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Transposable element: mobile genetic elements of a chromosome that, have the capacity to move from one location to another in the, genome., •, , Normal and ubiquitous components of prokaryote and eukaryote, genomes., Prokaryotes-transpose to/from cell’s chromosome, plasmid, or a, phage chromosome., Eukaryotes-transpose to/from same or a different chromosome., , •, , Nonhomologous recombination: transposable elements insert into, DNA that has no sequence homology with the transposon., , •, , Transposable elements cause genetics changes and make important, contributions to the evolution of genomes:, •Insert into genes., •Insert into regulatory sequences; modify gene expression., •Produce chromosomal mutations.
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Transposable Elements (Transposons), • DNA elements capable of moving ("transposing"), about the genome, • Discovered by Barbara McClintock (1940), largely, from, cytogenetic studies in maize, but since, found, in most organisms, • She was studying "variegation" or sectoring in, leaves and seeds, • She liked to call them "controlling elements“, because they affected gene expression in, myriad ways
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1. Nobelprize.org, (1983 Nobel Prize in, Physiology and Medicine), 2. profiles.nlm.nih.gov/LL/, , Corn (maize) varieties, , Barbara McClintock 1902-1992
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Corn evolution in 7000 yrs of domestication, cob of wild teosinte, , cob of Hopi Blue corn
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Maize (domesticated corn) kernel, structure
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Other Characteristics of McClintock's, Elements, • Unstable mutations that revert frequently but, often partially, giving new phenotypes., • Some elements (e.g., Ds) correlated with, chromosome breaks., • Elements often move during meiosis and, mitosis., • Element movement accelerated by genome, damage.
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Transposable elements:, Two classes of transposable elements/mechanisms, of movement:, 1. Encode proteins that (1) move DNA directly to a, new position or (2) replicate DNA and integrate, replicated DNA elsewhere in the genome, (prokaryotes and eukaryotes)., a) Cut-and-paste mechanism, b) Copy-and-paste mechanism, 2. Retro-transposons encode reverse transcriptase, and make DNA copies of RNA transcripts; new, DNA copies integrate at different sites (in, eukaryotes only).
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Broad Institute, MIT
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Insertion sequence (IS) elements:, 1., , Simplest type of transposable element found in bacterial, chromosomes and plasmids., , 2., , Encode gene (transposase) for mobilization and insertion., , 3., , Range in size from 768 bp to 5 kb., , 4., , IS1 first identified in E. coli’s glactose operon is 768 bp long and is, present with 4-19 copies in the E. coli chromosome., , 5., , Ends of all known IS elements show inverted terminal repeats, (ITRs).
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Insertion sequence (IS) elements:, Integration of an IS element may:, •, , Disrupt coding sequences or regulatory regions., , •, , Alter expression of nearby genes., , •, , Cause deletions and inversions in adjacent DNA., , •, , Result in crossing-over.
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Transposition of insertion sequence (IS) elements:, 1., , Original copy remains in place; new copy inserts randomly., , 2., , Transposition requires transposase, coded by the IS element., , 3., , IS element otherwise uses host enzymes for replication., , 4., , Transposition initiates when transposase recognizes ITRs., , https://en.wikipedia.org/wiki/Transposase
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Transposition of insertion sequence (IS), elements:, 1. Site of integration = target site., 2. Staggered cuts are made in DNA at target site, by transposase, IS element inserts, DNA, polymerase and ligase fill the gaps (note--transposase behaves like a restriction, enzyme)., 3. Small direct repeats (~5 bp) flanking the, target site are created.
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Fig. 7.20, Integration of IS element in chromosomal DNA.
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Transposons (Tn):, •, , Similar to IS elements but are more complex, structurally and carry additional genes, , •, , 2 types of transposons:, 1. Composite transposons, 2. Non-composite transposons
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Composite transposons (Tn):, •, , Carry genes (example might be a gene for antibiotic resistance), flanked on both sides by IS elements., , •, , Tn10 is 9.3 kb and includes 6.5 kb of central DNA (includes a gene, for tetracycline resistance) and 1.4 kb inverted IS elements., , •, , IS elements supply transposase and ITR recognition signals.
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IS elements, , IS (Insertion sequence) elements: The simplest, transposons, are autonomous units, each of, which codes only for the proteins needed to, sponsor its own transposition., Inverted repeats + transposase genes
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Keywords, 4), , Transposase:, , an, , enzyme, , that, , binds, , to, , ends, , of, , transposon and catalyses the movement of the transposon, to another part of the genome by a cut and paste, mechanism or a replicative transposition mechanism., , 5) Resolvase or Recombinase (nuclease enzyme):, According to the binding residue, the recombinases are, grouped to Tyr- and Ser-recombinase. The enzyme activity, involved, , in, , site-specific, , recombination, , between, , two, , transposons present as direct repeats in a co-integrate, structure.
