Recombination, Transposition, Hfr, Transduction Flashcards
Site Specific Recombination
-occurs at particular DNA sequences recognized by enzymes that recognize said DNA sequences and catalyze recombination with a specific recipient DNA
Homologous Recombination
- occurs between DNA sequences that are homologous-the two DNA sequences are nearly identical
- requires RecA
Intramolecular Recombination
-recombination can occur looping out a piece of DNA
-results in circular piece of DNA and shorter piece
(degradation of shorter piece)
-common way for plasmids or phage DNA to excise
Recombination Frequency
- probability that recombination will occur at a site of DNA is fairly constant (although hot spots do exist)
- increasing the amount of homology will increase the chances for recombination (the more base pairs that are similar, the more recombination there is)
- the further apart 2 DNA positions are the greater the probability that there will be a recombination event between them
- DNA sequences that can be distinguished are called markers
- recombination frequencies are directly proportional to distance between 2 markers
Genetic Crosses to determine how close genes are to each other
- genetic crosses often use co-inheritance of markers to tell how close genes are to each other
- co-inheritance frequency is inversely proportional to the distance between 2 DNA markers
RecA
- recombination is mediated by RecA
- RecA’s job is to pair homologous DNA segments together so they can exchange strands
- RecA binds DNA (usually single stranded DNA)
- then more RecA monomers bind cooperatively to make a nucleoprotein filament
- this RecA filament then goes looking for homology to pair with and recombine
4 Important things to remember about recombination
- Homologous recombination requires substantial DNA sequence homology between the donor and the recipient
- Recombination between any 2 sequences is a relatively low frequency event (genetic selection is required to find a specific recombination event in vivo)
- the co-inheritance of 2 genetic markers is inversely proportional to the distance between them
- homologous recombination can be prevented in a mutant
F plasmid
- conjugative plasmid
- can integrate into the chromosome
- cells that have F plasmid are F+
- cells that lack the F plasmid are F-
F’ plasmid
- in some cases excision can be imprecise and a piece of the chromosome is included in the plasmid
- the plasmid that now carries the extra DNA is F’
- F plasmids useful for constructing partial diploids (merodiploids)
Partial Diploid
-cell that has some genes that are diploid but not all
Hfr strains
- bacteria that have integrated the F plasmid are called Hfr strains “high frequency of recombination”
- when F plasmid excises it tries to transfer entire chromosome
- by using donor and recipients with different genetic backgrounds, Hfr strains can be used to map the location of genes
Transfer and Incorporation of donor Hfr DNA into recipient chromosome
- have Hfr plasmid and F- plasmid
- transferred fragment converted to dsDNA
- donor DNA inserted by double crossover
Hfr Mapping
- the entire chromosome of an Hfr strain may be transferred, rare
- the donor and recipient chromosomes can recombine to repair mutated genes
- by mating Hfr strains with various markers, you can determine the order of genes and direction of transfer
- the transfer of the chromosome can be stopped by shearing the mating cells apart
The transfer of genes from Hfr donor as a function of time
- the conjugative bridge is broken and the Hfr F- mating is stopped a various intervals
- while the bridge is intact, chromosome transfer occurs at a constant rate
- the order and timing of gene transfer directly reflects the order of genes on the chromosome
Insertion of F plasmid-orientation
- the F plasmid can insert at multiple sites and in either orientation
- this dictates the order of genes that will be transferred during conjugation
