View metadata, citation and similar papers at core.ac.uk brought to you by CORE R244 Dispatch provided by Elsevier - Publisher Connector Zebrafish genetics: Harnessing horizontal gene transfer Eric S. Weinberg The promiscuous spread of Tc1/mariner transposons involvement in the particular developmental process across species implies that host factors are relatively defective in the mutant. More systematic global unimportant for their transposition. Heterologous approaches are of course necessary. Positional cloning will elements can integrate on expression of the undoubtedly be effective, as demonstrated by the use of a corresponding transposases, an approach that should map-position-based approach for the recent identification greatly facilitate genetic analysis in the zebrafish. of one eyed pinhead as a gene encoding a ligand related to epidermal growth factor (EGF) [5]. But such approaches Address: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. are labor intensive and time consuming, even when a high- E-mail: [email protected] resolution map is available. Current Biology 1998, 8:R244–R247 A promising alternative to positional cloning is the use of http://biomednet.com/elecref/09609822008R0244 a pseudotyped retroviral vector which can be transmitted © Current Biology Ltd ISSN 0960-9822 at high efficiency through the germline after integration into zebrafish DNA [6]. Such a vector has recently been The zebrafish, Danio rerio, has become a favored organism shown to be effective in causing mutations by insertion for attempts to identify genes that are important in devel- and allowing identification of the affected locus by isola- opmental and physiological processes in vertebrates. tion of the vector tag [7,8]. And a second insertional muta- Among the advantages of this organism [1,2] are the genesis method may be close at hand, thanks to the optical clarity of the embryos, the ease of breeding fish exploitation of a class of transposable elements that are and obtaining gametes, a generation time short enough to known to be promiscuous in their transmission from host allow practical genetic experiments, and amenability to to host during evolution [9,10]. the induction of mutations at high frequency. These fea- tures of the zebrafish have permitted large-scale mutant Gene transfer between species, a phenomenon known as hunts after treatment of fish with the chemical mutagen horizontal gene transmission, appears to have played an ethyl-N-nitrosourea (ENU) [3,4]. important role in the evolution of the Tc1/mariner super- family of transposons [11–14]. These DNA elements, Several thousand mutants were initially identified in two found in most, if not all, animal phyla, transpose by a cut- separate screens, and many of these mutants have already and-paste mechanism via a DNA intermediate, using an been grouped into several hundred genes by complemen- element-encoded transposase of the D,D(35)E superfam- tation analysis [3,4]. The observed phenotypes included ily [14,15]. They have short, inverted terminal repeats defects in development of a number of organs and tissues, and duplicate a TA target site upon insertion. Although disturbances in axonal pathfinding, defects in cell motil- the amino-acid identities within the transposase open ity, and abnormal gastrulation and cleavage patterns. The reading frame between the mariner and Tc1 subgroups are finding that most of the complementation groups had only only 10–20%, particular amino acids scattered throughout one allele indicates that each of the screens was far from the protein are conserved in virtually all family members saturation. A number of the mutants were due to new [14]. Horizontal transmission is inferred from the occur- alleles of genes previously identified by gamma-ray- rence of very similar transposon sequences in distantly induced or spontaneous mutations [1]. At this time, many related species [11–14]. other groups are employing variations of these screening methods to identify additional mutants. It is expected that The extremely broad range of these elements, an indica- that, within a year, hundreds of additional zebrafish tion of the lack of importance of species-specific host mutants will be identified with defects in developmental factors in the transposition process, has led to the sugges- processes such as gastrulation, pattern formation, organo- tion that Tc1/mariner elements could be used as general- genesis and cell differentiation. ized DNA vectors [12]. The first demonstration that a Tc1/mariner element could function in a foreign species The obvious next step is to identify the genes in which was the use of Mos1, an autonomous mariner-like element these mutations have occurred. A surprisingly large from Drosophila