Chapter 8: Transposition Study Notes

Chapter 8: Transposition

Assays of Transposition

  • Insertion Elements

    • Originally discovered due to their ability to create mutations when they insert into a gene.

    • Convenient Method?

    • If the transposon carries an antibiotic resistance marker, it simplifies selection.

    • However, to confirm if transposition has occurred requires specific methods.

  • Mechanisms for Assaying Transposition

    • Two mechanisms include:

    • suicide vectors

    • mating out assay

  • Suicide Vectors

    • Defined as any DNA, including plasmid or phage DNA, that cannot replicate in a particular host

    • A transposon with an antibiotic resistance gene can be added to a suicide vector for delivery into a host where transposition is analyzed.

    • Requirements:

    • Efficient delivery (conjugation or transduction) is crucial since transposition is rare.

    • Requires integration of the transposon into a replicative DNA molecule such as a chromosome or a plasmid.

  • Evidence of Transposition

    • The appearance of resistant colonies suggests transposition has occurred, but caution is needed as mutations can occur independently.

    • Mating-out Assay

    • A transposon present in a non-transferable plasmid (or chromosome) is not transferred unless it hops into a self-transmissible plasmid.

    • Example: Tn10 (Tetr)

      • Inserted into a small plasmid that cannot self-transfer.

      • The plasmid is used to transform cells containing an F plasmid, allowing the Tn10 to hop into the F plasmid and facilitating transfer.

    • Mixing Recipients:

      • The donor cells mixed with streptomycin-resistant recipient cells will show growth of transconjugates on tetracycline and streptomycin plates.

Identifying the Mechanism of Transposition

  • Questions to Explore:

    • How many gene products are required for Tn3 transposition?

    • Where do these gene products act?

    • Location of the genes for these products on the transposon?

    • Do intermediates of transposition accumulate with inactivation of any gene product?

Tn3 Example: Mechanism of Transposition

  • Initial Steps:

    • Isolate mutations affecting the transposon.

    • Utilize mating-out assay to test the effects of these mutations.

  • Outcome Analysis:

    • No ampicillin-resistant transconjugates:

    • Indicates that mutation prevented transposition.

    • High number of transconjugates:

    • Suggests mutation increased transposition frequency.

Results of Tn3 Mutation Effects

  • Mutations in Inverted Repeats or tnpA ORF:

    • Prevent transposition entirely.

  • Mutations in tnpR ORF:

    • Result in elevated transposition rates and formation of cointegrates.

  • Mutations in resolution sequence (res):

    • Leads to cointegrates but normal transposition rates.

  • Cis vs. Trans-acting Functions:

    • Investigation into which mutations affect functions versus sites.

Utilizing Complementation and Mating-out Tests

  • Experimental Setup:

    • Use a cell containing another Tn3-related transposon in the chromosome, lacking Ampr.

  • Results Observation:

    • Mutations disabling tnpA or tnpR could restore normal transposition, while mutations in IR sequences halted transposition.

    • Mutations in res permitted transposition yet resulted in cointegrates.

Interpretations on Mechanisms

  • Key Takeaways:

    • Mutations in tnpA block transposition due to its role as the transposase enzyme.

    • IR sequence mutations prevent transposition because TnpA acts at these sites for successful transposition.

    • Mutations in tnpR can be complemented and result in higher transposition rates and cointegrates.

    • TnpR performs dual roles:

    • Acts as a repressor of tnpA transcription.

    • Functions as a recombinase that resolves cointegrates by promoting site-specific recombination between the res sites.

Observations on Replicative Transposition (Tn3)

  1. Target DNA short sequences are duplicated during insertion.

  2. Cointegrate intermediates observed where donor and target DNA fuse, containing two copies of the transposon.

  3. Resolution of cointegrates occurs via enzymes like resolvase at the res sequences.

  4. Donor and target DNA each retain a copy of the transposon post-transposition.

Mechanism of Replicative Transposition (Tn3)

  1. Transposase creates ss breaks at junctions between Tn3 and donor DNA, forming staggered ds breaks in target DNA.

  2. Junctions between Tn3 and donor DNA become ligated, allowing integration.

  3. Replication proceeds bidirectionally, resulting in a cointegrate.

  4. Resolution occurs through recombination between res sites in the cointegrate facilitated by resolvase.

Evidence for Other Types of Transposition (Tn10 Example)

  • Differences Noted in Tn10 Mechanism:

    • No cointegrate intermediates formed.

    • Mutants of Tn10 do not accumulate cointegrates, suggesting a distinct mechanism.

    • Unlike Tn3, artificially formed cointegrates rely on the host's recombination system for resolution, lacking a specific resolvase.

    • Both strands of the transposon are mobilized during transposition.

Mechanism of Cut-and-Paste Transposition

  1. Transposase makes ds breaks at the ends of the transposon from donor DNA.

  2. Staggered cuts in the target DNA facilitate integration.

  3. DNA polymerase fills in gaps created by these breaks, duplicating the target sequence.

  4. The donor DNA is ultimately destroyed during this type of transposition.

Comparison: Replicative vs. Cut-and-Paste Transposition

  • Key Comparisons:

    • Tn10: Cut and Paste

    • Replicative: - Resolution leads to two copies of transposon, loss of phage.

    • Cut-and-Paste: - Direct insertion without generating cointegrates.

Similarities Between Replicative and Cut-and-Paste Transposition

  1. Both mechanisms involve the transposon and target integration processes.

  2. Both types can encode functional genes, although they operate differently.

  3. Both exhibit a degree of gene fidelity during transposition.

  4. Cut-and-paste transposition (Tn7) can convert to replicative transposition with a single amino acid change.

Differences:

  • replicative has copy of donor in both strands

  • cut and paste does double stranded break in donor

  • cut and paste has no resolvase because it has no cointegrates