with pairs of mutants or single mutants
- He next prepared phage stocks and infected the non-permissive (strain K) and determined the frequency of phage that could infect
- the frequency of virulent phage from single mutants was 1 in 10^7-these were due to reversion
- the frequency of virulent phage recovered from coinfection was much higher (the phage DNA had recombined)
5
Q
Seymor Benzer’s T4 mutant mapping
A
- infection with single rll mutant gives reversion at a certain frequency
- mixed infection with mutations at different positions allows for recombination
- for mutations far apart the recombination frequency is higher (more homology)
- some coinfections resulted in no lytic phage because one of the mutants was a deletions
6
Q
Seymor Benzer’s T4 complementation
A
- Benzer found all A mutations could complement B mutants but not A mutants
- mutants that cannot complement each other are in the same complementation group (Benzer called them cistrons)
7
Q
Complementation in Bacteria
A
- mutations hisA1 and hisB3 are in different genes-both genes are His-
- crossing mutations together results in His+ cells
8
Q
Non-complementation in bacteria
A
- hisA1 and A2 are in the same genes
- crossing the mutations results in His- cells
- no way to get a functional hisA since neither bacteria has a functional copy of the gene
9
Q
Cis and trans mutations
A
- cis acting locus- a genetic region affecting the activity of genes on the same DNA molecule (e.g. lac operator)
- trans acting locus-encodes for a factor that can act elsewhere (e.g. LacI)
10
Q
Non complementation- dominant mutations (lacIs)
A
- lacIs: insensitive to lactose
- dominant to wild type repressor (acts antagonistically to wild type)
- can bind to operator
11
Q
Non-complementation-regulatory site
A
- dominant mutation
- Oc: repressor binding site damaged
12
Q
New Advances in Genetics (4)
A
- deletion collections
- signature tagged mutagenesis (barcodes)-allows the identification of mutants in pools
- next gen sequencing
- chemical genetics
13
Q
Chemical genetics
A
- most mutations are not conditional
- specific molecules (drugs) can be used to study effects of inhibiting a protein in real time
- can specifically target one activity of a protein
- especially important for essential genes (can’t make a mutation in an essential gene, needed for cell to exist)
- also for organisms where there are poor genetics (slow doubling time, no transformation, diploid)
14
Q
Small molecule study
A
- Develop robust assay-the more specific the better
- apply high throughput format
- add small molecule library
- investigate “lead compounds” for specificity, activity. etc
15
Q
Chemical Genetics Forward
A
- plate with yeast cells
- add 1 compound per well
- select compound that produces phenotype of interest
- identify protein target -this way usually used by drug companies, normally fails at this step
