Transposons I

Question 1

WHAT IS A TRANSPOSON?
- what is it? Other names? Found in?

Answer 1

1 * A segment of DNA or a genetic element THAT MOVES TO NEW CHROMOSOMAL LOCATIONS = A NEW LOCATION IN THE GENOME, BY A PROCESS NAMED TRANSPOSITION.

2 * other names - controlling elements, cassettes, jumping genes, mobile genes, mobile genetic elements & transposons

3 * transposons are found in BACTERIA, FUNGI, PLANTS, ANIMALS AND VIRUSES.

Question 2

WHAT IS TRANSPOSITION?

Answer 2

1 * A SPECIFIC FORM OF GENETIC RECOMBINATION that MOVES certain genetic elements – transposons – from one DNA site to another.

2 * transposition CAN CAUSE MUTATION by insertion into other genes or causing chromosomal rearrangements

Question 3

Discovery of transposable elements: 7

Answer 3

1 ▪ Barbara McClintock - American scientist

2 ▪ 1940s, McClintock studied coloured kernels of maize and found that colouration is linked to two genetic loci called ! Ac and Ds !

3 ▪ Predicted that Ds IS A MOBILE GENETIC ELEMENT THAT CAN TRANSPOSE

4 ▪ In non-coloured kernels, Ds is located within a colour gene.

5 ▪ When Ds moves out of the colour gene, the gene becomes active and we observe the colour.

6 ▪ This happens at random and not in all cells ➔ colour mosaicism.

7 ▪ Nobel prize in Physiology or Medicine in 1983

Question 4

Acceptance of McClintock’s transposon theory came with the discovery of BACTERIAL TRANSPOSONS:

Detection of Transposable elements in E. coli = 3

Answer 4

1 ▪ Comparing transducing phages λdgal+ (wild-type) and λdgal –

2 ▪ λdgal - contains gal gene from E. coli gal - mutants, which have insertions of a genetic element called IS in the gal gene

3 ▪ λdgal - DNA carrying IS mutation is longer than wild- type λdgal+ DNA

Question 5

Experimental demonstration of transposon presence in a bacterial genome:

Answer 5

*Electron micrograph of a λdgal+ / λdgal- DNA heteroduplex provides proof that IS mutation is due to insertion of a piece of DNA

1 * mix denatured DNA of wild- type and mutant phage

2 * hybrid DNA molecules renature

3 * single-stranded loop corresponds to inserted IS element in mutant

Question 6

Classification of transposons
By Kingdom, Structural, Mechanistic, Outcome for the genome:

Answer 6

1 * By kingdom
– Prokaryotic, Eukaryotic

2 * Structural
– Simple = non-composite transposons, composite transposons

3 * Mechanistic
– Cut and paste, copy and paste, reverse transcription
– DNA transposon, class II transposons ➔ all bacterial transposons, also found in eukaryotes
– Retrotransposon, class I transposons: viral, non-viral➔eukaryotes

4 * Outcome for the genome
– Movement: without duplication = conservative, with duplication = replicative

Question 7

The simplest transposon:
Insertion Elements or Insertion Sequences (IS)

(size)?

Answer 7

▪ simplest transposons are called Insertion Sequences or IS elements

▪ ~ 1kb

Question 8

IS elements belong to the simple transposons =

Answer 8

“conservative transposons“

Question 9

IS Element Structure (4):

Answer 9

1 ▪ Each IS element has a different sequence but all have the same overall structure.

2 ▪ Ends composed of SHORT INVERTED REPEATS - usually 15-25 bp long

3 ▪ Between ends is TRANSPOSON GENE ➔ IS ELEMENTS ENCODE FOR ONE PROTEIN ➔ NEEDED FOR TRANSPOSITION.

4 ▪ IS elements are surrounded by direct repeats which are NOT part of the IS element

Question 10

IS ELEMENT

Answer 10

Direct Repeats….. Inverted Repeats… Transposase gene….Inverted Repeats….Direct repeats

IS ELEMENT = inverted repeats,m Transposase gene

Question 11

Many DNA transposable elements have common characteristics: 2

Answer 11

1 * Most DNA transposons are FLANKED BY DIRECT SEQUENCE REPEATS.
- The direct repeats are NOT PART OF THE TRANSPOSON.
- These direct repeats have something to do with the mechanism of transposition.

