Transposons

Dr Derakhshandeh

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Mobile Genetic Elements

Transposons or Transposable elements (TEs)

move around the genome

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Transposable elements in prokaryotes

Insertion sequence (IS) elements

Transposons (Tn)

Bacteriophage Mu

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Insertion sequence (IS) elements

Simplest type of transposable element found in bacterial chromosomes and plasmids

Encode only genes for mobilization and insertion

Range in size from 768 bp to 5 kb

IS1 first identified in E. coli’s glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome

Ends of all known IS elements show inverted terminal repeats (ITRs)

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Integration of IS element in chromosomal DNA

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Three different mechanisms for transposition

Conservative transposition

Replicative transposition

Retrotransposition

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Three different mechanisms for transposition

Conservative transposition: The element itself moves from the donor site into the target site

Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate

Retrotransposition: The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted (cDNA)

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Conservative transposition

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Replicative transposition

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Retrotransposition

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Generation of short direct repeats flanking the newly inserted element

This results for a staggered cut being made in the DNA strands at the site of insertion

common feature of mobile elements

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Transposons (Tn)

Similar to IS elements but are more complex structurally and carry additional genes

2 types of transposons:

Composite transposons

Noncomposite transposons

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Composite transposons

1616IS10R is an autonomous element, while IS10L is non-autonomous

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Composite Transposons

Tetracycline resistance is carried by a transposable element

The transposon is a composite transposon, composed of IS-elements flanking an included sequence, in this case containing an antibiotic resistance gene

IS10R is an autonomous element while IS10L is non-autonomous Composite transposons probably evolved from IS

elements by the chance location of a pair in close proximity to one another. Inactivation of one element by mutation would not harm ability to transpose and would assure continued transposition of the entire transposon

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Noncomposite transposons

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Noncomposite transposons (Tn)

Carry genes (e.g., a gene for antibiotic resistance)

Ends are non-IS element repeated sequences

Tn3 is 5 kb with 38-bp ITRs and includes 3 genes; bla (-lactamase), tnpA (transposase), and tnpB (resolvase, which functions in recombination)

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Examples of DNA-intermediate mobile elements

Insertion Sequences (IS) elements in bacteria

P elements in Drosophila AC/DS (dissociation) elements in maize

AC is a full-length autonomous copy DS is a truncated copy of AC that is non-

autonomous, requiring AC in order to transpose At least seven major classes of DNA

transposons in the human genome (3% of total genome)

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Methods for Generation of Mutant Populations

The most reliable method to ascertain gene function is to disrupt the gene and determine the phenotype change in the resulting mutant individual

Two most popular methods to generate mutants: 1. Insertional mutagenesis 2. Deletion mutagenesis

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Two main methods

1. Transposon insertion

2. T-DNA insertion

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Transposon mutagenesis

Transposable elements or transposons

sections of DNA (sequence elements)

move, or transpose, from one site in the genome to another

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All transposable elements fall into one ofthe following two classes

1. DNA elements

2. Retroelements

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DNA elements

These elements transpose via DNA intermediates such as:

Ac/Ds and Spm in plants, P elements in animals, Tn in bacteria

A common feature of DNA elements is the flanking of the element by short inverted repeat sequences

The enzyme transposase recognizes these sequences, creates a stem/loop structure

excises the loop from the region of the genome The excised loop can then be inserted into another region of the genome

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DNA-Immediate Mobile Genetic Elements

The Short inverted repeats at the ends of the element

These inverted repeats act as the substrates for recombination reactions mediated by the transposase

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Structure and transposition of a transposable element

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Retroelements

transpose via RNA intermediates The RNA is copied by reverse

transcriptase into DNA the DNA integrates into the genome Retroelements are found in all

eukaryotes such as Tos in rice, copia in animals

and Ty1 in yeast

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Retrotransposon transposition

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Retorviruses

The basic structure is an LTR = long terminal repeat which flanks three genes,

A complete retroviruses also contains three genes:

gag = structural gene for capsid Pol = reverse transcriptase env = envelope gene for the virus

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How do we use a transposonfor mutagenesis?

