Transposons ask
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Transcript of Transposons ask
Transposons
A. Arunsaravanakumar,
Veterinary Immunology
Overview
Introduction History Nomenclature General characteristics Mechanism of transposition Bacterial transposons Eukaryotic transposons Applications Conclusion
Introduction Transposable or mobile genetic elements/Jumping
genes/ Molecular parasite “Piece of DNA that can move from one location to
another in a cell's genome” Recombination b/w transposon and another DNA –
target site Bacteria – target site within chromosome or from
plasmid to chromosome or b/w plasmid (vice versa) Jump from one location to another (cut-and-paste
translocation)
Identified in eukaryotes, prokaryotes etc. More abundant in eukaryotic genomes than prokaryotes High proportion of species genome
10% of several fish species 12 % of the Caenorhabditis. elegans genome 37% of the mouse genome 45% human genome up to >80% - some plants like maize
History 1940s-cytogenic studies in maize
kernels 1960s - James Shapiro and
others found transposable elements (TE) in bacteria and other species
1st TE found in bacteria is called insertion sequences (IS)
Recombinant technology – TE in all organisms
Barbara McClintock (1983)- Nobel Prize in Physiology or Medicine
Nomenclature Campbell et al., 1977 – nomenclature in prokaryotes Named as insertion sequences – IS1, IS2, IS3 etc. Antibiotic resistance genes named as Tn (Tn1-ampr,
Tn2, Tn3 etc). Eukaryotes – nonstandard way
Drosophila- copia, p-elements Yeast- Ty, maize- Ac&Ds, Human- Alu
General characteristics of TE DNA sequences – code for enzymes – insertion of
identical copy – new DNA site Recombination and replication process – lead to two
daughter copies (Parent site and target site) TE cannot replicate apart from the host chromosome Most have inverted repeats (IR) – EM Transposition-not require extensive areas of
homology b/w the transposon & target site
Transposons – IIIr lysogenic prophages – except –
originate in one chromosomal location – move - different location in the same chromosome
TEs differ from phages (lack virus life cycle) & plasmids (unable to reproduce autonomously and exist apart from the chromosome)
Multiple copies - antibiotic resistance - single R plasmid by moving genes - different plasmids
• Movement of TE from one location to another
Mechanism of transposition
Class I: retrotransposons
Retrotransposition: The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then insertedIIIr to viral retroelement except RNA from exogenous Eukaryotic genomes
Class II: DNA Transposons
No RNA intermediate
Move directly from one position to another within the genome using a transposase to "cut and paste" them within the genome
Conservative transposition: The element itself moves from the donor site into the target site
Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate
"cut and paste"
"copy and paste"
Class III: Miniature-inverted-repeats-transposable elements ( MITEs)
Too small to code for any protein Transposition- unknown Human, plants and animals
Bacteria Vary in structure
1. Insertion sequences
2. Composite transposons
3. Tn3-type transposon
4. Transposable phage Mu
Insertion sequence (IS) Simplest TE 750 -1600 bp (0.3 kb)
1. Transposase genes – enzymes required for transposition
2. Both ends – identical or very similar sequences of nt in reversed orientation – inverted repeats (9 to 41 bp – vary)
Replicatively or conservatively IS-cause mutations - within the coding sequence of a gene
or within a DNA region that regulates gene expression Single E.coli may contain 20 types-IS1, IS2, IS3 etc.
• Transposase – staggered cuts in DNA – IIIr to restriction enzymes
• ITRs – recognition sites for binding to enzymes
Composite transposons 1st characterized in E.coli contains genes other than those required for transposition
– IS with ARG (2.5-10kb) Often reside on plasmids and conservative may help bacteria adapt to new environments
Tn 5 & Tn903 kanamycin
Some prokaryotic transposons
schematic map of a plasmid carrying many resistance genes
Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome
Non composite transposons
Lack flanking insertion sequences (IS) Inverted repeats + Transposase gene + antibiotic
resistance gene (s) + Resolvase (site specific recombination)
Tn3 & Tn7 (5kb)
Bacteriophage Mu
• Bacterial viruses transpose replicatively as part of their normal infection cycle
• DNA-based transposition• Temperate (lysogenic) bacteriophage• Longest transposon so far• Carries genes for viral head & tail formation• Giant mutator transposon - 100 target sites
Simplified cartoon of the Mu genetic map
Life cycle of phage mu
(A) Mu infects a sensitive host, - linear DNA enters the cell – inserted "cut and paste" mechanism
(B) Lysogens of wild-type Mu – stable - not induced by UV or DNA damaging agents.
but temperature sensitive repressor - Mu c(Ts) - induced - 42°C
(C) Repressor – inactivated - A & B proteins are expressed and Mu transposes – Replicative 50 - 100 new sites on the chromosome.
Late phage gene products - phage heads, tails, lysis proteins, etc
Human genome
• LINEs- long interspersed nuclear elements
• SINEs-short interspersed nuclear elements
• Approx. 350, 000 DNA transposons – all with transposase and inverted repeats – majority inactive
Plants
Active transposons Eg. Ac/Ds transposon – 1st
McClintock Spm element – maize
Work as family Ac have transposase
not Ds
Variegated pigmentation in maize kernals caused by transposition in somatic cells
Effects
Gene inactivation Mutation Gene alterations (Insertions, excisions, Duplication or
translocation) Moderate gene expression or induce recombination Mutations results in diseases
Hemophilia A & B, X-linked severe combined immunodeficiency Porphyria, cancer etc.,
Applications
Induce mutations (Insertional mutagenesis) TE – insert - specific site - genome of an organism give
it a high potential to be used for genetic modification Gene therapy (Non-viral alternative) Act – probe for cloning genes that are mutated by
insertion of a particular element. Initially genetic screening is used to identify the mutation caused by transposon
Genes are isolated from given species due to transposon tagging (preferred choice if one specific gene is desired)
DNA transposons, PiggyBac, Tol2 and Sleeping Beauty have been evaluated for gene therapy in animal experiments, primary cell gene delivery and a few pre-clinical trials
Several DNA transposons, such as P element, PiggyBac, Tol2, Hsmar1 and Sleeping Beauty, have been utilized for mutagenesis in invertebrate and vertebrate cells
Characteristics of DNA Tranposons used in Genomics
Cargo capacityTransposition efficiencyGene Silencing and Stability of Gene InsertionInsertion Site Targeting
• All the organisms – present – huge fraction of total DNA sequences
• Major cause of mutations and genome rearrangement
• Used in genetic studies • Mutation – used to produce different colors of
grapes, corn and other fruits
Thank you