2 * Many DNA transposable elements also possess TERMINAL INVERTED REPEATS.
- These inverted repeats are PART OF THE TRANSPOSON.

3. Flanking direct repeat, Terminal inverted repeat, ………., Terminal inverted repeat, Flanking direct repeat.

Transposable element: (Terminal inverted repeat……terminal inverted repeat)

Question 12

Understanding Simple or conservative transposons: 5

Answer 12

1 * A SIMPLE TRANSPOSON = “conservative transposon” , or non-composite transposon

2 * Minimum: TRANSPOSASE ENCODING GENE BETWEEN SHORT, INVERTED, REPEATED SEQUENCES 50 bp.
– Enzyme for transposition

3 * CAN ALSO ENCODE OTHER GENES, e.g. resolvase - responsible for resolution of the transfer (see later) or/and antibiotic resistance genes

4 * IS element can be considered the simplest simple transposon

5. Example Tn3

STRUCTURE:
…IR..TRANSPOSASE…RESOLVASE… AM^R (Ampicillin resistance)..IR…
= Transposon Tn3.

Question 13

Understanding Composite transposon: SIMILARITY TO SIMPLE TRANSPOSON AND IS ELEMENTS:

Answer 13

They ALLHAVE PROTEIN CODING GENES FLANKED BY INVERTED REPEAT SEQUENCES that can be RECOGNISED BY TRANSPOSASE ENZYMES.

Question 14

Understanding Composite transposon:

Difference to simple transposons and IS elements: 3

Answer 14

1. Composite transposons are FLANKED BY TWO SEPARATE IS ELEMENTS which may or may not be exact replicas.

– Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to the other is transposed as one complete unit using the ‘external’ IR sequences.

2 * One of the IS elements provides the transposase gene / function

3 * Composite transposons will also often carry one or more genes conferring antibiotic resistance.

Question 15

How does transposition work - Overview

Answer 15

1 * Movement of a transposon occurs through RECOMBINATION between DNA sequences at the very ends of transposons with a DNA sequence at a NEW GENOMIC LOCATION.

2 * WITH OR WITHOUT DUPLICATION of the transposon

Genomic DNA (old site) –> movement without duplication –> excised from old site, and inserted in new site.

Question 16

Conservative versus replicative transposition

Answer 16

• Conservative – excision and insertion, e.g. cut and paste transposition
• Replicative - new copy generated

look at image slide (14)

Question 17

Cut and paste or conservative transposition - overview

Answer 17

1 * They transpose by EXCISION (cutting) of the transposable sequence from one position in the genome and its INSERTION (pasting) to another position within the genome

2 * The cut-and-paste transposition involves TWO TRANSPOSASE SUBUNITS. Each transposase submit BINDS to the SPECIFIC SEQUENCES AT THE TWO ENDS of transposon. These subunits of transposase protein then COME TOGETHER and lead to the EXCISION of transposon.

3 * This excised ‘Transposon-Transposase Complex’ then gets INTEGRATED to the target recipient site. In this manner, the transposon is cut from one site and then pasted on other site by a mechanism mediated by transposase protein

Question 18

Cut-and-Paste Transposons: Examples in prokaryotes are

Answer 18

IS-elements, Tn10

• also in eukaryotes

Question 19

Cut-and-Paste Transposons

• Consequence for the genome:

Answer 19

transposon number does NOT INCREASE.

A direct repeat duplication remains at the donor site.

Question 20

Cut and paste transposition mechanism: 3

Answer 20

• Also called NON-REPLICATIVE transposition

➔transposes without making a new copy

➔transposon number does not change in the genome

Question 21

Cut and paste transposition mechanism STEPS =7

Answer 21

1) The transposase gene is transcribed. The resulting mRNA is translated to synthesise the transposase protein

2) Multimers of the transposase bind to the inverted repeats of the donor DNA

3) Transposase proteins interact forming a SYNAPTIC OR PAIRED END’ COMPLEX with the Donor DNA.

4) Transposases make DOUBLE-STRANDED CUT in the DONOR DNA at the ends of the transposon

4a) Transposon is EXCISED from donor DNA

5) Transposase makes a STAGGERED CUT in
the RECIPIENT / target DNA.

