TRANSPOSON TAGGING

Transposon tagging for gene discovery: Transposon mutagenesis in plants has become an increasingly useful tool for gene discovery as well-marked, versatile transposons have been developed. Most of the transposon tagging systems that have been developed for plants other than maize are based on the maize Activator (Ac) transposable element family.

Transposon tagging with maize transposable elements: Ac, isolated from maize, transposes in dicots as well as other monocots and has been used as the basis for transposon tagging systems in a variety of plants, including tobacco, tomato, flax and Arabidopsis.

Advantages of transposon mutagenesis:

1. Introduction of the insertional mutagen can be separated from mutagenesis by controlling the supply of transposase to the transposon.

2. Remobilization of the element provides a rapid means of establishing that a mutation is caused by the insertion.

3. Because excision is imprecise, transposition of an element out of a gene often yields further mutations.

4. Transposons of the Ac family exhibit a marked preference for short-range transposition, potentially reducing the labor of identifying an insertion in a target gene by use of a closely linked transposon.

Use of Transposable Elements - Transposon Tagging - The transposable elements (TEs), in some cases, have been effectively utilized for isolation of genes, when the gene product is unknown. In this case a transposon works as a mutagen and therefore as a gene tag.

Following steps which are involved in this procedure:

(i) Clone a known gene with a scorable phenotypic effect.

(ii) TE is transposed to this gene to get an unstable allele.

(iii) This unstable allele is cloned and TE is isolated from this unstable allele (this is 10 select a TE which can produce unstable allele).

(iv)This TE is transposed to a gene of interest with known phenotypic effect, to produce unstable allele.

(v) The DNA is extracted from this mutant.

(vi) TE sequence is used as a probe to isolate and clone the mutant gene (carrying inserted TE), so that we can then isolate the gene of interest.

In maize, TE like Ac/Ds, En/Spm and Mu 1 have been isolated using the genes Wx, C2 and Adh1. Similarly, TEs like Tam3 and Tam7 have been isolated from snapdragon (Antirrhinum majus). These TEs have been used for gene tagging experiments leading to isolation of genes. In maize, several genes like Bz1, P, A1, Cl

and C2 have been isolated successfully using gene tagging method.

  For transposon tagging, often transposable elements endogenous to species like maize and snapdragon have been used However, rarely transposons available from one plant species can be moved into the genome of another plant species, whose gene is to be isolated.

For instance Ac, element of maize has been transferred to tobacco, where it can integrate into any lacus permitting transposm tagging and gene isolation

Transposon Tagging

The molecular isolation of transposable elements now permits the cloning of genes in which the element resides. The major advantage of this system is that genes whose function is not known can be cloned. The first step in this procedure is to identify a plant stock that is mutant for a specific trait because a transposable element has been inserted into and inactivated the gene. Next, a genomic library (often in bacteriophage lambda) of the plant stock is created. This library is then screened with a clone for the transposable element. Any clone that is selected from the screening will contain the element. In the clone, sequences for the mutated gene will lie adjacent to the element. A sublcone containing sequences from the gene is then developed from the non-transposable element DNA of the original clone. This clone is then used to screen a genomic library containing DNA from a normal plant. In this manner, any clone that is selected should contain a full, normal copy of the gene.

Because this system is so powerful, scientist have begun introducing elements from corn and Antirrhinum into other species using transformation techniques. It has been demonstrated that these elements can be induced to move from one location to another in the new species. If this movement is coupled with the appearance of mutant phenotype, then the gene responsible for the phenotype can cloned in that particular species. These techniques have now allowed the use of transposon tagging in plant species in which active transposable elements have not been identified.

Advantages of transposon tagging in Arabidopsis

Avoid unintentional mutagenesis. When a T-DNA (with or without a transposon) is introduced into Arabidopsis, mutations arise in the resulting lines. However, when transposons are used, mutagenesis can be separated from the transformation process that introduces the mutagen. This is because the transposase and the transposon T-DNAs are transformed into different plants. Transposition occurs only after the transposon line is crossed by the transposase line.

Get stable mutations that will revert. A mutation caused by a transposition-defective transposon is stable as long as the plant does not contain an active transposase gene. The transposon can be remobilized by reintroducing the transposase gene by a genetic cross. Revertants arise by excision of the transposon from the mutant gene. When the a plant transposon inserts, it creates a short duplication. Transposons of the Ac-Ds family create an 8-bp

duplication. When the transposon excises, it leaves behind either the entire duplication or some part of it, often with a few base pairs deleted or added at the former insertion site. If the insertion was in an exon, the reading frame must be restored to obtain a revertant, so revertants of exon insertions generally have some multiple of a 3-bp insertion left behind. Detecting this so-called "footprint" proves that the mutation was caused by the insertion.

Get new mutations. If a transposon is inserted in an exon, excision of the transposon can change the sequence at the insertion site, resulting in a change, insertion or deletion of one to several amino acids. Changes in the activity of the gene caused by such mutations can provide insight into protein function.

Target your gene for insertion. Transposons of the Ac family have a marked preference for short-range transposition. This can greatly reduce the amount of work involved in identifying an insertion in a target gene if there is a closely linked transposon which can be mobilized.

Pick the right tagging system. Transposon tagging systems for Arabidopsis have been developed in several laboratories (see reference list). The efficiency of transposon tagging has been improved by using transposons that 1) are immobile and maintained in separate lines from the transposase, 2) carry selectable markers, and 3) disrupt a selectable marker gene whose function is restored upon excision, providing a means of identifying plants in which transposition has occurred. The source of transposase is either an immobile Ac element or cDNA for the element-encoded ORFA protein. Recent transposons carry reporter genes which are activated upon insertion into a gene or promoter, facilitating analysis of the gene's expression pattern.