Jumping Genes A Buyers' Guide

Courtesy of Ivan Rayment CAUGHT IN MID HOP: Structure of the Tn5 transposase/DNA complex No one believed Barbara McClintock in 1951 when she first described DNA that "jumped" from site to site within maize chromosomes, altering the expression of genes near the sites of integration. In due course, these transposable elements, or transposons, were found to be ubiquitous in nature, and 30 years later McClintock won the Nobel Prize. Today transposons have gone from molecular oddity to molecular workhorse, finding use in academic labs and large sequencing centers. All transposons possess at least two characteristics: They encode transposases, which catalyze the transposition reaction; and they are flanked by short, inverted repeats that are recognized by, and a signature of, the encoded enzyme. Some also carry additional genes, such as antibiotic-resistance markers and regulatory sequences. Each transposase has a unique catalytic mechanism. Some transposons (Tn5, Tn7, and Tn10, for example) literally hop from place to place, removing themselves from the donor site before integrating into the recipient. Other elements (Tn3 and Mu, for example) are replicative; they copy (and thus propagate) themselves. For Tn5, the transposition reaction involves binding of monomeric transposase molecules to each of the element's flanking inverted repeats, followed by dimerization of the two complexes to form a "synapti complex." The transposon sequence is then excised, releasing the synaptic complex, which then captures the target molecule and inserts the transposable DNA. In the process, the sequence of nine nucleotides at the insertion site is duplicated.1 Any DNA between the transposon' terminal repeats will jump, so transposons make useful research tools. Recognizing this, several companies now offer transposon-based reagents and kits for use in sequencing and mutagenesis projects. Transposon-based sequencing, an alternative to primer "walking" approaches, employs transposons modified to contain outward-facing, sequencing primer binding sites (enabling bidirectional sequencing reads into the insertion point) and selectable markers. One benefit of this method: It eliminates the need for custom primers and subcloning. This approach has found use at large sequencing facilities. When Mark Stapleton, project director of full-length cDNA sequencing for the Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, needed a flexible system to sequence fruit fly transcripts, he turned to Finnzymes' Template Generation System. And, when researchers at The Institute for Genomic Research (TIGR) are polishing up their sequencing projects, they turn to New England Biolabs' GPS-1 system, says John Gill, TIGR's research and development manager. Transposons also can be used to create gene knockouts, since the insertion of a transposon into a gene usually will create a null mutation. This feature extends the application of transposon-based products into the realm of functional genomics and possibly even gene therapy (see sidebar at bottom). Qijing Zhang, assistant professor of veterinary preventive medicine, Ohio State University, used Epicentre's EZ::TN Transposome to generate a library of transposon mutants in Campylobacter jejuni, a human pathogen that causes food-borne illnesses. Similarly, Luis Actis, associate professor of microbiology, Miami University, Oxford, Ohio, used Transposomes to study another human pathogen, Acinetobacter baumannii. Paradigm Genetics, a Research Triangle Park, NC-based biotech company, has merged the sequencing and mutagenesis applications of transposons in an approach called TAG-KO™, or transposon-arrayed gene knockouts. The company creates a library of transposition events in a cosmid library to make mutations, and then sequences the library to create "an annotated collection of insertional gene-disruption vectors," for use in creating gene knockouts.2 Though most commercially available kits employ the bacterial transposons, Tn5 or Tn7, or the bacteriophage Mu transposon, eukaryotic counterparts also exist, such as the yeast Ty1 and Drosophila P elements. Evidence suggests that HIV-1 and other retroviral integrases are related to bacterial transposons, and even the process of immunoglobulin class switching could be distantly related.1 APPLICATION NOTES The accompanying table lists the four major suppliers of transposon-based tools and kits: Epicentre of Madison, Wis.; Finnzymes of Espoo,Finland; Invitrogen of Carlsbad, Calif.; and New England Biolabs of Beverly, Mass. These companies offer reagents based on Tn5, Tn7, or phage Mu, but generally, they also have tweaked the systems to make them more robust and efficient. Epicentre's EZ::TN system, for instance, employs a hyperactive transposase and modified inverted repeat sequences to boost transposition efficiency 1,000-fold. Most