6) Each 3’-END of the DONOR DNA is then COVALENTLY JOINED to an overhanging 5’-END of the RECIPIENT DNA.

6a) This leaves a GAP between the transposon 5’-end and the recipient DNA’s 3’-end

7) DNA POLYMERASE FILLS IN in the short, overhanging sequences
➔ results in a SHORT, DIRECT REPEATS on the sides of the transposon insertion in the recipient DNA

Question 22

Cut and paste transposition mechanism.. SIMPLIFIED

Answer 22

1. mRNA transposase IR Transposase gene IR
2. binding to multimer of transposase
3. synaptic complex
4. DNA cleavage of both strands

4a. excised transposon

5. Target DNA
   5’ to 3’ 3’OH
6. DNA strand stransfer
   6a.
7. DNA-repair synthesis to fill gaps.
   Ligation of nicks
   Element in new DNA location

Question 23

UNDERSTANDING:Cut and paste transposons are surrounded by short direct repeats = 5

Answer 23

1 ▪ At the site of insertion, IS element and many other transposons are flanked by very short direct repeats➔not part of transposon

2 ▪ During cut and past transposition: Insertion requires NICKING of the two recipient DNA strands at the insertion site

3 ▪ The nicks are SEPARATED by a FEW BASE PAIRS

4 ▪ This generates TWO SHORT COMPLEMENTARY SINGLE STRANDED DNAs stranded DNAs from the insertion site surrounding the transposon

5 ▪ Single stranded DNAs will be FILLED IN ➔ now a direct repeat flanking the transposon

SLIDE 19

Question 24

What are Copy and paste or Replicative Transposons?

Answer 24

• Replicative transposons transpose by a mechanism which involves replication of transposable sequence, i.e. they make a TRANSPOSON COPY.

Question 25

Copy and paste or Replicative Transposons PROCEDURE:

Answer 25

1 * The copy of the transposon is INSERTED into the recipient / target site

2 * The original transposon REMAINS located in the donor site

3 * The consequence for the genome is a DUPLICATION of the transposon ➔ the number of transposons increases in the genome

Question 26

Copy and paste or Replicative Transposons:

Example in prokaryotes:

Answer 26

Tn3

Question 27

Copy and paste or Replicative Transposons:

Consequence for the genome

Answer 27

1. Both, the donor and the recipient DNA molecule are
   having a transposable sequence each, after transposition.
2. Both transposons can transposase again ➔ this could lead to a large INCREASE IN TRANSPOSON NUMBERS IN THE GENOME

Question 28

Copy and paste or Replicative transposition of Tn3:
Proposed by who?

4 steps? RESULTS?

Answer 28

Mechanism proposed by James A. Shapiro in 1979:

1) Transposase makes FOUR SINGLE-STRAND CLEAVAGES
– one on each strand of the DONOR DNA
– one on each strand of the TARGET DNA

2) LIGATION of the donor and target DNA leads to formation of a COINTEGRATE also called a Shapiro intermediate or a THETA FIGURE after the Greek letter:

2a) The cointegrate has two regions made up of SINGLE STRANDED DNA. These regions represent the two strands of the ORIGINAL TRANSPOSON.

3) Using the single stranded DNA as template, DNA REPLICATION synthesises the second strand.
* ➔ transposon is replicated = two copies (original and new)

4) The donor and target DNAs are separated by an enzyme called RESOLVASE

• Resolvase recognises and binds to IRS or RES elements in the transposon and executesSITE SPECIFIC RECOMBINATION between the old and the new transposon copy
• This results in TWO DNAs, ONE WITH THE OL AND ONE WITH THE NEW TRANSPOSON insertion site.

Question 29

Transposition can increase genome size: EXPLAIN (3)

Answer 29

• Copy and paste or Replicative transposition creates a new transposon in addition to the original transposon
• This increase the amount of DNA in a genome
• If this occurs many times, the increase in DNA can be significant.

Question 30

Understanding The C-value Paradox:

SOLUTION?

Answer 30

• The term C-values WERE CONSIDERED AS A MEASURE FOR GENOME SIZE.
  It was thought that C-values (genome size) reflect the complexity of genomes, i.e. how many genes the genomes contain.