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co-integrate structure, , A, , number, , of, , bacterial, , transposons, , encode, , two, , distinct, , recombinases that participate in their transposition to other DNA, molecules., , In, , the, , initial, , step, , mediated, , by, , the, , element's, , transposase, a cointegrate is formed between the transposoncontaining, , donor, , DNA, , and, , the, , target, , molecule., , In, , this, , transpositional intermediate, the donor and target DNAs are joined, together by copies of the duplicated transposon, one copy, occurring at each donor–target junction. The second step in the, pathway, cointegrate resolution, is a site-specific recombination, performed by the transposon's resolvase protein, acting at a site,, called res, located within the transposon.
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Composite transposons, Composite transposons: Composite genetic elements, are larger than IS elements and contain one or more, protein-coding genes in addition to those required for, transposition e.g. Tn5, Tn9, Tn10., , Two IS elements + antibiotic resistance gene(s).
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Structure of Composite transposons, , Inverted, , Genes for, , Repeats, , Transposition, , Inverted, Structural genes, Repeats, , antibiotic resistance genes, , Inverted IS
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Non composite transposons, , Non-composite mobile genetic elements are those which, lack IS elements on its ends e.g. Tn3 and Tn7., Inverted, , repeats, , resistance gene (s)., , +, , transposase, , gene, , +, , antibiotic
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Mechanism, All transposons use a common mechanism in which, staggered nicks are made in target DNA, the transposon is, joined to the protruding ends, and the gaps are filled., , The order of events and exact nature of the connections, between transposon and target DNA determine whether, transposition is replicative or nonreplicative.
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Replicative Transposon, • Replicative transposon is first replicated and then one of the copy, will move to the another location in the genome. Thus, the, transposon will remain on its original position. “Copy and Paste”, , • Replicative, , transposition, , involves, , two, , acts, , the, , types, , of enzymatic, , activity:, a) Transposase, , that, , on, , ends, , of, , the, , original, , transposon and b) Resolvase that acts on the duplicated, copies., , • A, , group, , of transposons, , replicative transposition., , related, , to, , TnA, , move, , only, , by
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Replicative Transposon, •, , Replicative transposition occurs through a cointegrate formation., , •, , A co-integrate structure is produced by fusion of two replicons, one, originally possessing a transposon, the other lacking it; the co-integrate, has copies of the transposon present at both junctions of the replicons,, oriented as direct repeats., , •, , Resolution occurs by a homologous recombination reaction between the, two copies of the transposon in a co-integrate., , •, , The reaction generates the donor and target replicons, each with a copy of, the transposon., , •, , Resolvase is the enzyme activity involved in site-specific recombination, between two transposons present as direct repeats in a co-integrate, structure.
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Replicative Transposon, • The reactions involved in generating a cointegrate have been, defined in detail for phage Mu., •, , The process starts with the formation of the strand transfer, complex (sometimes also called a crossover complex)., , • The donor and target strands are ligated so that each end of the, transposon sequence is joined to one of the protruding single, strands generated at the target site., • The crossover structure contains a single-stranded region at each of, the staggered ends. These regions are pseudoreplication forks that, provide a template for DNA synthesis. (Use of the ends as primers, for replication implies that the strand breakage must occur with a, polarity that generates a 3 ′ –OH terminus at this point.)
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Non-replicative Transposon, • Non-Replicative transposon leaves its original place and, move to the another location in the genome. “Cut and Paste”, • This type of mechanism requires only a transposase., • The insertion elements and composite transposons like Tn5, and Tn10 use this mechanism., • Non-replicative transposons leave a break in the donor, molecule which is lethal to the cell unless it is repaired.
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Non-replicative Transposon, Tn5. Transposition initiated by Tnp binding to the transposonspecific ESs and the formation of a highly ordered nucleoprotein, complex (synaptic complex, SC) through a process called synapsis., The SC contains two protomers of Tnp, which exist as a dimer, and, two ESs. Catalytic cleavage occurs when an activated H2O, coordinated by Mg2+ nicks the transferred DNA strand (TS) on both, sides of the transposon, through a nucleophilic attack, forming a 3′hydroxyl group. The free 3′-hydroyxl group acts as a nucleophile, and cleaves the non-transferred DNA strand (NT), forming a, hairpin. A second activated water molecule resolves the hairpin,, resulting in a double-stranded DNA cleavage product. The postcleavage synaptic complex is now free to bind to target DNA, through target capture. The 3′-hydroxyl group of the transposon, end attacks the phosphodiester backbone of target DNA during, strand transfer. A 9-bp duplication in the target results, due to the, staggered strand transfer reactions followed by DNA repair by host, enzymes., , ME- Mosaic Ends
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Models of transposition:, •, , Similar to that of IS elements; duplication at target sites, occurs., , •, , Transposition may be replicative (duplication = copy and, paste), but it can also be non-replicative (transposon lost, from original site = cut and paste)., , •, , Movement of a transposon from one genome (e.g., plasmid), to another (e.g., chromosome)can cointegrate transposon to, both genomes (duplication) in the case of replicative., , •, , Result in same types of mutations as IS elements:, insertions, deletions, changes in gene expression, or, duplication.
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Fig. 7.22, Recombination, crossing-over, and duplication of a, transposable element.
