Transposition and transposable elements

Transposable elements
mobile genetic elements comprise 45% of human chromosomal DNA middle repetitive DNA contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) cells must depress transposition to insure genetic stability

Types of transposable elements

DNA vs. RNA viral vs. nonviral replicative mechanism vs. excision mechanism

transposon

insertion mutation transposon

Discovery of transposons
Barbara McClintock 1950 s Ac Ds system in maize influencing kernel color unstable elements changing map position promote chromosomal breaks Rediscovery of bacterial insertion sequences source of polar mutations discrete change in physical length of DNA inverted repeat ends: form lollipops in EM after denaturation/reannealing

Composite bacterial transposons

repeated ends, usually inverted, sometimes direct repeated ends themselves are IS elements and can independently transpose ends mobilize all intervening DNA often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline) often reside on plasmids

Basic minimal insertion sequence structure

tnp ORF ends: genetically required, in cis tnp (transposase): genetically required, trans-acting

Element 1 wt endstrpendstnptnp-

Element 2 wt endstnpwt wt ends-

Transposition? 1 and 2 neither neither only 2 1 and 2 only 1

Structure of Tn3
4957 bp 3 trans-acting genes:

transposase tnpA

repressor tnpR Tn3 resolvase

ampcillin-resistance bla

2 cis-acting sites: 38 bp inverted repeat ends 120 bp IRS or res internal resolution site

tnpR and res mutations cause accumulation of co-integrate structure

direct repeat of Tn

cointegrate

2 types of DNA tranposons

excisive mechanism examples: Tn5, Tn10, P elements replicative mechanism examples: Tn3, bacteriophage Mu

Replicative transposons
orignal cut of transposon is only nick and only one strand is initially ligated element replicates through itself produces as intermediate a co-integrate structure co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

Excisive transposons
cut-and-paste mechanism cut themselves out of original site, producing double strand break cut target site and ligate to element ends, thereby inserting at new site original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

Source of target site duplication TSD

GAC CTG Staggered cleavage of target GAC CTG Ligation of transposon DNA GAC CTG GAC CTG

Repair replication generates short direct repeats

degenerate transposons
many naturally occurring transposable elements have suffered mutation and are no longer active some of these may have cis-acting end mutations and cannot be mobilized others may have intact ends but no transposase: these can be mobilized by a element that is tnp+ ( autonomous element) Ac Ds system is an example of latter: Ac (activator) can mobilize Ds (dissociator) MITEs (minature inverted repeat transposable elements) are nonautonomous DNA elements SINEs are retrotransposon version (LINEs)

Comparison of transposition reactions

Direct transesterification reactions DDE motif transposase (integrase)

Comparison of tranposase structural organization

Mechanism of transposases and retroviral integrases

Classification of retroelements
Have obligate RNA intermediate, use reverse transcriptase (RT, RNA-dependent DNA polymerase) LTR-retroelements: long terminal repeats Ty1/copia, Ty3/gypsy, retroviruses Non-LTR-retroelements retroposons LINES

Characteristics of LTR retroelements

Long terminal repeats: required for replication cycle Genes: gag, pol, (viruses also have env) Pol is polyprotein which gives rise to RT (reverse transcriptase), IN (integrase) RH (RNase H), PR (protease) Forms VLPs virus-like particles Integrase is functionally and structurally similar to transposase of DNA transposons, DDE motif Integration gives characteristic TSD

LTR element replication

tRNA primer

Multiple template jumps

Characteristics of non-LTR retroelements

2 ORFs, orf1, orf2 Variable TSD ORF2 gives rise to EN, endonuclease, (similar to APE) and RT Uses target primed reverse transcription TPRT Can transduce 3 downstream non-element segments

Human L1 (LINE-1) retroelement

15% of human DNA 520,000 copies, only 3-5,000 are full-length Associated with human disease loci Transpose specifically in germ line

Methods for Generation of Mutant Populations The most reliable method to ascertain gene function is to disrupt the gene and determine the phenotype change in the resulting mutant individual Two most popular methods to generate mutants:
1. Insertional mutagenesis 2. Deletion mutagenesis
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Two main methods

1. Transposon insertion 2. T-DNA insertion

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Transposon mutagenesis
Transposable elements or transposons sections of DNA (sequence elements) move, or transpose, from one site in the genome to another

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All transposable elements fall into one of the following two classes

1. DNA elements 2. Retroelements

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DNA elements
These elements transpose via DNA intermediates such as: Ac/Ds and Spm in plants, P elements in animals, Tn in bacteria A common feature of DNA elements is the flanking of the element by short inverted repeat sequences The enzyme transposase recognizes these sequences, creates a stem/loop structure excises the loop from the region of the genome The excised loop can then be inserted into another region of the genome
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DNA-Immediate Mobile Genetic Elements

The Short inverted repeats at the ends of the element These inverted repeats act as the substrates for recombination reactions mediated by the transposase

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Structure and transposition of a transposable element

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Retroelements
transpose via RNA intermediates The RNA is copied by reverse transcriptase into DNA the DNA integrates into the genome Retroelements are found in all eukaryotes such as Tos in rice, copia in animals and Ty1 in yeast

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Retrotransposon transposition

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Retorviruses
The basic structure is an LTR = long terminal repeat which flanks three genes, A complete retroviruses also contains three genes: gag = structural gene for capsid Pol = reverse transcriptase env = envelope gene for the virus

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How do we use a transposon for mutagenesis?