mauritiana, to direct integration of a non- number of the affected loci (perhaps a score at this point) autonomous target element into the genome of Drosophila have already been identified using a candidate gene melanogaster [16]. The Mos1 transposon was recently approach. However, such identifications depend on having shown to transpose in the genome of the trypanosomatid cloned genes with expression patterns suggestive of protozoan Leishmania major, resulting in the inactivation of Dispatch R245 Figure 1 (a) Constructs used for Sleeping Beauty- (a) pT/neo (b) pTc3GFP mediated genomic integration [9]. pT/neo is neo unc-22 unc-22 the substrate construct composed of inverted repeats of the salmonid Tc1-like element L48685 from Tanichtlys albonubes flanking a SV40 EF1α enhancer/ promoter promoter neo gene under the control of the SV40 SV40 poly(A) enhancer/promoter. pSB10 contains the GFP SV40 poly(A) pSB10 Tc3GFP resurrected Sleeping Beauty transposase SB transposase open reading frame under the control of the human CMV immediate early (IE) gene (b) CMV IE enhancer/promoter. Constructs used for enhancer/ promoter Tc3 element insertion in the zebrafish [10]. Tc3A mRNA pTc3GFP is the substrate construct Cap AAAA containing the inverted repeats from Current Biology transposon Tc3 flanking a GFP open reading frame expressed from a Xenopus EF1α Also shown is the Tc3GFP element after sequences; thick blue lines, flanking zebrafish promoter. As the Tc3 used to make this integration into zebrafish DNA. The elements sequences; red arrows, transposon inverted construct was derived from an integration in from both of these contexts were mobilized by repeats; green arrows, enhancer/promoter C. elegans, the inverted repeats are flanked injection of Tc3A transposase mRNA elements; other elements are labelled and by unc-22 gene sequence which was the prepared in vitro. Grey, transposon variously coloured. integration site of that particular insertion. sequences; thick black lines, flanking plasmid at least one specific gene [17]. This was the first demon- between terminal inverted repeats from a salmonid-type stration that a Tc1/mariner transposase from one species element (Figure 1a). Integration of the donor element was can function in species of a different order. These results enhanced over background (a certain level of integration are consistent with the finding that recombinant purified was probably due to non-legitimate integrations typical in transposase is the only protein factor necessary for the transfected cells) in all three cell types, most notably in transposition of Tc1/mariner elements in vitro [18,19]. the HeLa cells which showed a 20-fold increase in G-418- resistant colonies. A promising system for genetic transformation and insertional mutagenesis that works in vertebrates has The enhancement of integration required a full-length recently been developed by Ivics et al. [9] using a Tc1-like transposase, as proteins that were incompletely changed to transposase encoded by a sequence reconstructed from a the Sleeping Beauty consensus, or that lacked the catalytic group of fish transposons of the salmonid Tc1-like sub- domain, did not promote integration. The presence of two group. The salmonid transposons, like other vertebrate inverted repeats in the donor substrate was essential for Tc1/mariner elements cloned and sequenced thus far, integration. Southern blots of DNA from the G-418 contain transposase pseudogenes and so cannot function selected cells showed that transgenes were integrated into autonomously. Aligning the sequence of 12 partial different locations of the human genome, and sequencing salmonid-type elements, Ivics et al. [9] restored an open of junction fragments revealed that they had the expected reading frame by removing premature translational stop duplicated flanking TA dinucleotides and intact inverted codons and frameshifts and systematically changing the repeat sequences. These findings indicated that the amino acids at 24 positions, creating a putative full-length Sleeping Beauty transposase can function faithfully in the transposase gene that matched the aligned 340 amino acid heterologous cultured cells. Thus, the transposase is a consensus sequence. This procedure resulted in the resur- potentially effective reagent for introduction of DNA for rection of an active transposase gene that was endearingly transgenesis and transposon tagging in vertebrates, includ- named Sleeping Beauty. ing the zebrafish. After showing that the reconstructed transposase had an In a recent issue of Current Biology, Raz et al. [10] reported active nuclear localization signal domain and could specifi- a direct demonstration that a heterologous Tc1/mariner cally bind inverted repeat