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BACTERIALBACTERIAL TRANSPOSONSTRANSPOSONS

S.FEROSEKHANS.FEROSEKHAN

FNB-41FNB-41

TRANSPOSONS

“Transposable elements”

“Jumping genes”

Mobile DNA

– able to move from one place to another within a cell’s genome

– sometimes a

moves – insertion requires target DNA sequences

copy is made

and

the copy

Transposon

Transposon inverted terminal repeat (ITR)

inverted terminal repeat (ITR)

• In the process, they may

- cause mutations.

- increase (or decrease) the amount of DNA in the genome.

- promote genome rearrangements.

- regulate gene expression.

- induce chromosome breakage and rearrangement.

Discovery of transposons

• Barbara McClintock 1950’s Ac Ds system in maize influencing kernel color unstable elements changing map position promote chromosomal breaks.

• Rediscovery of bacterial insertion sequences source of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation.

These mobile segments of DNA are sometimes called "jumping genes"

There are two distinct types of transposons:

1) DNA transposons -transposons consisting only of DNA that moves directly from place to place

2) Retrotransposons - first transcribe the DNA into RNA and then - use reverse transcriptase to make a DNA copy of the RNA to insert in a new location

Classification of Transposons into two classes

Classification of Transposons into two classes In both cases ds DNA intermediate is integrated into the

In both cases ds DNA intermediate is integrated into the target site in DNA to complete movement

BACTERIAL TRANSPOSONS

● In bacteria, transposons can jump from

chromosomal DNA to plasmid DNA and back.

● Transposons in bacteria usually carry an

additional gene for function other than transposition---often for antibiotic resistance.

● Bacterial transposons of this type belong to

the Tn family. When the transposable elements

lack additional genes, they are known as insertion sequences.

BACTERIAL TRANSPOSONS - TYPES

1. Insertion sequence 2.Composite transposon 3.Tn3-type transposon 4.Transposable phage

1.Insertion sequences

Insertion sequences – IS1 and IS186, present in

the 50-

kb segment of the E. coli DNA, are examples of DNA transposons.

Single E. coli genome may contain 20 of them.

Most of the sequence is taken by one or two genes for transposase enzyme that catalyses transposition.

IS elements transpose either replicatively or conservatively.

IS elements

cont….

Study of E. coli mutations resulting from insertion of 1-2 kb long sequence in the middle of certain genes.

Inserted stretches or insertion sequences – could be visualized by EM.

IS - molecular parasites in bacterial cells.

Transposition of IS is very rare – one in 105-107 cells per generation.

Higher rates result in greater mutation rates.

Bacterial IS element

Bacterial IS element Central region encodes for one or two enzymes required for transposition. It is

Central region encodes for one or two enzymes required for transposition. It is flanked by inverted repeats of characteristic sequence.

The 5’ and 3’ short direct repeats are generated from the target-site DNA during the insertion of mobile element.

The length of these repeats is constant for a given IS element, but their sequence depends upon the site of insertion and is not characteristic for the IS element.

Arrows indicate orientation.

Insertion sequences in E.coli

Elements

Size (bp)

No.of.copies/

genome

IS1

768

8

IS2

1327

5

IS3

1300

1

or more

IS4

1426

1

or more

2.Composite transposons

Bacteria contain composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition:

Composite transposons - are basically the pair of IS elements flanking a segment of DNA usually containing one or more genes, often coding for AB resistance.

They use conservative method of transposition.

2.Composite transposon

Cont…

- Antibiotic resistant gene

- Flank by IS element (inverted or directed repeat)

gene - Flank by IS element (inverted or directed repeat) - Terminal IS can transpose by

- Terminal IS can transpose by in self Ex. Tn5, Tn9, Tn10

3. Tn 3 transposon family

- 5000 bp

- code for Transposase, β-lactamase, Resolvase

- Function of resolvase Decrease Transposase production Catalyse the recombination of transposon

Cont…

ITR ITR transposase resolvase β-lactamase
ITR
ITR
transposase
resolvase
β-lactamase

Tn3 – type transposon --- 5kb

ITR - inverted terminal repeat

• β- lactamase – Resistance gene

4.Transposable phage

Transposable phages – bacterial viruses which tranpose replicatively as a part of their normal infectious cycle.

Integrate into E. coli chromosome at regulatory element

Eg. Mu phage

Transposable phage

ITR ITR Integration and Replication genes Lysis genes Protein coat genes
ITR
ITR
Integration and
Replication genes
Lysis genes
Protein coat
genes

Transposable phage – 38kb

ITR - inverted terminal repeats

Transposable phage - Mu phage

Transposable phage - Mu phage

Mechanism of transposition

Two distinct mechanisms of transposition:

Replicative transposition – direct interaction between the donor transposon and the target site, resulting in copying of the donor element

Conservative transposition – involving excision of the element and reintegration at a new site.

Mechanism of transposition

1. Replicative transposition

Copy of transposon sequence

1. Replicative transposition Copy of transposon sequence Transposase enzyme cut target DNA Transposition Duplication

Transposase enzyme cut target DNA

transposition Copy of transposon sequence Transposase enzyme cut target DNA Transposition Duplication of target sequence

Transposition Duplication of target sequence

Replicative transposition

Replicative transposition

2. Non-replicative (conservative) transposition

- Cannot copy transposon sequence - Transposition by cut and paste model Cut transposon sequence from donor molecule

- Transposition by cut and paste model Cut transposon sequence from donor molecule attach to target

attach to target site Ex. IS10, Tn10

Non-replicative (conservative) transposition

Non-replicative (conservative) transposition

Mechanism of transposition

Mechanism of transposition

Evolution of Transposons

• Transposons are found in all major branches of life.

• It arisen once and then spread to other kingdoms by horizontal gene transfer.

• Duplications and DNA rearrangements contributed greatly to the evolution of new genes.

Cont…

• Mobile DNA most likely also influenced the evolution of genes that contain multiple copies of similar exons encoding similar protein domains (e.g., the fibronectin gene).

• The evolution of an enormous variety of antibiotic resistance transposons and their spread among bacterial species. example of genetic adaptation via natural selection.

Transposons causing diseases

• Transposons are mutagens. They can damage the genome of their host cell in different ways:

1. A transposon or a retroposon that inserts itself into a functional gene will most likely disable that gene.

2.After a transposon leaves a gene, the resulting gap will probably not be repaired correctly.

3.Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication.

Cont…

• Diseases caused by transposons include

-hemophilia A and B -severe combined immunodeficiency -Porphyria -Cancer -Duchenne muscular dystrophy

Applications

• The first transposon was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds).

• Likewise, the first transposon to be molecularly isolated was from a plant (Snapdragon).

• Transposons have been an especially useful tool in plant molecular biology.

• Researchers use transposons as a means of mutagenesis.

Cont…

• To identifying the mutant allele.

• To study the chemical mutagenesis methods.

• To study gene expression.

• Transposons are also a widely used tool for mutagenesis of most experimentally tractable organisms.

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