The insertion and excision of transposable elements result in changes to the DNA at the transposition site The transposition can be identified when a known DNA sequence or selection markers are inserted within the elements

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Transposomics
EZ::TN Transposomes provide an efficient and reliable method for generating a library of random gene knockouts in vivo Gene inactivation and examination of the resulting phenotype will identify the function of the interrupted genes

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Transposon-Mediated Homologous Recombination Gene Knockout in Fungi

Hamer et al. 2001. Proc Natl Acad Sci U S A. 2001 24;98(9):5110-5

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T-DNA insertion mutagenesis

T-DNA is a segment of the tumor-inducing (Ti) plasmid of Agrobacterium delimited by short imperfect repeat border sequences

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T-DNA transfer from Agrobacterium to plant cell

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Temperate bacteriophage Mu (Mu = mutator)

37 kb linear DNA with central phage DNA and unequal lengths of host DNA at each end Mu integrates by transposition replicates when E. coli replicates During the lysogenic cycle, Mu remains integrated in E. coli chromosome

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bacteriophage Mu

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The advantages / disadvantage of Mu

The advantages of the use of Mu are: it is not normally found in the bacterial genome therefore there are few problems with homology to existing sequences in the chromosome; in contrast to most other transposons Mu does not need a separate vector system since it is itself a vector A wide variety of useful mutants of Mu have been generated The disadvantage of Mu: it is a bacteriophage and therefore can kill the host cell
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Drosophila transposons
~15% of Drosophila genome thought to be mobile
2 different classes:

Copia retrotransposons Conserved, 5-100 scattered copies/genome Structurally similar to yeast Ty elements Use RNA and reverse transcriptase Eye Color in Drosophila (white apricot wa)
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ITR(17bp) DTR

ITR(17bp) DTR

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P elements
Hybrid dysgenesis, defects arise from crossing of specific Drosophila strains Occurs when haploid genome of male (P strain) possesses ~40 P elements/genome P elements vary in length from 500-2,900 bp P elements code a repressor, which makes them stable in the P strain in male (but unstable when crossed to the wild type female/; female lacks repressor in cytoplasm)
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Ac (activator)/Ds (dissociation) System

discovered by B. McClintock (Noble Prize Winner in 1983)

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Ac/Ds System

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Ac/Ds System

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Schematic Diagram of the Ds Donor Site and Possible Transposition Events

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 Open arrowheads indicate the 5' and 3' ends of th transposon The Ds element carries the NPTII gene, which confers resistance to kanamycin (KanR) and a modified GUS reporter gene (Sundaresan et al. 1995 ) Possible transposition events include the following:

(1) unlinked or loosely linked transposition to the same chromosome; (2) transposition to a different chromosome; (3) closely linked transposition; and (4) closely linked transposition disrupting theIAAH gene
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Ac/Ds Transposon tagging system

Advantages: Efficient and cost-effective method to generate a large mutant population Disadvantages: Secondary transposition complicates gene identification And transposon system is not available in many species

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Transposition elements in Human

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Mobile Genetic Elements and Other Families of Repetitive DNA

The genome is littered with large families of repetitive sequences have no apparent function in the cell Mobile Genetic Elements Tandemly repeated simple sequence DNAs Satellite DNAs Short simple repeats (microsatellites)
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LINEs (Long interspersed elements) LINEs are one of the most ancient and successful inventions in eukaryotic genomes In humans, are about 6 kb long encode two open reading frames (ORFs) Most LINE-derived repeats are short, with an average size of 900 bp - 1,070 bp The LINE machinery is believed to be responsible for most reverse transcription in the genome
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SINEs (Short interspersed elements)

short (about 100-400 bp) A single monophyletic family of SINEs (ALU) This family is the only active SINE in the human genome The human genome contains three distinct monophyletic families of SINEs: the active Alu, and the inactive MIR and Ther2/MIR3
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Identification of a human specific Alu insertion in the factor XIIIB gene Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies An Alu Insert as the Cause of a Severe Form of Hemophilia A (factor VIII)
Acta Haematol 2001;106:126 129

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