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Transposable elements in eukaryotes:, Barbara McClintock (1902-1992), Cold Spring Harbor Laboratory, NY, Nobel Prize in Physiology and Medicine 1983, “for her discovery of mobile genetic elements”, •, , Studied transposable elements in corn (Zea mays), 1940s-1950s, (formerly identified as mutator genes by Marcus, Rhoades 1930s), , •, , Also known for work demonstrating crossing over as, part of the chromosomal basis of inheritance., , •, , Biographical sketch, pp. 155-156
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General properties of plant transposons:, •, , Possess ITR sequences and generate short repeats at target sites., , •, , May activate or repress target genes, cause chromosome, mutations, and disrupt genes., , •, , Two types:, , •, , •, , Autonomous elements transpose themselves; possess, transposition gene., , •, , Nonautonomous elements do not transpose themselves; lack, transposition gene and rely on presence of another Tn, , McClintock demonstrated purple spots in otherwise white corn (Zea, mays) kernels are results of both these types of transposable, elements.
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McClintock’s discovery of transposons in corn:, •, , c/c = white kernels and C/- = purple kernels, , •, , Kernal color alleles/traits are “unstable”., , •, , If reversion of c to C occurs in a cell, cell will produce purple, pigment and a spot., , •, , Earlier in development reversion occurs, the larger the spot., , •, , McClintock concluded “c” allele results from a non-autonomous, transposon called “Ds” inserted into the “C” gene (Ds =, dissassociation)., , •, , Autonomous transposon “Ac” controls “Ds” transposon (Ac =, activator).
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Fig. 7.24, Transposon effects on corn kernel color.
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McClintock’s discovery of transposons in corn (cont.):, •, , Ac element is autonomous/Ds element is nonautonomous., , •, , Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an, 807 amino acid transposase., , •, , Ac activates Ds; Ds varies in length and sequence, but possesses, same ITRs as Ac., , •, , Many Ds elements are deleted or rearranged version of Ac; Ds, element derived from Ac., , •, , Ac/Ds are developmentally regulated; Ac/Ds transpose only during, chromosome replication and do not leave copies behind.
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Fig. 20.12 2nd edition, Structure of Ac autonomous and Ds nonautonomous transposable elements in corn.
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Fig. 7.25, Ac transposition mechanism during chromosome replication.
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Ty elements in yeast:, •, , Similar to bacterial transposons; terminal repeated sequences,, integrate at non-homologous sites, with target site duplication., , •, , Ty elements share properties with retroviruses, retrotransposons:, •, , Synthesize RNA copy and make DNA using reverse transcriptase., , •, , cDNA integrates at a new chromosomal site., , Fig. 7.26
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Drosophila transposons:, •, , ~15% of Drosophila genome thought to be mobile., 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 present in the cytoplasm, which, makes them stable in the P strain (but unstable when crossed to, the wild type female; female lacks repressor in cytoplasm)., •, Used experimentally as transformation vectors.
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Female, No P elements (M elements), DNA + cytoplasm, No repressor, Male, P elements, DNA only, , Offspring, P elements, No repressor, Unstable germ line, , Female, P elements, DNA + cytoplasm, Repressor, Male, Stable, No P elements, DNA only
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Fig. 7.28 Illustration of the use of P elements to introduce genes, into the Drosophila genome
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•, , The structure of Ty1 is analogous to that of retroviral proviruses., , •, , The most highly characterized Ty1 element is Ty1-H3, which was isolated following its, retrotransposition into plasmid DNA., , •, , 5918 base pairs (bp) in length with 334 bp direct repeats, or LTRs, at each end., , •, , Ty1 LTRs, have the dinucleotide inverted repeat, 5′-TG…CA-3′ at their termini, and are, composed of three distinct domains-U3, R and U5., , •, , These domains are defined by their position in the major sense-strand transcript expressed, from Ty1 DNA., , •, , The 38-nucleotide U5 region and 240-nucleotide U3 region are unique to the 5′ and 3′ end, of the Ty1 RNA, respectively, while the R region of 56 nucleotides is repeated at both ends, of the processed transcript., , •, , Functional Ty1 elements encode two partially overlapping open reading frames: GAG, (historically known as TYA1) and POL (TYB1).
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•, , The last 3 nucleotides of the R region of the 5′ LTR encode the first codon of GAG., , •, , The GAG ORF encodes a single functional protein with capsid and nucleic acid, chaperone functions., , •, , The POL ORF is in the +1 frame relative to GAG and overlaps the last 38 base pairs, of GAG. POL encodes three proteins with catalytic activity: protease (PR), integrase, (IN), and reverse transcriptase/RNase H (RT/RH)., , •, , Ty1 does not contain an equivalent of the retroviral ENV gene or any remnant of, one.
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Biological Significance of, Transposons, • They provide a means for genomic, change and variation, particularly in, response to stress (McClintock’s, "stress" hypothesis), (1983 Nobel lecture, Science 226:792), , • or just "selfish DNA"?, • No known examples of an element, playing a, normal role in, development.
