Transposon Tagging
Transposon Tagging
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.
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.
(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.
(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.
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.