Tn10

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Tn10 is a transposable element, which is a sequence of DNA that is capable of mediating its own movement from one position in the DNA of the host organism to another. There are a number of different transposition mechanisms in nature, but Tn10 uses the non-replicative cut-and-paste mechanism. [1] The transposase protein recognizes the ends of the element and cuts it from the original locus. The protein-DNA complex then diffuses away from the donor site until random collisions brings it in contact with a new target site, where it is integrated. To accomplish this reaction the 50 kDa transposase protein must break four DNA strands to free the transposon from the donor site, and perform two strand exchange reactions to integrate the element at the target site. This leaves two strands unjoined at the target site, but the host DNA repair proteins take care of this. The target site selection is essentially random, but there is a preference for the sequence 5'-GCTNAGC-3'. The 6-9 base pairs that flank the sequence also influence selection of the insertion site. [2]

Cut-and-paste transposition does not cause an increase in the number of transposons per se: there is one copy at the start and one copy at the end. If this was the end of the matter the transposon would perish by genetic drift and the loss of copies owing to the occasional failure to achieve successful integration at the target site. However, the transposon has a mechanism to favor transposition immediately after a replication fork passes through, leaving a hemimethylated copy of Tn10 on each sister chromosome. Since transposition is favored when Tn10 is hemimethylated, the transposon on one sister chromosome can hop somewhere onto the other chromosome so that two copies of the transposon end up on one chromosome. [3]

Tn10 has a composite structure and it is composed of a pair of insertion sequence elements (IS10) flanking five genes. Only one of the IS10 elements encodes a functional transposase. [4] Since the ends of the IS10 element contain the transposase recognition sites, Tn10 has a total of four such sites. If the transposase binds the two recognition sites flanking an IS10 element, the IS10 element undergoes transposition independently of the larger composite structure. If the transposase binds the two outermost recognition sites, the whole composite Tn10 structure undergoes transposition.

Two of the five genes encoded by the central portion of Tn10, tetA and tetR , confer resistance to the antibiotic tetracycline. The TetA protein is an efflux pump. It has served as a model system for such proteins and has accumulated hundreds of publications indexed in PubMed. The functions of the other three genes, jemA, jemB and jemC, are unknown but they may implicated in heavy metal resistance or oxidative stress. [5]

The Tn10/IS10 transposase is closely related to another composite transposon, Tn5/IS50, which harbors a gene for kanamycin resistance in the unique (i.e. non-repeated) central region of the transposon.

The Tn10 transposon is often used in genetics to transfer and select-for genes of interest from one organism into the chromosome of another.

The mechanism of Tn10 transposition has served as a model system and the archetype for the cut-and-paste mechanisms. However, the transposase is difficult to work with in vitro and the Tn5 transposase was the first to be crystallized. Tn10 was one of the great work-horses of bacterial genetics for many years during which it served as a useful tool. A commercial kit for Tn5 transposition is commercially available and is extensively used in post-genomic technologies.

Two comprehensive reviews of Tn10 biology are available as chapters in the book Mobile DNA and Mobile DNA II. [6] [7]

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Insertion sequence

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Knockout rat

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Ac/Ds transposable controlling elements was the first transposable element system recognized in maize. The Ac Activator element is autonomous, whereas the Ds Dissociation element requires an Activator element to transpose. Ac was initially discovered as enabling a Ds element to break chromosomes. Both Ac and Ds can also insert into genes, causing mutants that may revert to normal on excision of the element. The phenotypic consequence of Ac/Ds transposable element includes mosaic colors in kernels and leaves in maize.

Transposable elements are short strands of repetitive DNA that can self-replicate and translocate within the eukaryotic genome, and are generally perceived as parasitic in nature. Their transcription can lead to the production of dsRNAs, which resemble retroviruses transcripts. While most host cellular RNA has a singular, unpaired sense strand, dsRNA possesses sense and anti-sense transcripts paired together, and this difference in structure allows an host organism to detect dsRNA production, and thereby the presence of transposons. Plants lack distinct divisions between somatic cells and reproductive cells, and also have, generally, larger genomes than animals, making them an intriguing case-study kingdom to be used in attempting to better understand the epigenetics function of transposable elements.

Conservative transposition

Transposition is the process by which a specific genetic sequence, known as a transposon, is moved from one location of the genome to another. Simple, or conservative transposition, is a non-replicative mode of transposition. That is, in conservative transposition the transposon is completely removed from the genome and reintegrated into a new, non-homologous locus, the same genetic sequence is conserved throughout the entire process. The site in which the transposon is reintegrated into the genome is called the target site. A target site can be in the same chromosome as the transposon or within a different chromosome. Conservative transposition uses the "cut-and-paste" mechanism driven by the catalytic activity of the enzyme transposase. Transposase acts like DNA scissors; it is an enzyme that cuts through double-stranded DNA to remove the transposon, then transfers and pastes it into a target site.

DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. It is important to note that DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.

Tc1/mariner is a class and superfamily of interspersed repeats DNA transposons. The elements of this class are found in all animals, including humans. They can also be found in protists and bacteria.

References

  1. ↑ Bender J, Kleckner N (June 1986). "Genetic evidence that Tn10 transposes by a nonreplicative mechanism". Cell. 45 (6): 801–15. doi:10.1016/0092-8674(86)90555-6. PMID   3011280. S2CID   43227252.
  2. ↑ Bender J, Kleckner N (September 1992). "Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence". Proceedings of the National Academy of Sciences of the United States of America. 89 (17): 7996–8000. doi: 10.1073/pnas.89.17.7996 . PMC   49842 . PMID   1325639.
  3. ↑ Roberts D, Hoopes BC, McClure WR, Kleckner N (November 1985). "IS10 transposition is regulated by DNA adenine methylation". Cell. 43 (1): 117–30. doi:10.1016/0092-8674(85)90017-0. PMID   3000598. S2CID   31933078.
  4. ↑ Foster TJ, Davis MA, Roberts DE, Takeshita K, Kleckner N (January 1981). "Genetic organization of transposon Tn10". Cell. 23 (1): 201–13. doi:10.1016/0092-8674(81)90285-3. PMID   6260375. S2CID   35704714.
  5. ↑ Chalmers R, Sewitz S, Lipkow K, Crellin P (2000) Complete nucleotide sequence of Tn10" J Bacteriol 182: 2970-2972
  6. ↑ Kleckner N (1989) Transposon Tn10. In: Berg DE, Howe MM, editors. Mobile DNA. Washington, D.C.: American Society for Microbiology. pp. 227-268.
  7. ↑ Haniford DB (2002) Transposon Tn10. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington, D.C.: American Society for Microbiology. pp. 457 - 483.
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