transposons

content consist of brief study of transposons, its types, transpostion, its uses and application

1. TRANSPOSONS (TRANSPOSABLE ELEMENTS) “THE JUMPING GENE” “MOBILE DNA”

2. CONTENT Introduction General characteristics of TE Types of Transposable Elements Mechanism of Transposition Transposition Retrotransposons Mechanism of Retrotransposition Uses of Transposons

3. INTRODUCTION Segment of DNA that move from one genomic location to another. The simplest transposable elements are insertion sequences(IS), first TE discovered in bacteria. Comprises about 45% in humans.

4. A  transposable element  ( TE  or  transposon ) is a DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's  Genome size. Transposition often results in duplication of the TE. Barbara McClintock's discovery of these  jumping genes  earned her a Nobel Prize in 1983. Discovered largely from cytogenetic studies in maize, but since found in most organisms. BARBARA McCLINTOCK’S

5. GENERAL CHARACTERISTICS OF TE They were found to be DNA sequences that code for enzymes, which bring about the insertion of an identical copy of themselves into a new DNA site. Transposition events involve both recombination and replication processes which frequently generate two daughter copies of the transposable elements. One copy remains at the parent site and another appears at the target site. A transposable element is not a replicon. Thus, it can not replicate apart from the host chromosome.

6. Types of transposable elements:- Different type of transposable elements are present in both prokaryotes and eukaryotes. Insertion sequences Transposons Bacteriophage mu

7. INSERTION SEQUENCE IS were first transposable elements identified as spontaneous in some bacterial operon. The IS are shorter (800 to 1500 base pairs) and do not code for proteins. In fact, IS carry the genetic information necessary for their transposition (the gene for the enzyme transposase). There are different IS such as IS1, IS2, IS3 and IS4 and so on in E.coli.

8. Transposons Transposons are similar to IS elements but carry additional gene. Transposons are several thousand base pair long and have genes coding for one or more protein. On either side of transposon is a short direct repeat. The sequence into which the transposable element insert is called target sequence. Two types of transposons :- 1. COMPOSITE TRANSPOSONS 2. NON-COMPOSITE TRANSPOSONS

9. COMPOSITE TRANSPOSON A composite transposon is similar in function to simple transposons and insertional sequence(IS) in that it has protein coding sequence flanked by inverted, repeated sequences that can be recognized by transposase enzyme. A composite transposon , however e.g. carry gene for antibiotic resistance and is flanked by two separate IS elements which may or may not be exact replicas. Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to other is transposed as complete unit. Composite transposon will often carry one or more genes conferring antibiotic resistance.

10. Composite transposons may be thousands of base pairs long. The IS elements are both of the same types and are called IS-L (for “left”) and IS-R (for “right”). Depending upon the transposon, IS-L and IS-R may be in the same or inverted orientation relative to each other. Because the ISs themselves have terminal inverted repeats, the composite transposons also have terminal inverted repeats . Figure shows the structure of the composite transposon Tn 10 to illustrate the general features of such transposons. The Tn 10 transposon is 9,300 bp long and consists of 6,500 bp of central, nonrepeating DNA containing the tetracycline resistance gene flanked at each end with a 1,400-bp IS element. These IS elements are designated IS10L and IS10R and are arranged in an inverted orientation. Cells containing Tn 10 are resistant to tetracycline resistance gene contained within the central DNA sequence. Transposition of composite transposon occurs because of the function of the IS elements they contain. One or both IS element supplies the transposase. The inverted repeats of the IS elements at the two ends of the transposon are recognized by transposase to initiate transposition (as with transposition of IS elements).

11. Transposition of Tn 10 is rare, occurring once in 10 cell generations. This is the case because less than one transposase molecule per cell generation is made by Tn 10. Like IS elements, composite transposons produce target site duplications after transposition.

12. NON-COMPOSITE TRANSPOSON They like composite transposons, contain genes such as those for drug resistance. Unlike composite transposons, they do not terminate with IS elements. However, they do have the repeated sequences at their ends that are required for transposition. Tn3 is a non-composite transposon . Tn3 has 38 bp inverted terminal repeats and contains three genes in its central region. One of those genes, bla , encodes β-lactamase which breaks down ampicillin and therefore makes cells containing Tn3 resistant to ampicillin. The other two genes, tnpA and tnpB , encode the enzymes transposase and resolvase that are needed for transposition of Tn3 (Fig. 12.4). Transposase catalyzes insertion of the Tn into new sites, and resolvase is an enzyme involved in the particular re-combinational events associated with transposition. Resolvase is not found in all transposons. The genes for transposition are in the central region for non-composite transposons, while they are in the terminal IS elements for composite transposons. Non composite transposons also cause target site duplications when they move.

13. BACTERIOPHAGE mu The longest transposon k nown so far. Carries numerous gene for viral head and tail formation The vegetative replication of mu produces about 100 viral chromosomes in a cell arises from the transposition of mu to about 100 different target sites. Therefore considered as giant mutator transposon.

14. MECHANISM OF TRANSPOSITION Movement of transposon occurs only when enzyme tranposase recognizes and cleaves at either 5’ or 3’ of both ends of transposon and catalysis at either at 5’ or 3’of both both the ends of transposons and catalysis staggered cut at the target site. Depending on transposon, a duplication of 3 to 12 base of target DNA occurs at the site where insertion is to be done. One copy remains at each end of the tranposon sequence. After attachment of both ends of transposon to the target site, two replication forks are immediately formed. At this stage there starts two paths for carrying out onwards. 1. DIRECT or NON-REPLICATIVE 2. REPLICATIVE

15. TRANSPOSITION Mechanism of movement of TE from one location to another. In this process staggered cuts are made in the target DNA. The TE is joined to single stranded ends of the target DNA. Finally DNA is replicated in the single stranded gap.

16. There are two general pathway for transposition in bacteria :- 1. Direct transposition 2. Replicative transposition In direct (or simple) transposition cuts on each side of the transposon excise it, and the transposon moves to a new location. This leaves a double strand break in the donor DNA that must be repaired. At target site, a staggered cut is made the transposon is inserted into the break, and DNA replication fills in the gaps to duplicate the target site sequence. In replicated, transposition the entire transposon is replicated, leaving a copy behind at the donor location. A cointegrate is an intermediate in this process, consisting of the donor region covalently linked to DNA at the target site. Two complete copies of the transposons are present in the cointegrate , both having the same relative orientation in DNA

17. Retrotransposons Retrotransposons  (also called transposons via RNA intermediates) are genetic elements that can amplify themselves in a genome and are ubiquitous components of the DNA of many eukaryotic organisms. These DNA sequences use a "copy-and-paste" mechanism, whereby they are first transcribed into RNA, then converted back into identical DNA sequences using reverse transcription, and these sequences are then inserted into the genome at target sites . Retrotransposons are particularly abundant in plants, where they are often a principal component of nuclear DNA . In maize, 49–78% of the genome is made up of retrotransposons .  In wheat, about 90% of the genome consists of repeated sequences and 68% of transposable elements . Around 42% of the human genome is made up of retrotransposons .

18. Mechanism of retrotransposition

19. USES OF TRANSPOSONS As cloning vehicle. Transformation vectors for transferring genes between organisms. Also drug resistance genes encoded by many transposons are useful in the development of plasmids as cloning vehicle. Transposon mutagenesis. Use of transposon is to increase rate of mutation due to insertional inactivation(e.g. production of different colour of grapes, corn and other fruits). Used in genetic studies

20. THE END THANK YOU