MBB 331 Lecture 10

Learning Objective 1

Describe how a transposon creates a duplicated target sequence

What happens to the target site after transposition

The target sequence becomes duplicated

What enzyme performs transposition

Transposase

What type of cuts are made in the target DNA?

Staggered cuts

Why do staggered cuts create target-site duplications

Gap filling after insertion copies the target sequence on both sides of the transposon

original target sequence now appears where

on both sides of the transposon

Step One

transpose cuts where arrows are

Step Two

overhangs produced by cuts

Step Three

Insert transposon between overhangs

Step four

fill in gaps to make it double stranded

Learning Objective 2

Understand the importance of genes carried within transposable elements

What gene is essential in most transposons

Transposase

What happens if transposase is missing

The transposon cannot move

What additional genes are often found in composite transposons

Antibiotic resistance genes

What is a composite transposon

has transposons on either end of DNA sequence

Why are composite transposons important medically

They spread antibiotic resistance genes between bacteria

Why can Ac transpose independently

Ac contains a functional transposase gene

Why can't Ds transpose independently

Ds lacks a functional transposase and depends on Ac

Learning Objective 3

Understand the structure of transposons

What is the simplest bacterial transposon?

Insertion sequence (IS)

What does an IS contain

Transposase gene and inverted repeats

If IS was single stranded, what would happen

base pairs with itself

What are inverted repeats (IRs)

Short DNA sequences at opposite ends that are reverse complements

What is a composite transposon

Two IS elements flanking additional genes

What genes are commonly found between the IS elements

Antibiotic resistance genes

What is LTR retrotransposon

mobile genetic element that moves to a new location in the genome by making an RNA intermediate and has directed repeats

What are Non-LTR retrotransposon

no repeats

Learning Objective 4

Site-Specific Recombination

What is site-specific recombination

DNA exchange occurring between specific recombination sites

Does it require extensive homology

No

What enzyme performs site-specific recombination

Recombinase

What genomic feature is required

Matching recombination sites

Where do we want to insert phage recombiation site

into the bacterial recombination sites

How do we know recombination is occuring

through crossover (X) between the two sites

What makes up the recombination site

two inverted repeats (two blue arrows facing each other) and a core (yellow arrow moving 5-3 facing right)

How do the inverted repeats look like

same sequence on either side different directionally

When does inversion occur

when the inverted sequences (FRT) face each other (different directions)

When does insertion/deletion occur

When FRT are facing the same way

When the pathway goes straight througha molecule it cause

deletion

What are the steps in recombination by tyrosine

theres 2 cleavage steps and 2 recombination steps

Learning Objective 5

Cut-and-Paste Transposition

Is cut-and-paste transposition replicative

No

What happens to the donor site

The transposon leaves, creating a break

What enzyme performs the reaction

Transposase

Step One

cut donor site on either side of transposon and expose 3' OH

Step Two

3' OH attacks opposite strand, generating hairpin low

Step Three

Transposase cleaves hairpin generating 3'OH

Step Four

OH attacks and creates strand transfer complex

Step 5

Replication and ligation to fill and seal gaos

Learning Objective 6

Replicative Transposition

What happens with the donor

remains unchanged and is copied into recipent

Donor and Recipient have what

the same copy of transposon

Step One

Phosphodiester bond is cleaved on each side of the transposon exposing 3'

Step Two

OH attatck two strands of target DNA creating strand transfer complex

Step Three

Use 3' ends as primer and create a cointegrate

Which protein forms the cointegrate

TnpA

Which protein resolves the cointegrate

TnpR (resolvase)

Step Four

cointegrate is resolved by homologous recombination

Learning Objective 7

RNA-Mediated Transposition

What key enzyme is required

Reverse transcriptase

Step 1

have a transposon and transcribe into RNA

Step 2

RNA is translated to proteins such as:

Reverse transcriptase

Step 3

Reverse transcriptase copies the RNA into cDNA

Step 4

Cleavage exposing 3' OH

Step 5

OH attacks, inserts and repairs

What is the difference

extra step creating 2nd strand using cDNA

Learning Objective 8

EP Retrotransposition (LTR)

What does EP stand for

Extra-Chromosomally Primed (LTR) retrotransposition

What primes reverse transcription

tRNA and anneals to transposon to create a dsDNA

Which enzyme inserts the new copy

Integrase will insert DNA transposon copy into DNA target site

What is it closely related to

Retroviruses

Learning Objective 9

TP Retrotransposition (Target-Primed)

What does TP stand for

Target-primed retrotransposition

What happens first

Target DNA is nicked

What primes reverse transcription

The exposed 3' end of target DNA to convert to dsDNA

Examples

LINEs and SINEs

Step One

Cleavage

Step Two

reverse transcription

Step Three

Elongation

Step Four

Second cleavage and DNA duplication of transposon

Step Five

Integration

Learning Objective 10

Ac/Ds Elements of Maize

What does Ac stand for

Activator.

What does Ds stand for

Dissociation

Is Ds autonomous

No

Why can Ac move by itself

It encodes transposase

Why does Ds require Ac

Ds lacks functional transposase

Pathway Step One

Ds gets inserted into C (colour gene) inactivating it and cause kernel to go white

Step Two

Ac will help get Ds out of C and bring back purple

Learning Objective 11

Cesium Chloride Gradient Assay

What was the purpose of the CsCl gradient experiment

To detect insertion sequences

Why did λgal⁻ DNA have a different density than λgal⁺ DNA

λgal⁻ contained extra DNA from an insertion sequence

What conclusion was drawn from the experiment

Some mutations were caused by insertion of extra DNA rather than point mutations

In eukaryotes what's the three main transposition strategies

Ac/Ds = DNA transposons

LINEs/SINEs = retrotransposons

LTR retrotransposons = retrovirus-like elements

Two types of Non- LTR

Lines and Sines