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Transposon Tagging

Transposon Tagging

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Published by: navkir on Sep 19, 2010
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12/10/2012

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TRANSPOSON TAGGING
y
 
Transposon tagging for gene discovery:
Transposon mutagenesis in plants has becomean 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 elementfamily.
y
 
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
.
y
 
A
dvantages of transposon mutagenesis:
 1.
 
I
ntroduction of the insertional mutagen can be separated from mutagenesis by controllingthe supply of transposase to the transposon.2.
 
Remobilization of the element provides a rapid means of establishing that a mutation iscaused by the insertion.3.
 
Because excision is imprecise, transposition of an element out of a gene often yieldsfurther 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 aclosely linked transposon.
Use of Transposable Elements - Transposon Tagging
- The transposable elements (TEs), in somecases, have been effectively utilized for isolation of genes, when the gene product is unknown. In this case atransposon 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 whichcan 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 wecan 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 beenused 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 snapdragonhave been used However, rarely transposons available from one plant species can be moved into thegenome 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 lacuspermitting transposm tagging and gene isolation
Transposon Tagging
The molecular isolation of transposable elements now permits the cloning of genes inwhich the element resides. The major advantage of this system is that genes whosefunction is not known can be cloned. The first step in this procedure is to identify a plantstock that is mutant for a specific trait because a transposable element has beeninserted into and inactivated the gene. Next, a genomic library (often in bacteriophagelambda) of the plant stock is created. This library is then screened with a clone for thetransposable element. Any clone that is selected from the screening will contain theelement. 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 agenomic library containing DNA from a normal plant. In this manner, any clone that isselected should contain a full, normal copy of the gene.Because this system is so powerful, scientist have begun introducing elements fromcorn and Antirrhinum into other species using transformation techniques. It has beendemonstrated 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 mutantphenotype, then the gene responsible for the phenotype can cloned in that particular species. These techniques have now allowed the use of transposon tagging in plantspecies in which active transposable elements have not been identified.
 Advantages of transposon tagging in Arabidopsis
y
 
 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.
y
 
G
et stable mutations that will revert.
 A mutation caused by a transposition-defectivetransposon is stable as long as the plant does not contain an active transposase gene. The

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