Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line

Bessereau, Jean-Louis; Wright, Ashley P.; Williams, Daniel C.; Schuske, Kim; Davis, Matthew L.; Jørgensen, Erik M.

doi:10.1038/35092567

Cited by 145 publications

(143 citation statements)

References 21 publications

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“…1). An expression construct for the QQR nuclease was prepared with the coding sequence under the control of the well-characterized C. elegans 16-48 heat-shock promoter (36).…”

Section: Resultsmentioning

confidence: 99%

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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169

124

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Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...

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“…1). An expression construct for the QQR nuclease was prepared with the coding sequence under the control of the well-characterized C. elegans 16-48 heat-shock promoter (36).…”

Section: Resultsmentioning

confidence: 99%

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

169

124

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“…We compiled and categorized, from all available sources, more than 2,000 endogenous transposon insertion alleles for which the exact insertion site in the C. elegans genome is known. In addition, a heterologous transposon insertion system has been introduced in C. elegans 31 . The advantage of this system is the absence of a background of endogenous transposons.…”

Section: Discussionmentioning

confidence: 99%

Targeted gene alteration in Caenorhabditis elegans by gene conversion

Barrett¹,

Fleming²,

Göbel³

2004

Nat Genet

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“…Using exogenous transposon systems would be a way to circumvent these limitations. Indeed, the mariner element isolated from D. mauritiana has been demonstrated to be mobile in C. elegans (Bessereau et al, 2001), and has served as a useful tool for tagged mutagenesis.…”

Section: Significance In Biotechnology Applicationsmentioning

confidence: 99%

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

Takagi

Koga

2008

Heredity

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Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/ Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.

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Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line

Cited by 145 publications

References 21 publications

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Targeted gene alteration in Caenorhabditis elegans by gene conversion

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

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