Transposable genetic elements Flashcards by Yasmin Yau

what is a replicon

DNA that is replicated in a cell

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examples of replicons

chromosome

plasmid

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what are transposable elements

DNA sequences which can ‘hop’ from a site on one replicon to a site on another (or same) replicon

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what did transposable elements used to be called

Insertion Sequences (IS) or transposons (Tn)

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what is the simplest are smallest transposable elements

insertion sequences

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how many insertion sequences are there in a bacterial chromosome

May be multiple copies of IS in a bacterial chromosome

Used to “type” bacteria

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what is a transposase

Code only for the peptide responsible for their transposition

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do transposases have an advantage

do not confer any particular phenotypic trait, (don’t have any advantage)

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what are ITRs

inverted terminal repeats

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what do ITRs do

flank the coding region

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what do most transposable elements have

an ITR

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what is the IS1 structure

host DNA
inverted repeats
base pairs
central region ‘base pairs’ encodes transposase

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what is the target site in transposition of IS elements

site of integration

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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)

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what does transposition of IS elements create

Small direct repeats (~5 bp) flanking the target site are created

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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

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what is a composite transposon

Code for selectable marker gene(s)

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why are new composite transposons readily formed

As IS may be in either orientation

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what flanks marker gene(s) in composite transposon

IS elements

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what is the function on transposon carried by

- one may be defective

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what are selectable marker examples

lux genes (glows)
antibiotic resistance
ability to metabolise certain sugars

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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

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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

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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

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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

Transposable genetic elements Flashcards

(62 cards)