Copyight 0 1996 by the Genetics Society of America

Germline Transformation of Virilis Mediated by the Transposable Element hobo

Elena R. Lozovskaya,* Dmitry I. Nurminsky,* Daniel L. Had* and David T. Sullivan’ *Department of Organismic and Euolutionaly Biology, Haruard University, Cambridge, Massachusetts 02138 and tDepartment of Biology, Syracuse University, Syracuse, New York 13244-0001 Manuscript received July 17, 1995 Accepted for publication September 21, 1995

ABSTRACT A laboratory strain of Drosophila uirilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp7Odriven hobo transposasecoding sequence. The rate of transformation was 0.5% per fertile GO . Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known wide- spread distribution of hobo in diverse , suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of other than D. melanogaster.

HERE is at present considerable interest in the Thusfar, most transformation experiments have T application of germline transformation to the ge- been carried out with D. melanogaster as a model organ- netic manipulation of genomes, especially ism. One advantage is that the experimental procedures those of agricultural pests and vectors of human dis- for injection of DNA and the treatment of embryos are ease (KIDWELL 1993; WARRENand CRAMPTON 1994). well established (SPRADLING1986). Another advantage For a number of reasons, transposable elements are is that any one of a number of genetic marker systems strong candidates as potential vectors. An experimen- can be used in vector construction and transformation. tal paradigm already exists in thegermline transforma- A third advantage is that the method is known to work, tion of (RUBINand SPRADLING which means that the injected DNA must persist long 1982; BLACKMANet al. 1989; LIDHOLMet al. 1993). enough to get into the nucleus and become incorpo- Some types of transposable elements are distributed rated into the genome. widely among diverse insects andother organisms It is not known, however, how representative D. mela- (LIDHOLMet al. 1991; ROBERTSON1993), suggesting nogaster may be of in general, or even how that whatever genetic functions are required of the representative it may be of other drosophilids. There host genome are either minimalor highly conserved. is considerable variation even among strains of D. mela- In vivo assays based on excision from or transposition nogaster in their tolerance of the experimental proce- between transposon-bearing plasmids affords addi- dures and their ability to be transformed. In species tional evidence for a wide host range for some trans- otherthan D. melanogaster, the embryos may be less posable elements (O’BROCHTAet al. 1991,1994;ATKIN- tolerant of the experimental manipulations necessary SON et al. 1993; HANDLERand GOMEZ1995). Some for transformation. In some organisms, the injected transposable elements found in distantly related spe- DNA may be degraded by nucleases in the cytoplasm cies give evidence of horizontal transmission, includ- and rendered ineffectual. In others, the injected DNA ing mariner-like elements (MARUYAMA and HARTL1991; may be less likely to get into the nucleus or to become ROBERTSONand MACLEOD1993; LOHEet al. 1994; GAR- incorporated into the chromatin.These are all key vari- CIA-FERNANDEZet al. 1995), P elements (DANIELSet al. ables that, in the absence of direct experimental evi- 1990; KIDWELL 1992; CLARKet al. 1994),and hobo (WAR- dence, can only be objects of speculation. REN et al. 1994, 1995). Finally, transposable elements The potential problems caused by variability among of various typeshave been shown to be capable of species provides a strong incentive to investigate the supporting the incorporation of exogenous DNA into feasibility of germline transformation in a variety of the germline of arthropods other than their species insects other than D. melanogaster. Among drosophi- of origin (BRENNANet al. 1984; SCAVARDAand HARTL lids, a strongcase can be made for theutility of germ- 1984; DANIELSet al. 1985; GARZAet al. 1991; SIMONELIG line transformationin D.virilis. With a divergence time and ANXOLABEHERE1991; LIDHOLMet al. 1993; LOUK- of 40 million years (RUSSOet al. 1995), D. virilzs and EMS et al. 1995; D. A. O’BROCHTA,W. D. WARREN,K. J. D. melanogaster have an evolutionary separation that is SAVILLEand P. W. ATKINSON,unpublished results). greatenough that most nonconservednucleotides have had at least one opportunity to become substi- Corresponding authort Daniel L. Hartl, Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Ave., Cam- tuted, yet the separation is not so great that the identi- bridge, MA 02138. E-mail: [email protected] fication of homologous genes is compromised. Fur-

