Genet 270 lec 9- transposition
What are transposons?
Transposons are mobile segments of DNA that can “hop” or transpose from one location in the genome to another.
🧠 Memory Tip: Think “Trans-posons” = “Transfer positions” (DNA pieces that change position).
Who discovered transposons and in what organism?
Barbara McClintock discovered them in corn (maize) in the 1950s.
🧠 Memory Tip: “McClintock’s Corn Hops” — imagine kernels jumping on a cob.
How is transposition different from homologous recombination?
Transposition is RecA-independent, meaning it doesn’t require the RecA protein or homologous DNA sequences.
Homologous recombination requires RecA and extensive sequence similarity between DNA regions.
🧠 Memory Tip: “Transposons travel alone — no RecA, no homology, no help.”
Are transposons found only in bacteria?
No — transposons are now known to exist in all organisms, from bacteria to humans.
🧠 Memory Tip: “Jumping genes jump everywhere.”
What is the biological importance of transposition?
It enables gene transfer between bacteria even when little or no DNA sequence homology exists, promoting genetic diversity and evolution.
What are the two major mechanisms of transposition?
Conservative (Cut-and-Paste) transposition
Replicative transposition
How do conservative and replicative transposition differ?
Conservative (Cut-and-Paste): The transposon is excised from the original site and inserted into a new site → only one copy exists before and after.
Replicative: The transposon is copied into the new site while retaining the original → results in one or two copies after transposition.
🧠 Tip: “Cut = one copy, Copy = two copies.”
What is site-specific recombination in the context of transposition?
It refers to transposons that insert at specific DNA sequences. Though often drawn as free-floating, in reality they likely remain attached to the original DNA while scanning for a target site in the new DNA.
🧠 Tip: Think of it as “anchored scanning” — still tethered, just looking for a landing pad.
What do all transposons have in common?
All transposons share inverted repeat sequences (IRs) at their ends and contain a transposase gene (tnp) inside, which catalyzes the transposition process.
🧠 Tip: “IRs on the ends, tnp in the middle” → think of it like DNA “bookends” around a “scissors enzyme.”
What enzyme do transposons encode, and what is its function?
They encode transposase (tnp), which recognizes the inverted repeats and cuts and joins DNA to move the transposon to a new site.
🧠 Tip: “Tnp = transport enzyme; transposase transfers.
What surrounds a transposon after insertion into DNA, and why?
Each transposon becomes flanked by short direct repeats of the target DNA. These direct repeats are not part of the transposon itself, but a byproduct of the insertion process.
🧠 Tip: “Inverted inside, direct outside” — IRs belong to the transposon; DRs belong to the host.
What are insertion sequence (IS) elements?
IS elements are the smallest transposons, typically 750–2000 base pairs long. They consist of inverted repeats at each end, a transposase gene, and regulatory sequences, but no selectable marker (no antibiotic resistance or other gene).
🧠 Tip: “IS = It’s Small” — only the essentials: IRs + tnp + regulation.
What do IS elements lack compared to other transposons?
They lack selectable markers (like antibiotic resistance genes). They only code for transposase and its regulators, not for extra traits.
🧠 Tip: “IS elements are minimalist — no extras, no markers.”
How were IS elements first discovered?
The IS3 element was found through mutations in the gal genes that had unusual traits:
Highly polar,
Could not revert,
Transferred gal⁺ plasmids became gal⁻ after acquiring the mutation,
The mutated plasmids were larger than wild-type ones.
These clues indicated DNA insertion events.
How common are IS elements in bacteria?
They are nearly universal in bacteria. For example, E. coli contains several different IS elements in its genome.
🧠 Tip: “If it’s bacterial, it’s got IS.”
What are non-composite transposons?
Non-composite transposons are transposable elements that have:
Short inverted repeats (IRs) at each end (but not IS elements),
A transposase gene,
A regulatory gene for transposase activity, and
Additional genes, often antibiotic resistance (Abᴿ) genes.
🧠 Tip: “Non-composite = No IS parts, but still carries resistance.”
How do non-composite transposons differ from composite transposons?
Composite transposons are flanked by IS elements at both ends.
Non-composite transposons have only short inverted repeats, not IS elements, but still contain functional genes like Abᴿ.
🧠 Tip: “Composite = IS on each side; Non-composite = short IRs only.”
What are composite transposons?
Composite transposons are larger transposable elements that consist of two insertion sequence (IS) elements—in the same or opposite orientation—that bracket one or more additional genes, often antibiotic resistance (Abᴿ) genes.
🧠 Tip: “Composite = composed of 2 IS + 1 cargo gene.”
What is special about the IS elements in a composite transposon?
The flanking IS elements can transpose independently (by themselves) or together with the entire composite transposon as a single unit.
🧠 Tip: “The IS can go solo or take the whole crew.”
How can composite transposons affect plasmids?