The insertion and excision of transposable elements

result in changes to the DNA at the transposition site

The transposition can be identified when a known DNA sequence or selection markers are inserted within the elements

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Transposomics

EZ::TN Transposomes provide an efficient and reliable method for generating a library of random gene knockouts in vivo

Gene inactivation and examination of the resulting phenotype will identify the function of the interrupted genes

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Transposon-Mediated Homologous RecombinationGene Knockout in Fungi

Hamer et al. 2001. Proc Natl Acad Sci U S A. 2001 24;98(9):5110-5

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T-DNA insertion mutagenesis

T-DNA is a segment of the tumor-inducing (Ti) plasmid of Agrobacterium

delimited by short imperfect repeat border sequences

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T-DNA transfer from Agrobacterium to plant cell

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Temperate bacteriophage Mu (Mu = mutator)

37 kb linear DNA with central phage DNA and unequal lengths of host DNA at each end

Mu integrates by transposition replicates when E. coli replicates During the lysogenic cycle, Mu remains

integrated in E. coli chromosome

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bacteriophage Mu

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The advantages / disadvantage of Mu

The advantages of the use of Mu are: it is not normally found in the bacterial genome therefore there are few problems with homology to

existing sequences in the chromosome; in contrast to most other transposons

Mu does not need a separate vector system since it is itself a vector A wide variety of useful mutants of Mu have been

generated The disadvantage of Mu: it is a bacteriophage and therefore can kill the host

cell

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Drosophila transposons

~15% of Drosophila genome thought to be mobile 2 different classes:

Copia retrotransposons

Conserved, 5-100 scattered copies/genome

Structurally similar to yeast Ty elements Use RNA and reverse transcriptase Eye Color in Drosophila (white apricot wa)

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DTR DTR

ITR(17bp) ITR(17bp)

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P elements

Hybrid dysgenesis, defects arise from crossing of specific Drosophila strains

Occurs when haploid genome of male (P strain) possesses ~40 P elements/genome

P elements vary in length from 500-2,900 bp

P elements code a repressor, which makes them stable in the P strain in male (but unstable when crossed to the wild type female/; female lacks repressor in cytoplasm)

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Ac (activator)/Ds (dissociation) System discovered by B. McClintock (Noble Prize Winner in 1983)

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Ac/Ds System

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Ac/Ds System

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Schematic Diagram of the Ds Donor Site andPossible Transposition Events

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Open arrowheads indicate the 5' and 3' ends of th transposon

The Ds element carries the NPTII gene, which confers resistance to kanamycin (KanR)

and a modified GUS reporter gene (Sundaresan et al. 1995 ) Possible transposition events include the following:

(1) unlinked or loosely linked transposition to the same chromosome;

(2) transposition to a different chromosome; (3) closely linked transposition; and (4) closely linked transposition disrupting theIAAH gene

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Ac/Ds Transposon tagging system

Advantages: Efficient and cost-effective method to generate a large mutant population

Disadvantages: Secondary transposition complicates gene identification

And transposon system is not available in many species

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Transposition elements in Human

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Mobile Genetic Elements and Other Families of Repetitive DNA

The genome is littered with large families of repetitive sequences

have no apparent function in the cell Mobile Genetic Elements Tandemly repeated simple sequence DNAs

Satellite DNAs Short simple repeats (microsatellites)

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LINEs (Long interspersed elements)

LINEs are one of the most ancient and successful inventions in eukaryotic genomes

In humans, are about 6 kb long encode two open reading frames (ORFs) Most LINE-derived repeats are short, with an

average size of 900 bp - 1,070 bp The LINE machinery is believed to be

responsible for most reverse transcription in the genome

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SINEs (Short interspersed elements)

short (about 100-400 bp)

A single monophyletic family of SINEs (ALU) This family is the only active SINE in the human

genome The human genome contains three distinct

monophyletic families of SINEs: the active Alu, and the inactive MIR and Ther2/MIR3

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Identification of a human specific Alu insertion in the factor XIIIB gene

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies

An Alu Insert as the Cause of a Severe Form of Hemophilia A (factor VIII)

Acta Haematol 2001;106:126–129

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