Transposable Element: Tool for Gene Tagging.
Authors: Neeraj Kumar and Kuldeep Jangid
Deptt. of Genetics And Plant Breeding.
COA, CCS Haryana Agricultural University, Hisar " 125004.
Corresponding author : jangidkuldeep09@gmail.com


Transposable elements ( TE or transposon) are DNA sequence those are to change position within a genome, due to change in position in DNA , they are able to change the function of phenotype in both direction, wild to mutant or mutant to wild. There is duplication of TE due to transposition. McClintok discovered that Ac and Ds element in maize and these are able to change its own location within genome and give term mobile element for these DNAsegment. Barbara McClintock's discovery of these jumping genes earned her a Nobel Prize in 1983 (McClintock, 1950). These elements are large fraction of the genome and this genome is named as junk DNA play no role but important in evolution (Bucher et al, 2012). On the basis of structure and movement Transposable elements can be divided into major two classes. The class I, consists of such elements those contain terminal inverted repeats and transpose through DNA intermediates. These activity results into flanking direct repeats at their points of insertion into DNA. All active forms of these transposable elements encode transposase, which is neccesary for their movement from one site to another site. Some also encode resolvase, repressors, and other proteins. Their transposition may be replicative or nonreplicative, but they never use RNA intermediates. Examples of transposable elements in this first class include insertion sequences and all complex transposons in bacteria, the Ac and Ds elements of maize, and the P elements of Drosophila.The class II of transposable elements are the retrotransposons, which transpose by RNA intermediates. This result into flanking direct repeats at their points of insertion when they transpose into DNA. Retrotransposons do not encode transposase, but some types are similar in structure to retroviruses and carry sequences that produce reverse transcriptase. Transposition takes place when transcription produces an RNA intermediate, which is then transcribed into DNA by reverse transcriptase and inserted into the target site. Examples of retrotransposons in this class include Ty elements in yeast, copia elements in Drosophila, and Alu sequences in humans. Retrotransposons are not found in prokaryotes. (Benjamin, 2012)

Characteristic of two major classes of transposable genetics element

Class Structure Gene encoded Transposition Example
I Long terminal direct repeats; short flanking repeat at target site Reverse transcriptase( and sometimes others) By RNA intermediates Ty (yeast) Copia (Drosophilla) Alu (human)
II Short terminal inverted ; shorts flanking direct repeats at target site Transposase gene (and sometime others) Through DNA (replicative or nonreplicative) IS1(E.coli) Tn3(E.coli) Ac,Ds (maize) P elements(Drosophilla)


Tranposon as a tool for gene cloning .

Transposon element have ability to move from one site to another within a genome. Sequence of these element is also known by using these two property we can clone the the gene. Trnsposon elements are used as probe for cloning of DNA sequence adjacent to the transposable element. By this it can be possible to isolate the mutant allele. This statargey used in maize very much but initially developed to clone the droshophilla white locus (Binghaam et al. 1981). We can use only those transposon as tag whose sequence is known such as Ac,Ds,En/Spm and Mu .There are mainly two approaches direct and nondirect.(Settles, 2009)

Direct approach:

This is used to tagging specific locus of interest. Plant those contain the active transposon are crossed with homozygous mutant phenotype. If this specific locus is mutated by transposon the resulted phenotype is unstable. The locus may be mutated at near-saturated level. Thses mutated phenotype were crossed with standard inbred or hybrid to separate the mutable allels. Segregating population is screened with transposon probe by using DNA gel blot or with PCR method to identify the site where tranposon is inserted and adjacent site is cloned. A stable muataion with always co segregate with the mutated phenotype is very helpful for cloning. Ac/ds ,en/spm and mu have been employed in direct tagging.

Non-direct or Random:

By this we tag any random locus. Plant with active transposon were crossed with a population to obtain mutation at any random site. After crossing we obtain M1 population , selfing of these M1 result into M2. In M2 we indentify the recessive phenotype and these M2 populations used to identification co-segregating insertion where transposn is inserted and clone the adjacent region for tagging of a locus as in random approach.

Proof of a tagged gene (Walbot, 1992)

  • Tagged phenotype is characterized by variegation or chimeric sector of wild and mutant
  • Conducting a segregation analysis with a TE probe
  • If a band is present in mutant homozygous and absent in wild homozygous is a evidence
  • Cloning of DNA fragment from mutant plant through I-PCR by using primer specific to TE
  • This fragment can be used as a probe for the DNA sample of wild type plant


References:

1. Bingham, Paul & Levis, Robert & M. Rubin, Gerald. (1981). Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method. Cell. 25. 693-704.
2. Bucher E, Reinders J, Mirouze M (2012). "Epigenetic control of transposon transcription and mobility in Arabidopsis". Current Opinion in Plant Biology. 15 (5): 503-510.
3. McClintock, Barbara (June 1950). The origin and behavior of mutable loci in maize. Proc Natl Acad Sci U S A. 36 (6): 344-55.
4. Pierce, B. A. (2012). Genetics: A conceptual approach. New York: W.H. Freeman.
5. Settles AM (2009) Transposon tagging and reverse genetics. In: Kriz A, Larkens B (eds) Molecular genetic approaches to maize improvement. Springer, Berlin, pp 143-160.

6. Walbot, V. (1992). Strategies for mutagenesis and gene cloning using transposon tagging and T-DNA insertional mutagenesis. Annu. Rev. Plant Physiol. Plant MOI. Biol. 43, 49-82.



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