When a composite transposon inserts into a plasmid, the DNA between the inverted repeats (including other genes or plasmid regions) can also transpose, enabling plasmid DNA to move between cells.
🧠 Tip: “Composite on a plasmid = portable plasmid parts.”
What is a suicide vector and what is it used for in transposition assays?
A suicide vector (or plasmid) is a DNA molecule that cannot replicate in the host cell. It carries a transposon with a selectable marker (e.g., antibiotic resistance gene) used to detect transposition events.
🧠 Tip: “Suicide vector = one-time delivery—dies unless it jumps.”
How does a transposition assay using a suicide vector work?
Introduce the suicide vector into the bacterial host.
Plate the bacteria on antibiotic-containing media.
Only cells where the transposon has jumped (transposed) into the chromosome will survive.
This process is known as transposon mutagenesis.
🧠 Tip: “Jump or die” — growth = successful transposition.
What is transposon mutagenesis?
It’s a genetic technique that uses a transposon’s random insertion to inactivate genes and identify their function based on the resulting mutant phenotype.
🧠 Tip: “Mutagenesis by jumping genes.”
What is the purpose of a mating-out assay in transposition studies?
It’s used to detect transposition by observing the acquisition of a selectable marker (e.g., antibiotic resistance) on a conjugative plasmid after mating between bacteria.
🧠 Tip: “Mating-out = marker moves out.”
Describe the setup of a mating-out assay.
Donor: has a plasmid carrying an Ampᴿ transposon.
Recipient: has a Kanᴿ transposon on its chromosome.
After mating, Ampᴿ + Kanᴿ exconjugants indicate possible transposition of Kanᴿ from the chromosome onto the plasmid.
🧠 Tip: “Amp on plasmid, Kan on chromosome — look for both together.”
How is transposition confirmed in a mating-out assay?
Use the Ampᴿ, Kanᴿ exconjugants as donors in a second mating with Ampˢ, Kanˢ, transposon-free recipients.
Exconjugants that become both Ampᴿ and Kanᴿ confirm that Kanᴿ transposed onto the plasmid, proving a transposition event.
🧠 Tip: “Two matings to prove the jump.”
What does hybridization analysis detect in transposition studies?
It detects new transposon insertions in plasmid DNA by identifying regions that fail to hybridize (non-homologous DNA) with the original parent plasmid.
🧠 Tip: “If it doesn’t match—it’s a new transposon patch.”
How is hybridization analysis performed?
Obtain plasmids (e.g., from a mating-out assay).
Denature DNA and allow single strands to hybridize with the parent plasmid DNA.
Regions where a new transposon has inserted will not hybridize, forming loops.
How are new transposon insertions visualized in hybridization analysis?
The non-hybridized single-stranded loops are observed using electron microscopy (EM), where they appear as looped-out DNA regions—a direct sign of new transposon DNA.
🧠 Tip: “Loop = new landing site.”
What is the end result of a transposition event?
The transposon becomes inserted between two nucleotide pairs in the recipient DNA molecule, creating a new integration site in the genome.
🧠 Tip: “Every hop ends with an insertion.”
What DNA features are required for transposition?
Inverted repeats (IRs) at both ends of the transposon, recognized by transposase.
Low or variable target specificity — transposons can insert into many different DNA sites.
🧠 Tip: “IRs guide, target’s wide.”
What happens to the target DNA when a transposon inserts?
The insertion always produces a duplication of a short nucleotide sequence at the target site (called a target site duplication).
This duplication flanks the inserted transposon and is a hallmark of transposition.
🧠 Tip: “Jump in → target twins.”
What is replicative transposition, and which transposons use it?
Replicative transposition is a mechanism where the transposon is copied during transfer, so a new copy inserts into the target DNA while the original remains in place.
It is used by non-composite transposons, such as Tn3.
🧠 Tip: “Replicative = copy-and-paste; Tn3 likes to clone itself.”
What enzymes and sites are required for replicative transposition?
Transposase: catalyzes the transposition event (cutting/joining DNA).
Resolvase: mediates site-specific recombination to separate joined DNA molecules.
Res site: the specific DNA site where resolvase acts.
🧠 Tip: “Transposase moves, resolvase resolves.”
What is the overall outcome of replicative transposition?
A replicated copy of the transposon is inserted into a new DNA site, leaving the original in place, resulting in two copies of the transposon per event.
🧠 Tip: “One in, one stays — replication pays.”
What happens to the transposon sequence during replicative transposition?
The original transposon is duplicated — one copy stays at the original site, and a new copy inserts at the new target site.
🧠 Tip: “Copy here, copy there — replicative plays fair.”
What is a cointegrate, and when does it form?
A cointegrate forms during replicative transposition when the donor and recipient DNA molecules become temporarily fused into one large molecule.
It is later resolved by resolvase acting at the res site, separating the DNAs but leaving a copy of the transposon in each.
