Recombination, Transposition, Hfr, Transduction Flashcards
1
Q
Site Specific Recombination
A
-occurs at particular DNA sequences recognized by enzymes that recognize said DNA sequences and catalyze recombination with a specific recipient DNA
2
Q
Homologous Recombination
A
- occurs between DNA sequences that are homologous-the two DNA sequences are nearly identical
- requires RecA
3
Q
Intramolecular Recombination
A
-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
4
Q
Recombination Frequency
A
- 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
5
Q
Genetic Crosses to determine how close genes are to each other
A
- 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
6
Q
RecA
A
- 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
7
Q
4 Important things to remember about recombination
A
- 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
8
Q
F plasmid
A
- conjugative plasmid
- can integrate into the chromosome
- cells that have F plasmid are F+
- cells that lack the F plasmid are F-
9
Q
F’ plasmid
A
- 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)
10
Q
Partial Diploid
A
-cell that has some genes that are diploid but not all
11
Q
Hfr strains
A
- 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
12
Q
Transfer and Incorporation of donor Hfr DNA into recipient chromosome
A
- have Hfr plasmid and F- plasmid
- transferred fragment converted to dsDNA
- donor DNA inserted by double crossover
13
Q
Hfr Mapping
A
- 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
14
Q
The transfer of genes from Hfr donor as a function of time
A
- 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
15
Q
Insertion of F plasmid-orientation
A
- the F plasmid can insert at multiple sites and in either orientation
- this dictates the order of genes that will be transferred during conjugation
16
Q
Specialized Transduction
A
- bacterial chromosome with phage DNA
- induction of the lytic cycle
- prophage incorrectly excised from the bacterial chromosome
- phage DNA incorporating some bacterial genes
- phage replicates
- bacterial cell is lysed, phages are released
- phage infects new host cell
- phage incorporated into chromosome
- host cell acquires phage DNA and genes from previous host
17
Q
Generalized Transduction
A
- occurs during lytic cycle
- host DNA is degraded and bits are mistakenly packaged along with the phage DNA
- host DNA can be transferred to new host
- not very efficient since only a single phage is getting packaged with the DNA from the host
- useful way to move around mutations
- common transducing phage include P1, P22, mu
18
Q
Insertion Sequences
A
- encodes a transposon flanked by inverted repeats
- transposases duplicate a target sequence and inserts the element
- insertion sequences insert themselves randomly
- IS elements can promote recombination
19
Q
Transposons
A
-like insertion sequences but they have a selectable marker
2 types
-non composite
-composite
20
Q
Composite Transposons
A
-contain 2 copies of insertion sequence flanking genes for antibiotic resistance
21
Q
Non-composite transposons
A
-do not terminate with IS elements but contain internal inverted repeats
22
Q
Regulation of transposition (7)
A
- unregulated transposition would be lethal
- occurs upon integration to a naive host or follows replication
- transposase binds poorly to fully methylated DNA
- hemimethylation occurs at replication and allows transposase to act
- transposase preferentially acts in cis
- unstable and does not accumulate to high levels
- binds DNA sequences as soon as its made, more likely to binds its own site
23
Q
Barbara McClintock
A
- reported genetic element Ac and Ds that could control the activity of genes
- in 40s, developed notion of gene regulation
- stopped reporting her results in 50s because geneticists didnt get it
- lac operon opened doors for her to explain her work in 60s
24
Q
Transposon Mutagenesis
A
- useful way to create mutants (random insertion ensures that you can inactivate all genes)
- transposition occurs at low frequency
- ways to deliver a transposon: encode transposon on a plasmid or phage that cannot replicate in the recipient
25
Q
Suicide Plasmid
A
- conjugative plasmid with a R6K origin of replication-required Pi protein for replication
- Pi protein supplied by temperate phage
- plasmid contains a transposon with antibiotic selection
- mate donor (where plasmid replication is permitted) to recipient (plasmid cannot replicate)
- select for antibiotic resistance due to transposon hopping
