What happens if there is only one homologous recombination after transfer
- nonviable
- linear will not survive
- NEED TWO CROSS OVERS TO HAPPEN
How many crossovers are required to incorporate transferred DNA
EVEN NUMBER
Will you see recombination ?
When Hfr X F- = High frequency of recombinant, what happens to the recpeient bacteria?
Remains F-
When F+ X F- what happens to recipient bacteria ?
F- converted to F+
Formation of F prime factors
- they are generated by excision by recombination with a distant IS element (IS2 on the diagram look)
- carry chromosomal DNA
- “in vivo” cloning vectors
- useful for doing complementation tests
- used before “modern” recombinant teachinques
Is homologous recombination a one directional process?
No - F prime factors can occur
How to figure out the direction of transfer and what is transferred first
5 Hfr strains with different F insertion points and/or orientations of insertion
What did W.Hayes (Wollman-Jacob) do?
W.Hayes isolated HfrH and showed that it donated Thr and Leu at high frequencies compared to other markers
THIS MEANS Thr and Leu are close to the origin of transfer
- you select for F- by using Stremptomysin
Results of the wollman- JAcob mating experiment
Why are there less recombinations in the genes further away from the point of origin after a fixed time period?
- due to the collapse of bridges
Data from the interrupted mating experiment
- each donor gene first appears in recipient at a specific time after mating begins
- donor genes appear in a specific sequence
- the maximal yield of cells containing a specific donor marker is lower for the donor markers that enter later (due to breakage of mating bridges)
Conclusions from interrupted mating experiment
- Hfr chromosome is transferred in linear fashion
- begins at a specific origin
- the further from OriT, the later a gene will be transferred
- transfer more likely to be interrupted before later genes transferred
- “time of entry” of each marker can be used to construct a genetic map, using “minutes” as the measure of distance
- now know that transfer of the entire chromosome takes about 100 minutes
- Genetic map of E.coli, expressed in units of minutes, is 100 units long
Map example
How do we determine the distance between markers?
Using high resolution mapping by recombination frequency
What is the purpose of High resolution mapping by recombination frequency:
To determine the !distance! between markers
(Interrupted mating will determine the order of markers)
In high resolution mapping by recombination frequency why do you have to select for last marker to enter then screeen for earlier markers?
- other wise will have more recombination with markers closer to the origin
(- with eukaryotes, if we’ve got two linear genes, two genes that are further apart they are less likely to get recombination
- if u select for the last gene there is an equal change that bacteria has got recomabinaiton anywhere between last gene and origin of transfer
Which cross over in high resolution mapping by combination frequency is selected?
How do u figure out which marker to use in High resolution mapping
Interrupted mating - (last one)
How many possible recombinant classes are there in a three-factor cross?
4
How do you figure out the frequency of different recombinant classes in high resolution mapping?
- the frequency of different recombinant classes will depend on the order of the genes and the distance between each pair of genes
(Bigger distance between genes = higher number of recombinations )
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