Genetics 142: 173-177 (January, 1996) 174 Lozovskaya E. R. et al.

thermore, the genetic transformation of genes from Sau3A and ligation with the XhoI arms of the replacement D. uirilis into D. melanogaster is coming into increasing vector LambdaScan after half-filling the cohesive ends with the Klenow fragment of DNA polymerase. Lambdascan is a prominence for studies of gene regulation and func- specialized derivative of Lambdam (Stratagene) designed tional divergence. It would contribute significantly to to cloneinserts in thesize range from 8 to 12 kb (NURMINSKY the power of this approach if the reciprocal transfor- and HARTL 1995). The ligated DNA molecules were pack- mation of genes from D. melanogaster into D. virilis aged in vitro using Gigapack XL extracts (Stratagene). The could also be carried out. resulting libraries were screened with a hobo probe obtained byPCR amplification of nucleotides 290-860 in the hobo In this paper, we report that D. virilis can be geneti- sequence. From the positive lambda clones, plasmids were cally transformed with vectors based on the transpos- obtained by in vivo excision with the ExAssist/SOLR system able element hobo. These vectors have previously been (Stratagene) and sequenced with the DyeDeoxyTerminator demonstrated to support germline transformation of D. reagents on an Applied Biosystems373A automated DNA melanogaster (BLACKMANet al. 1989). Thegenetic marker sequencer using the hobwpecific primers Sul-5a (5”CAGCA- GGCCTCGGGTGGTTTT-3’), Sul-7 (5’-ACTCGTAACAAAA- usedin the transformation was a miniwhite cassette, ACACAACG3‘), and SUI-8 (5”CCGAGTATTTTTGGAAAC- which we reasoned would be able to complement a ACC-3’). The unique insertion in strain Dv[hawll-12 was white mutation in D. virilis. Three transgenic insertions recovered and the ends sequenced.Two different insertions were examined at themolecular level and foundto have were recovered fromthe multiply transformed strain the sequencecharacteristics expected of a conventional Dv[hawl] -131. hobo insertion. RESULTS MATERIALSAND METHODS Efficiency of transformation:A total of 1804 D. virilis Drosophila strains: The X-linked w mutation in D. virilis w embryos were injected with the hobo miniwhite trans- was obtained from JEAN DAVID.The eye-color phenotype is formation vector Ei[w+, hawl] (Caw et al. 1991) and bleached white. Based on its linkage to the X chromosome the helper elementHSH2. Among these, 477 GO adults and the phenotypeof the mutantflies, the mutationhas been presumed to be homologous to the X-linked w gene in D. were obtained, of which 398 were fertile. Two fertile melanogaster (ALEXANDER 1976). Further evidence for this in- GO parents yielded progeny that were putative trans- ference was obtained by isolation of a D. vidis P1 clone, using formants, for a rate of 0.5% transformants per fertile a probe from the D. melanogaster w gene, and demonstrating GO. Strains derived from the putative transformants that the wbearing PI clone undergoes in situ hybridization were designated Dv[hawl] -12and Dv[hawl] -131.The with the polytene chromosome region containing theD. virilis w gene (LOZOVSKAYAet al. 1993). The