Transposon mutagenesis 629
- 1. INDIAN INSTITUTE OF TECHNOLOGY INDORE Department Of Biosciences and Biomedical Engineering Autumn Semester 2021 Presentation Topic- Transposon Mutagenesis Presented By- Sushma Ahirwar Roll no. – 2103171011 M.Sc. Biotechnology, 1st semester BSBE Department, IIT Indore Course Instructor Dr. Avinash Sonawane
- 2. Points of discussion Introduction to transposon. Structural Elements of Transposon History of discovery Classification of transposon Transposon Mutagenesis Application of Transposon mutagenesis
- 3. Introduction to Transposons Transposon- A DNA sequence that is able to move or insert itself at a new location in the genome.” The phenomenon of movement of a transposon to a new site in the genome is referred to as transposition. Also called jumping DNA /mobile DNA/junk DNA/selfish DNA. Transposase is the key enzyme for transposition.
- 4. Short direct repeat Transposable element IS element contains single open reading frame (ORF) which encodes for the enzyme transposase, catalyzing its own transposition. Short direct repeat are important hallmark of IS (insertion) elements. Reference image -https://www.onlinebiologynotes.com/wp-content/uploads/2020/04/insertion-sequences.png
- 5. The enzyme transposase is like restriction endonuclease which binds to terminal inverted repeats (IR) of IS element which is the restriction site. Then the enzyme cut and excise IS elements from chromosome or plasmid. The excised IS element is mobile in nature and moves along the length of chromosome to recognizes the target site for insertion on same or different chromosome or plasmid. Once recognizing the target site, it generate staggered cleavage (cut the single strand of DNA) generating sticky and itself get inserted. As IS element get inserted, the proofreading mechanism of DNA results in duplication of the DNA sequence at the target site of the insertion such that one copy of target DNA is located on each side of IS element. Reference -https://www.onlinebiologynotes.com/wp-content/uploads/2020/04/Transposition-of-IS-element-1024x576.jpg
- 6. Presence of transposable elements was first predicted by Barbara McClintock in maize (corn) in late 1940s. She was awarded a Nobel prize in 1983. History She found Ac and Ds elements, which changed their position and cause variation in colour of kernels in maize plant. Ac element is capable in autonomous movement but ds element cannot move by itself. She named Ac and Ds as controlling elements.
- 8. Autonomous elements (Ac element) Transposons Transposon family Retro transposon family Encodes own transposase enzyme Terminal inverted repeats Do not encode own transposase enzyme Terminal inverted repeats Encodes own transposase enzyme Terminal inverted repeats Do not encode own transposase enzyme Terminal inverted repeats Non-autonomous elements(Ds-element) Autonomous elements (Ac element) Non-autonomous elements(Ds-element)
- 9. Transposon (cut and paste mechanism) Retro Transposon (copy and paste mechanism) Classification of transposons on the basis of difference in transposition mechanism Donor DNA Donor DNA RNA polymerase RNA intermediate Reverse transcriptase DNA intermediate Target DNA Flanking DNA Transposed mobile element DNA intermediate
- 10. Transposon mutagenesis Transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Any gene of interest Transposon Mutation in gene due to transposon insertion
- 11. Yfg 1 Transposon insertion into yeast genomic DNA Bacterial Tn 7 transposon Yeast genomic DNA Transposon mediated mutagenesis in yeast Disruption in Yfg 1 gene function
- 12. In vivo Mutagenesis In Vitro mutagenesis
- 13. Inverse PCR is performed in order to know the transposon insertion site. Transposon Then we can compared sequence PCR products against sequence database
- 14. Advantages of Transposon mediated mutagenesis Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. transposon insertions can be easily identified in mutants of interest by inverse PCR Limitation of transposon mutagenesis In in-vivo mutagenesis there is less no. of transformed are obtained.
- 15. Application of transposon mediated mutagenesis Transposon mutagenesis can be used to study a particular gene function. It can also be Used to control the gene expression by altering the enhancer or silencer sequences by transposable element insertion and transposon can also used as gene transfer vehicle. It also has therapeutic utility – transposon mediated mutagenesis can be used as a effective tool to inactivate or mutate the disease causing gene. Be can create desired mutation in order to achieve desired output .