🧠 Tip: “Cointegrate = combined DNAs during the copy step.
What is nonreplicative transposition?
Nonreplicative (or conservative) transposition occurs by a cut-and-paste mechanism in which the transposon is physically excised from the donor DNA and inserted into a new target site, without duplication.
🧠 Tip: “Cut it out, paste it in — no copies left behind.”
What distinguishes nonreplicative from replicative transposition?
Replicative: transposon is copied, both donor and recipient end up with one copy.
Nonreplicative: transposon is cut from donor and moved directly to new site — only one copy exists after.
🧠 Tip: “Replicative = copy-and-paste; Nonreplicative = cut-and-paste.”
Which transposon was used to prove nonreplicative transposition, and by whom?
Bender and Kleckner (1986) proved nonreplicative transposition using the Tn10 transposon.
🧠 Tip: “Tn10 was the 10/10 proof for cut-and-paste.”
How did Bender and Kleckner design their experiment?
They created a heteroduplex Tn10 with:
One strand carrying a lacZ⁻ mutation,
The other carrying the wild-type lacZ⁺ gene.
This setup allowed them to track which DNA strand moved during transposition.
🧠 Tip: “Two lacZ versions—see which one jumps.”
How was transposition detected in Bender and Kleckner’s experiment?
After transposition, only one lacZ version (not both) appeared in the recipient DNA, showing physical movement without replication.
Lactose utilization was tested with X-gal, which turns blue when the lacZ⁺ gene is active.
🧠 Tip: “Blue = active jump; white = no move.”
What were the expected results for replicative vs. nonreplicative transposition in Bender & Kleckner’s Tn10 experiment?
Replicative: Cells receive both lacZ⁺ and lacZ⁻ copies → colony contains mixed Lac⁺/Lac⁻ sectors.
Nonreplicative: Each cell gets only one (lacZ⁺ or lacZ⁻) → colony is uniformly Lac⁺ or Lac⁻.
🧠 Tip: “Copy = mixed colors; Cut = pure colors.”
What result did they actually observe, and what did it prove?
They observed sectored (mixed Lac⁺/Lac⁻) colonies, indicating both lacZ⁺ and lacZ⁻ copies were present → replicative transposition had occurred.
🧠 Tip: “Blue-and-white = replicated right.”
What is one major genetic consequence of transposition?
Mutation via insertional inactivation — when a transposon inserts into a gene, it disrupts its coding sequence, producing a loss-of-function mutation.
🧠 Tip: “Jump in → gene off.”
How can deletions and inversions arise from transposition?
A second transposition can occur to another site within the same circular DNA molecule.
Recombination between the two transposons (via resolvase or RecA) can produce either a deletion or an inversion, depending on their orientation.
🧠 Tip: “Same direction → deletion; Opposite → inversion.”
What determines whether recombination between transposons causes a deletion or inversion?
The relative orientation of the transposons:
Direct (same) = deletion of intervening DNA.
Inverted (opposite) = inversion of intervening DNA.
🧠 Tip: “Parallel = delete; Opposite = flip.”
What is unusual about the replication of Phage Mu?
Short answer:
Phage Mu replicates its DNA by transposition rather than standard viral DNA replication.
Expanded explanation:
Phage Mu is a transposable bacteriophage, meaning its life cycle depends on transposition events.
It inserts randomly into the host genome and replicates by duplicating itself through transposition, not by using a separate viral replication origin.
Each new copy of Mu is generated when its DNA transposes to a new site in the bacterial chromosome during infection.
This mechanism causes multiple insertions and sometimes host mutations—which is unique among phages.
🧠 Tip: “Mu multiplies by moving.”
How would you determine whether a newly discovered transposon integrates randomly into DNA?
Short answer:
Map multiple independent insertion sites in the genome to see if they are distributed randomly or show sequence bias.
Expanded explanation:
You could introduce the transposon into a bacterial population, isolate many independent insertion mutants, and sequence or map the insertion sites.
If insertions occur throughout the genome without pattern, it’s random.
If they cluster near specific sequences or features (like promoter regions, GC-rich sites, or consensus motifs), then integration is site-preferential.
Techniques: PCR mapping, Southern blot, or whole-genome sequencing of insertion junctions.
🧠 Tip: “Random = everywhere; Specific = somewhere.”
How did transposon Tn3 and similar transposons spread throughout the bacterial kingdom?
Short answer:
Through horizontal gene transfer, especially via plasmids and conjugation.
Expanded explanation:
Transposons like Tn3 are often located on conjugative plasmids, which can transfer between bacteria during mating (conjugation).
Once inside a new host, Tn3 can transpose into that genome or onto other plasmids.
Over time, this mobile + transferable combination allowed Tn3 to spread widely across bacterial species and genera.
Other vehicles like bacteriophages can also facilitate indirect transfer.
🧠 Tip: “Tn3 travels by plasmid passport.”