transformation experi- eye-color phenotype of Dv[hawl] -12is very weak and ments described here demonstrate the homology directly by appears variegated. The phenotype of Dv[hawl] -131 is complementation. dark red but distinct from wild type. As shown below, Transformation vector: The hobebased transformation vec- Dv[hawl] -12has a single insertion of the hobo-miniwhite tors have been described previously. The helper elementwas construct whereas Dv[hawl] -131has three different in- a plasmid containing the hsp70:hobo fusion HSHP described in Guw and GELBART(1994; their Figure 1, p. 1637). The sertions. transformation vector was H[w+, hawl] described in Gum et Evidencefor hobo-mediated transformation: The al. (1991; their Figure 4B). Theminiwhite insert in this plasmid first line of evidence for the authenticity of the putative is oriented with the transcriptional start near the 3’ end of transformants was the observation that genomic DNA hobo. In D. melanogaster, the miniwhite gene confers an interme- from both Dv[hawl]-l2 and Dv[hawl]-131, butnot diate w+ phenotype (PIRROTTAet al. 1985). Germline transformation: Plasmid DNA was purified twice from untransformed controls, could support PCR am- in cesium chloride density gradients and used for injection plification using oligonucleotide primers to amplify spe- of embryos. D. virilis w embryos were prepared for injection cifically across the unique hobo-miniwhitejunction (data in the usual manner with manual dechorionation (SPRADLING not shown). 1986). No heat shock was employed. The injected DNA solu- A second line of evidence that the transformations tion contained 450 pg/ml of the transformation vector along were mediated by hobo insertion was in situ hybridization with 150 pg/ml of the helper element. Hybridization in sitw Hybridization to D. virilzs polytene with polytene chromosomes using El[w+, hawl] as chromosomes was carried out as described (LOZOVSKAYAet al. probe. Strain Dv[hawl]-12 yielded a single site of hy- 1993) using the probe mw+, hawl]. bridization at 4°F (Figure 1). Strain Dv[hawl] -131 PCR amplification: Presence of the Nw+, hawl] construct yielded three distinct sites of hybridization at 23C, in the putative transformants was verified by PCR amplifica- 25M, and49C (data not shown). Thesites of hybridiza- tion using the primers Sul4b and Sull spanning the junction between hobo and the miniwhite cassette. Primer Sul4b, in the tion in both strains are euchromatic. 3‘ end of hobo,is 5’-CGTATGGGTGAGTGGACGCAG3’; Cloning and characterization of insertion sites: To primer Sull, near the 5‘ end of the miniwhite gene, is5’- verify that the sites of insertion of H[w+,haw11 were GTTCAGATGCTCGGCAGATGG3’.PCR conditions were 30 mediated by hobo activity, three of the insertions were cycles with 1 min denaturation at 94”, 1 min annealing at58”, isolated fromgenomic libraries preparedfrom the and 3 min extension at 72”. Recovery of ends of hobo insertions: Genomic libraries transformed strains. From the cloned genomic inser- were constructed by partial digestion of genomic DNA with tions, the DNA sequences flanking thesite of insertion Transformation of D. vin'li.5 175