systems employ in vitro transposition reactions to create a random library of insertion events. Epicentre, however, also offers an in vivo transposition reagent called a Transposome™, which is a stable transposase-transposon complex that can be introduced into cells by electroporation. All systems include a selectable marker--kanamycin, chloramphenicol, tetracycline, or dihydrofolate reductase (DHFR)--and a few include oriV, an inducible origin of replication that permits higher yields of low-copy-number DNA. Still others contain the conditional Escherichia coli origin of replication, R6K*ori, for use in "rescuing" insertion sites. Simply shear genomic DNA for clones of interest, self-ligate, and transform into cells expressing the pir gene product. In some cases, the transposon is designed so that the bulk of the insertion can be excised to leave a short segment for linker-scanning studies. Other applications include transcription, in which the transposon contains a bacteriophage T7 promoter, and selection of secreted and envelope proteins. Suppliers of Transposon ToolsClick for larger version (35K) New applications are forthcoming. "Now that the focus of the scientific community is gradually shifting from genome sequencing projects into analysis of the encoded proteins, new kinds of tools based on transposons are also emerging," says Atto Forsberg, export manager at Finnzymes. That company will soon be launching a transposon-based system designed for inserting translational stop codons into target genes in all reading frames, which can be used to study the effect of C-terminal deletions in the encoded target protein. At New England Biolabs, Fiona Stewart, product manager, announ-ces that the company plans to release a new system that will allow selection of transposition products in yeast. Hillary E. Sussman (hillary@sciwriter.com) is a freelance writer in Port Jefferson, NY. References 1. D.R. Davies et al., "Three-dimensional structure of the Tn5 synaptic complex transposition intermediate," Science, 289:77-85, 2000. 2. L. Hamer et al., "Gene discovery and gene function assignment in filamentous fungi," Proc Natl Acad Sci, 98:5110-5, 2001. "JUMPING" GENE THERAPY Gene therapy typically uses viral vectors to replace defective genes with functional ones. But though the process is generally efficient, it is also problematic: Viral vectors can elicit an immune response, induce toxicity, and cause insertional mutagenesis. Earlier this year a gene-therapy trial using retroviruses was halted when two of the participants developed T-cell leukemia as a result of insertion of the therapeutic gene into LMO-2, an oncogene.1 The Sleeping Beauty Transposon system™, the proprietary technology of Discovery Genomics (www.discoverygenomics.net), offers a nonviral alternative. Sleeping Beauty (so named because the transposase was "awakened from a long evolutionary sleep") was resurrected from ancient salmonid sequences in the Tc1/mariner family of transposable elements that were active roughly 10 million to 14 million years ago.2 The system contains the revived Sleeping Beauty transposase and a transposon bounded by a pair of inverted terminal repeats, between which researchers may insert any desired sequence. "The way to look at the transposon is as a molecular cargo truck into which you can put whatever gene you want," says Perry Hackett, chief science officer of Discovery Genomics, which is located in Minneapolis. According to Hackett, Sleeping Beauty is the first known, active vertebrate transposon; scientists have used it successfully in several cultured vertebrate systems, including human cells and mouse embryos. Long-term expression of a therapeutic gene has been observed in hepatocytes of hemophilic adult mice using Sleeping Beauty, suggesting potential gene-therapy benefits.3 Hackett says, "if we're successful soon in showing that it will be a good gene-therapy vector, then of course we can license the system for virtually any disease in which you'd want lifelong expression of a gene." And what about the promiscuous insertion of genes into undesirable chromosomal locations? "We have a patent pending where we designed some safeguards into our transposon that should prohibit that from happening," he replies. References 1. B.A. Maher, "More gene therapy trials halted," The Scientist, Jan. 16, 2003; available online at www.the-scientist.com/news/20030116/06 2. Z. Ivics et al., "Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells," Cell, 91:501-10, 1997. 3. S.R. Yant et al., "Somatic Integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system," Nat Genet, 25:35-41, 2000. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Jumping Genes A Buyers' Guide

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Courtesy of Ivan Rayment
CAUGHT IN MID HOP: Structure of the Tn5 transposase/DNA complex