– If this is correct, we would expect that more complex organisms have bigger genomes.

– This is not necessarily true, for example some salamander genomes may contain 40 x more
DNA than human genomes➔C value paradox

SOLUTION?
- the PRESENCE OF NON-CODING DNA in genomes: Most of eukaryotic DNA is non-coding and hence does not reflect genes. The human genome has less than 2% protein encoding regions

– A large proportion of this non-coding DNA can be made up of transposons

Question 31

UNDERSTANDING BEFORE/AFTER Transposon insertions can cause mutations:

Answer 31

Before transposition:
The target gene is active
promoter -> gene coding region – transposon inserted

After transposition:
1) The target gene may be inactive
promoter -> no transcription or transposon

2) The target gene may have altered transcription levels
- stronger transcription
- weak or no transcription
or transposon -> gene coding region

3) The transposition may have no effect
- promoter; 1. transcription or gene coding region -> transposon

Question 32

Transposition of an IS element can create a new composite transposon

Answer 32

IS element1…Gene A…Gene B……(transposition) IS element 2 inserteted

—> then : CREATES A NEW COMPOSITE TRANSPOSON

IS element 1…..Gene A… Gene B…IS element 2….

PAGE 26

Question 33

Bacterial transposons can be localised on plasmids:

Distribution of IS and Tn elements in E. coli …SLIDE 27

Answer 33

IS1 : 5 – 8 copies on chromosome

IS2 : 5 copies on chromosome, 1 copy on F PLASMID

IS3 : 5 copies on chromosome, 2 copies on F PLASMID

IS elements can jump from one location to another, eg chromosome to an F plasmid

Distribution of IS elements and Tns in one F plasmid

Because IS elements are regions of identical sequence, they are sites where RECOMBINATION occurs.

Question 34

Bacterial plasmids: self-replicating circular genetic elements that are separate from the bacterial chromosome

Answer 34

**Transposons play a major role in generating R plasmids

RESPONSIBLE FOR THE MULTI-DRUG RESISTANT BACTERIA – SERIOUS PROBLEM IN HOSPITALS

Question 35

Bacterial plasmids: self-replicating circular genetic elements that are separate from the bacterial chromosome

• e.g. F plasmid, R plasmids (resistance or R factors)

Answer 35

• e.g. F plasmid, R plasmids (resistance or R factors)

1 * resistance-transfer section mediates conjugation, contains Tn10

2 * resistance-determinant section has cluster of resistance genes on 3 Tns, can transpose as a whole or each Tn can transpose independently

3 * Tn3 (simple) is within Tn4 (composite)

Question 36

Examples of genetic determinants found on transposons - Tn3

Answer 36

ampicillin resistance

Question 37

Examples of genetic determinants found on transposons - Tn5

Answer 37

kanamycin resistance

Question 38

Examples of genetic determinants found on transposons - Tn9

Answer 38

chloramphenicol resistance

Question 39

Examples of genetic determinants found on transposons - Tn10

Answer 39

tetracycline resistance

Question 40

Examples of genetic determinants found on transposons - Tn501

Answer 40

mercury resistance

Question 41

Examples of genetic determinants found on transposons - Tn951

Answer 41

lactose utilization

Question 42

Examples of genetic determinants found on transposons - Tn1681

Answer 42

enterotoxin production

Question 43

Examples of genetic determinants found on transposons - Tn4451

Answer 43

chloramphenicol resistance
- C.perfringens

Question 44

Summary

Answer 44

1 * Bacterial IS elements - first transposable elements identified at the molecular level. They encode only transposase.

2 * IS element are very simple DNA transposons belonging to the simple or conservative transposons so do some Tn transposons (Tn3)

3 * Composite transposons are flanked by two IS elements, one of the IS elements encodes for the transposase. Example: Tn10

4 * Tn transposons are flanked by terminal repeat sequences and confer extra properties on their host cell such as antibiotic resistance

5 * Two modes of transposition – conservative = cut and paste and replicative = copy and paste

6* Tns insert into plasmids such as R plasmids and can be transferred by conjugation to recipient bacteria➔multiply-antibiotic-resistant pathogens

7 * Transposition can increase genome size or alter gene transcription or create new composite transposons.