Insertion Flanking sequence

DvfhawfJ-lZ S-TGTGTG~CG~~~~~~~~C~~CAATG-~

Gvfhawl1-131No. 1 S-GCTAACG~G~~~hawf~~~TGTGCmAGC-3

DV~~~W~J-~JINO. 2 S-GCCAAGT~~~~~~~~~TCACCGGTAGCA.~ FIGURE2.-Genomic sequences flanking three indepen- dent insertions of W7[w+,hawl]. The 8-bp direct duplication characteristic of hobo is boxed. The abbreviation hawl inser- tion refers to the presence of the intact 5' and 3' ends of hobo flanking the miniwhite cassette.

an extensive literature on thegenetics and evolution of D. virilis (reviewed in LOZOVSKAYAet al. 1993). The time of divergence of D. uirilis and D. melanogasteris -40 million years (RUSSOet al. 1995). Hence, therehas been ample time for sequencedivergence between D. melane gaster and D. uirilis, with an average rate of molecular FIGUREl.-h si/u hybridization with strain Ihj[hn71tl]-12 evolution of -10 X lo-" to 15 X nucleotide substi- showing (arrow) a single euchromatic site of hybridization at 47F (chromosome 4). tutions per synonymous site per year (NURMINSKYet al. 1995), the mean number of substitutions per synony- mous nucleotide site is 0.8-1.2.Likewise, there has were determined. Three independentinsertions were been ampletime for genomeevolution; with an average recovered and analyzed: one from Du[ hawZ] -12 and rate of genomic rearrangement of approximately 2 X two from Du[hawZ]-131. The results are shown in Fig- lo-" to 10 X fixed euchromatic breakpoints per ure 2. Each insertion site is flanked by the 8-bp direct genome per year (NURMINSKYet al. 1995), the mean duplicationcharacteristic of hobo targetsequences number of breakpoints per megabase of euchromatin (CAL.VIet al. 1991). is 1-6. Because its evolutionary divergence from D. melano DISCUSSION gasteris neither excessively closenor excessively distant, The demonstration that strains Du[hawZ]-Z2 and D. virilis has become the organism of choice for studies Du[ haw11 -131 contain authentic hobomediated inser- in comparative molecular genetics and genome evolu- tions with the hobo inverted repeats flanked by an 8- tion (reviewed in HARTL and LOZOVSKAYA1995). For bp direct duplication of target sequence is of some example, in studies of gene regulation, sequencemotifs importance in view of the observation that D. virilis, that are conserved between D. mlunogasterand D.uirilis but not D. melanogaster, can promote hobo excision in are strong candidates for putative regulatory domains the absence of hobo transposase (HANDLER andGOMEZ (for example, TREIERet al. 1989; FORTINIand RUBIN 1995). In the presence of transposase, the majority of 1990; HOOPERet al. 1992; LUKet al. 1994; NURMINSKY excision events appearto be transposase-mediated et al. 1995). Similarly, germline transformation of D. (HANDLER andGOMEZ 1995).Our data imply that the mlanogasterwith genes from D. uirilis yields important insertion of the ha7uZ fragment is also mediated by hobo data aboutconservation of patterns of gene expression transposase, originating most likely from the injected and functional complementation (for example, BRAY helper plasmid. and HIRSH1986; CURTISet al. 1995; MARTIN-MORRIS The rate of recovered transformants was 2/398 fertile and HENIKOFF1995). Thus far, the interspecific gene GO , or 0.5%. On the other hand, oneof the trans- transfers have been in only one direction. The recipro- formed lines carriedthree independent insertions. cal transformation of D. uirilis with genes from D. mla- Hence, the low rate of transformation may not be indi- nogasterwould yieldadditional insight into generegula- cative of the efficiency of the process but rather may tion and function. The opportunity to carry out such indicate that there is a relatively small proportion of experiments is now available. cells that are competent for transformation and that The hobo element is apparently widespread among competent cells are readily transformed. The experi- dipteran insects and perhaps otherinsect groups as well ments were carried out with a helper elementin which (WARRENet al. 1994, 1995).Hence, the finding that the hobo transposase was driven by the hp70 promoter; hobo can mediate germline transformation of D. uirilis use of other promoters, including the endogenoushobo suggests that it may also be useful as a vector for other promoter, may improve the yield. insects, perhaps even including nondrosophilids. The The choice of D. uirilk for germline transformation choice of genetic marker is important. In view of the was motivated by a number of considerations. There is frequent complementation of D. mlanogaster mutants 176 E. R. Lozovskaya et al. with homologous genes from D. virilis, we expected that MuNoz-M~MoI.,A. CAsAI.1 et al., 1995 High copy number of highly similar mariner-like transposons in planarian (Platyhel- the miniwhite gene would function in D. virilis, at least minthe): evidence for a trans-phyla horizontal transfer. Mol. Biol. well enough that its presence could be detected. Al- Evol. 12: 421-431. though the transformant Dv[hawl] -131has strong eye- GARZA,D., M. MEDHORA,A. KOCA and D. L. HARTI.,1991 Introduc- tion of the transposable element mariner into the germline of color pigmentation, it contains multiple inserts. The Drosophila melanogaster. Genetics 128: 303-310. single-insert transformant Dv[hawll-12 has very weak HANDLER, A.M., and S. P. GOMEZ,1995 The hobo transposable ele- pigmentation. We do not know whether the weak pig- ment has transposase-dependent and -independent excision ac- tivity in drosophilid species. Mol. Gen. 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