TRANSPOSON MEDIATED MUTAGENESIS

SWETHAPALB-1236Jr. MSc ,Plant biotechnology

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-Transposons-Types of transposon-Type on transposition-Examples of DNA-intermediate mobile elements--Transposon mutagenesis-Applications

TRANSPOSONS(Mobile DNA)

Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may cause mutations.

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

A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell.

The mechanism of transposition can be either "copy and paste" or "cut and paste".

Transposition can create phenotypically significant mutations and alter the cell's genome size.

Barbara McClintock's discovery of these jumping genes (Nobel prize in 1983).

There are two distinct types:

Class II transposons : These consist of DNA that moves directly from place to place.

Class I transposons : These are retrotransposons thatfirst transcribe the DNA into RNA and thenuse reverse transcriptase to make a DNA copy of the RNA to insert in a new location.

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 galactose 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)

INTEGRATION OF IS ELEMENT IN CHROMOSOMAL DNA

Three different mechanisms for transposition

Conservative transposition

Replicative transposition

Retrotransposition

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).

Transposons (Tn)

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

2 types of transposons:

Composite transposons

Noncomposite transposons

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)

There are three main orders of retrotransposons

Those with long terminal repeats (LTRs): encode reverse transcriptase, similar to retroviruses;

LINEs (Long interspersed elements ): encode reverse transcriptase, lack LTRs, transcribed by RNA polymerase II;

SINEs (Short interspersed elements) : do not code for reverse transcriptase, transcribed by RNA polymerase III.

Transposon mutagenesis

Transposable elements or transposons

sections of DNA (sequence elements)

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

Transposon – mediated mutation stratergy

Transposons cause rearrangement of DNA Homologous recombination between multiple copies of a transposon cause rearrangement of host DNA.

Homologous recombination between the repeats of a transposon may lead to precise or imprecise excision.

1. Deletion: sequences adjacent to a transposon are removed

2. Precise excision : The removal of a transposon plus one copy of the duplicated sequences.

3. Imprecise excision: Occurs when the transposon removes itself from the original insertion site, but leaves behind some of its sequence.

All transposable elements fall into one of the following two classes1. DNA elements

2. Retroelements

DNA elementsThese elements transpose via DNA intermediates such as:

Ac/Ds in plants, P elements in animals, Tn in bacteriaA 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

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

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

Retrotransposon transposition

How do we use a transposon for 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

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

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

Drosophila transposons

~15% of Drosophila genome thought to be mobile

Copia retrotransposons

Conserved, 5-100 scattered copies/genomeStructurally similar to yeast Ty elementsUse RNA and reverse transcriptaseEye Color in Drosophila (white apricot wa)

Repicative transposition proceds through a cointegrates

Cointedrates: A fusion of two original molecules

-Replication of a strand complex generated a cointegrate, which is a fusion of the donor and target replicons.-The cointegrates has two copies of the transpose, which is lies between the original replicons.--The recombination reaction is catalyzed by a resolvase coded by the transposon.

Tn A Transposition requires transposase and resolvase

Replicative transposition of TnA requires a transposase to the cointegrate structure and resolvase to release to release the two replicon.The two stages of TnA transposition are accomplished by the transposase and the resolvase , whole genes, trpA and tnpR, are identified by recessive mutation .The site of resolution is called res. Resolution occure by breakage and rejoining bonds without input of energy .

Non replicative transposition proceds by breakage and reunion

Non replicative transposition results when a cross over structure is released by niking . This insert the transposon into The target DNA, flanked by the direct repeats of the target, and the donor is left with in double standard break.

Controlling elements in maize cause breakage and rearrangements

Transposition in maize was discovered because of the effect of the chromosome breaks generated by transposition of controlling elements. The breakage generate one chromosome that has a centromere and broken end and one acentric fragment.

The acentric fragment is lost during mitosis , and this can be detected by a the disappearance of dominant alleles in a heterozygote .Fusion between the broken end of the chromosome generate dicentric chromosomes, which undergo further cycle of breakage and fusion.

The fusion –breakage- bridge cycle is responsible for the occurencce of somatic varigation .Clonal analysis identifies a group of cells descended from a single ancestor in which a transposition –mediated events altered the phenotype. Timing of the events during development is indicated by the number of cells ; tissue specificity of the event may be indicated by the location of the cells‘A break at a controlling element causes loss of an acentric fragments ; if the fragments carries the dominant marker of a heterozygote ,its loss change the phenotype.

Applications

Transposable elements as a genetic toolThe first TE was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds). Likewise, the first TE to be molecularly isolated was from a plant (Snapdragon).

TEs have been an especially useful tool in plant molecular biology. Researchers use them as a means of mutagenesis.

The insertion of a TE into a gene can disrupt that gene's function in a reversible manner, in a process called insertional mutagenesis;

transposase-mediated excision of the DNA transposon restores gene function. This produces plants in which neighboring cells have different genotypes. TEs are also a widely used tool for mutagenesis of most experimentally tractable organisms.

The Sleeping Beauty transposon system has been used extensively as an insertional tag for identifying cancer genes.

The Tc1/mariner-class of TEs Sleeping Beauty transposon system, awarded as the Molecule of the Year 2009 is active in mammalian cells and are being investigated for use in human gene therapy.

Case study

Transposon-mediated Insertional Mutagenesis in Gene Discovery and Cancer

Jun Kong

During experiment they tried to incorporate insertional mutagenesis tools for in vivo studies to generate mouse models of various cancer types. These experimental strategies are novel in that mutagenesis is taking place under a specific genetic background to trigger disease. These experiments are designed to keep a ‘right’ balance between the oncogenic fusion protein and mutagenesis rate, so that mutagenesis rate is not too high to override the oncogenic effect of the fusion protein, and is also not too low to lose the efficiency of transformation.

Similarly, another group showed that activation of the transposon in the gastrointestinal tract epithelium could giverise to neoplasia, adenomas and adenocarcinomas (188). These recent applications based on transposon mutagenesis highlighted the potential of this strategy to identify tissue-specific cancer genes that are relevant for human cancer.

P. Venkatasubramanian, S. K. Kumar, and S. N. Venugopal, “Use of ‘Kshiravidari’ as a substitute for ‘Vidri’ as per Ayurvedic descriptions,” Indian Journal of Traditional Knowledge, vol. 8, pp. 310–318, 2009.

R. N. Chopra, S. L. Nayar, and I. C. Chopra, Glossary of Indian Medicinal Plants, Council for Scientific and Industrial Research, Government of India, New Delhi, India, 1992.

G. Pandey, Dravyaguna Vijnana, Part III Reprint, Chowkhamba Krishnadas Academy, Varanasi, India, 2004.

Kasarskis, A., Manova, K. and Anderson, K.V. (1998) phenotype based screen for‐embryonic lethal mutations in the mouse. Proceedings of the National Academy of Sciences of the United States of America, 95, 7485 7490.‐

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