Lecture 18 - Genetics Of Homologous Repair Flashcards
Consequences of homologous recombination?
New genetic materials
The life cycle of the lamba bacteriophage?
Attaché to e.coli and injects DNA into bacterium and forces the bacteria to synthesis phage proteins and phage DNA. These then destroy the cell and are released to infect more
What does a lambda infection results in?
The formation of plaques
What are the characteristics of the lambda plaques?
They are turbid or clear, small or large
What are some ways the recombination of genes in e.coli can happen?
Breaking a parental strand
Presence of heteroduplex DNA at the site of crossing over
What leads to the observation of negative interference?
Crosses and the measurement o recombination frequencies
Early models for the mechanism of recombination - break join
Both double strands are broken and have religased with the wrong strand so the opposite strand binds with another one.
There is no DNA synthesis
Early models for the mechanism of recombination - copy-choice
Both DNA molecules are being replicated and at some point the replication templates are swapped over which creates a totally newly synthesised product.
Early models for the mechanism of recombination - break copy
One of the double strands breaks and uses the other as a template which leads to part of the DNA being newly synthesised.
The Meselson and Weigle experiment - They investigated the question: does recombination occur by breaking parental DNA? How did they do this - identifying regions of the genome?
They worked out the genome of the bacteriophage lambda and identified the region which coded for whether it was turbid or clear and the size of the molecules. When + the plaques are turbid c is clear. + = large, mi = small.
The Meselson and Weigle experiment - the two types of phages used in this experiment?
They took two populations of the lambda bacteriophage. One a wild type for the ++ genes = turbid large plaques and the second a population of mutants (clear, small plaques).
Wild type had heavy DNA due to it being cultivated in isotopes and the mutant had light DNA due to it being cultivated normally.
The Meselson and Weigle experiment - what did they do with the two phages?
They coinfected the same e.coli population. The E.coli had been cultivated normally making the DNA light.
The Meselson and Weigle experiment - what happened after the coinfection?
They collected the population and centrifuged them in caesium chloride which separates different phages by density. The bottom fraction is the heaviest and the DNA in these phages had to be wild type.
Medium phages had one strand wild type one strand mutant.
The Meselson and Weigle experiment - what did they do with the fractions?
Isolated them and infected a new population of E.coli to compare the phenotype of the newly obtained lysis plaques to the weight of the DNA in the phage
What did the results of the Meselson and Weigle experiment show?
That to get a turbid and a small plaque means that recombination must have happened. This experiment was not good enough to determine if there was no newly synthesised DNA or if there was only a little.
What was the final conclusion of the Meselson and Weigle experiment?
That is could not be a copy-choice model as both parent strands are not fully synthesised. It could be break join or the break copy model which means that recombination does occur by a double stranded break of a parental strand.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? How was this set up?
He only looked at the locus for turbidity and clear plaques. He made the wildtype (turbidity) DNA heavy and the mutant (clear) DNA light before co-infecting e.coli.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What happened after coinfection?
Collect all the progeny and centrifuge in caesium chloride before letting the fractions made reinfect a new population of E.coli
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? what did he see after reinfecting a new e.coli population?
clear plaques, turbid plaques and plaques that where mottled. These mottled plaques had both turbid and clear spots and therefore information from both the wild type and the mutant.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? Why did he see mottled plaques
The DNA in the turbidity/clear locus is a heteroduplex (each strand of ds DNA codes for different things) One codes for wild type (turbid) and the other mutant (clear). once heterodulex DNA is duplicated it creates a wild type and a mutant population.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What would happen to the amount of heavy DNA you would have if there was a ds break at the c-locus (due to scarring)
If a double stranded break occurs at the c-locus and the DNA is ligated by a break join or copied by break copy from the DNA in the mutant we will have DNA that it 3/4 heavy DNA from wildtype.
Or 1/4 heavy DNA and the rest light
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What would happen to the amount of heavy DNA you would have if there was a ss break at the c-locus (due to scarring)
You would have 3/8 of heavy DNA or 1/8 of heavy DNA
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What would happen to the amount of heavy DNA you would have if there was one recombination at the c-locus (due to scarring)?
You’d get one heavy strand and one light - if this was then to go on and recombine you would have a strand that is 3/8 heavy or 1/8 heavy
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What would happen to the amount of heavy DNA you would have if there was recombination happening on ++ break after several rounds of replication at the c-locus (due to scarring)
You would have 0 heavy DNA as newly synthesised DNA ++ is light.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA? What did centrifuging with caesium do?
Can correlate the presence of mottled plaques at lysis with the density of phages they creates and can isolate phages with contained 3/4 heavy atoms or 3/8 heavy DNA.
Another Meselson and Weigle experiment - does recombination leave a scar on DNA?
Yes in the form of heteroduplex
The Amati and Meselson experiment - Does recombination at one locus have an effect on recombination frequency at a nearby locus? How was this done?
They carried out a three factor cross and calculated recombinant frequency and then the level of interference and the coefficient of coincidence. They also created different phage populations and could place markers at different distances from one another.
The Amati and Meselson experiment - Does recombination at one locus have an effect on recombination frequency at a nearby locus? - they’re results?
The nearer the markers the higher the coefficient of coincidence instigating negative interference
How do you work out recombinational distance?
Rab + Rbc
If one recombination event happens at a close loci what happens at another loci?
A recombination event
How do you calculate recombinant frequency in a two factor cross?
Number of recombinant/ total number of progeny
What would you do when calculating recombinant frequency if there is a double mutant?
You would times the number of recombinant by 2
How do you calculate recombinant frequency in a 3 factor cross?
Number of Recombinant/ total progeny
How do you calculate R(double expected)?
RAB x RBC
