Transposable genetic elements Flashcards
what is a replicon
DNA that is replicated in a cell
examples of replicons
chromosome
plasmid
what are transposable elements
DNA sequences which can ‘hop’ from a site on one replicon to a site on another (or same) replicon
what did transposable elements used to be called
Insertion Sequences (IS) or transposons (Tn)
what is the simplest are smallest transposable elements
insertion sequences
how many insertion sequences are there in a bacterial chromosome
May be multiple copies of IS in a bacterial chromosome
Used to “type” bacteria
what is a transposase
Code only for the peptide responsible for their transposition
do transposases have an advantage
do not confer any particular phenotypic trait, (don’t have any advantage)
what are ITRs
inverted terminal repeats
what do ITRs do
flank the coding region
what do most transposable elements have
an ITR
what is the IS1 structure
host DNA
inverted repeats
base pairs
central region ‘base pairs’ encodes transposase
what is the target site in transposition of IS elements
site of integration
what happens in transposition of IS elements
Staggered cuts are made in DNA at target site by transposase
IS element inserts
DNA polymerase and ligase fill the gaps
(note—transposase behaves like a restriction enzyme)
what does transposition of IS elements create
Small direct repeats (~5 bp) flanking the target site are created
what can integration of IS element cause
- Disrupt coding sequences or regulatory regions
- Alter expression of nearby genes
- Cause deletions and inversions in adjacent DNA
- Result in crossing-over
what is a composite transposon
Code for selectable marker gene(s)
why are new composite transposons readily formed
As IS may be in either orientation
what flanks marker gene(s) in composite transposon
IS elements
what is the function on transposon carried by
IS
- one may be defective
what are selectable marker examples
lux genes (glows)
antibiotic resistance
ability to metabolise certain sugars
what happens in composite transposons
Two insertion sequences flanking internal DNA
Encode antibiotic resistance genes, etc.
IS can transpose independently, or composite Tn can transpose as a unit
what is the function of P out
can switch on chromosomal genes downstream of insertion site but is also used by Tn10 to regulate transposase activity
what can you insert if have a set of genes without promoter but want to turn them on
P out promoter and know works as this promoter has antibiotic
resistance so will survive
what enzyme is involved in tetracycline resistance
P out
prevents ribosomes binding to translation start on transposase mRNA, decrease transposase synthesis
what do non-composite transposonscarry
Tn carries at least three genes:
Marker gene e.g. antibiotic resistance
Transposase
Resolvase
what is the transposase in non-composite transposon for
for integration
what is the resolvase for in the composite transposon
resolves the co-integrate in the second step of the transposition process via res site
i.e. has 2 separate proteins that allows non-homologous recombination events to occur
what is resolvase
protein
what don’t the Tn3 family have
IS at termini
what do Tn3 family have
long inverted repeats at termini
where are Tn3 found
both Gram-negative and Gram-positive bacteria
where are the Tn insertion sites
they may not always be random
Tn5 and 7 have insertional bias
may favour GC rich
what is Tn insertional bias
At certain points there is some bias, likes having more of these (Tn insertions) than less of them
what does Tn insertions require
long inverted sequences
what are Mariner transposons
Mariner-family transposable elements are a diverse and taxonomically widespread group of transposons occurring throughout the Animal kingdom
what is the mariner transposable element known to yield
a random distribution of insertions
where are mariner elements inserted
at TA dinucleotide pairs in the target sequence and transpose independently of any host factors
what does the insertion of mariner transposons cause
adds another base
what are the types of transposition
conservative
replicative
what happens in conservative transposition
cut-and-paste
what happens in replicative transposition
leaves an original copy and replicates itselfTransposon duplicated during transposition
Results in formation of cointegrate intermediate
what resolves cointegrate
resolvase
what does does resolvase do
cuts four DNA strands, conformational change, put the right ones together
what can go wrong in replicative transposition
may not properly separate, DNA cannot be replicated and the organism dies (catenated DNA)
what are transposons used for
- Tn insertions can be found in large number of sites on the bacterial chromosome
- Tn insertion in a gene usually causes complete loss of function
- phenotype of the insertion mutation is completely linked to antibiotic resistance
what does Tn insertion cause
Disruption of open reading frame
Many Tns cause polar mutations
where can Tn insertions be found
In principle it’s possible to find Tn insertions in any non-essential gene in the genome
how is it possible to transfer a mutation into a new strain using transposons
by selecting for antibiotic resistance
how are transposons made mutant
Tn must be delivered into cell
how is Tn delivered into cell
Often achieved by transformation of plasmid containing Tn sequence
what is needed for Tn delivery into cell
Only feasible if we have an organism that we can transform with high efficiency
e.g. need a high efficiency plasmid
what must happen after plasmid has inserted DNA sequence in transposon delivery
Need to get the plasmid in to insert the DNA sequence, then need to lose the plasmid from the bacteria
what does losing the plasmid from bactiera after insertion allow us to see
Allows us to distinguish cells with Tn on plasmid and Tn in chromosome
what is a good method to use in transposon delivery
use special donor (mobilising) strains & conjugation
where must the plasmid go to in transposon delivery
plasmid with Tn can replicate in donor strain (correct oriV site)
what is used to transfer plasmid into cell in transposon delivery
Conjugation used to transfer the plasmid carrying Tn into target strains (broad host range)
how will the plasmid with Tn replicate in the cell
Plasmid with Tn cannot replicate in this strain cell)
Only cells with Tn in chromosome will be resistant to antibiotic
what do mini transposons have
transposase outside of the transposable element
what happens to a plasmid that is taken from E. coli and put into Pseudomonas
plasmid that carries transposon can be mated into Pseudomonas but can only replicate in E. coli
following conjugation to insert Tn plasmid into Pseudomonas
how are transposants selected for Pseudomonas when plasmid from E. coli insert
transposants selected for on Pseudomonas selective agar supplemented with kanamycin; so only Tn containing Pseudomonas can grow
what do all mutants in Tn library ideally have
a single Tn insertion so any change in phenotype is linked to a single locus
