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Insect population control using female specific pro-drug activation
Insect Biochemistry and Molecular Biology 34 (2004) 131–137 www.elsevier.com/locate/ibmb
Insect population control using female specific pro-drug activation Maria Markaki a, Roger K. Craig b, Charalambos Savakis a,b,c, a
c
Institute of Molecular Biology and Biotechnology, FoRTH, Heraklion, Crete, Greece b Minos BioSystems Ltd, Jubilee House Farm, Sandbach, CW11 2XB, UK Division of Medical Sciences, Medical School, University of Crete, Heraklion, Crete, Greece Received 22 December 2002; accepted 21 March 2003
Abstract A system for population control of insects is proposed. It is based on transgenic insects expressing an enzyme which converts an inactive pro-drug into an active, toxic form. A model system is presented which relies on transposon-mediated integration of a bacterial cytosine deaminase (CD) gene into the genome of Drosophila melanogaster. We demonstrate female-specific sterility and transgene-dependent lethality when flies carrying the CD gene under a Drosophila female-specific promoter/enhancer are treated with 5-Fluorocytosine, a low-toxicity nucleoside analogue which is converted to toxic 5-Fluorouracil by the enzyme. The approach can be used with existing pro-insecticides and appropriate converting enzymes in combination with established mass rearing technology, for targeted, environmentally acceptable control of insects of economic and public health importance. # 2003 Elsevier Ltd. All rights reserved. Keywords: Pro-insecticide activation; Transgenic insect; Drosophila; Cytosine deaminase; Genetic sexing
1. Introduction The control of insects which damage crops or act as vectors for human and animal diseases depends on the widespread application of often toxic insecticides. A strategy to minimize undesirable toxicity of insecticides to non-target organisms is to use pro-insecticides, i.e. compounds which are inactive unless converted into an active form by enzymes in the target species (Magee, 1982; Yamamoto et al., 1998). Insects or other organisms exposed to a pro-insecticide and lacking an enzyme capable of metabolizing the pro-drug into its active toxic form are not affected. A small number of pro-insecticides are currently in use and it has been demonstrated that toxicity can depend on bioactivation by the target species; as a result, selective toxicity has been documented in some cases (Kao and Fukuto, 1977; Bull, 1979; Mahajna et al., 1997; Mahajna and Casida, 1998). In principle, it should be possible to sensitize any refractory insect species to any given proinsecticide by integrating into the genome a gene Corresponding author. IMBB-FoRTH, P.O. Box 1527, Heraklion, Greece. Tel.: +30-810-391114; fax: +30-81-391950. E-mail address: [email protected] (C. Savakis).
0965-1748/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2003.03.001
expressing an appropriate converting enzyme, using standard genetic engineering methodologies. Introduction of the gene into a population would then render this population sensitive to the pro-insecticide. This approach can be used to improve the Sterile Insect Technique (SIT), a pest management method that involves mass rearing, sterilization and release of large numbers of insects into the field to compete with wild males for mating, thus drastically reducing the size of pest populations. Conventional SIT is generally carried out by releasing both sexes, and even though the sterilized females play no part in population suppression, they cause fruit damage. In addition, mass releases of females may present a serious risk since, in many cases, diseases vectored by insects are transmitted by the females (Robinson et al., 1999). Successful SIT programs for the eradication of the tsetse fly, Glossina spp., (Vreysen, 2001), the screwworm fly, Cochliomyia hominivorax (Wyss, 2000) and the Mediterranean fruit fly (Medfly), Ceratitis capitata, (Hendrichs et al., 1995) have been carried out. Use of genetic sexing strains, i.e. strains that produce predominantly males, results in a major improvement of SIT. Genetic sexing has obvious economic advantages, reducing the cost of SIT and enhancing the effectiveness of released
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sterile males, which disperse more widely in search of females and thus compete more intensely with wild males. Genetic sexing methods currently rely on translocations of selectable dominant genes to the maledetermining Y chromosome complementing an X-linked or autosomal recessive trait such as pupal color, temperature-sensitive lethality or insecticide resistance (Seawright et al., 1978; Robinson et al., 1986; Riva, 1987; Robinson et al., 1999). Efficient genetic sexing strains based on the use of a two component tetracycline-repressible expression system have independently developed in Drosophila. (Thomas et al., 2000; Heinrich and Scott, 2000). One component of the system is the tetracycline-dependent transactivator gene (tTA) controlled by a female-specific enhancer of a yolk protein gene. The other component consists of a cell-lethal effector gene under the control of a tetracycline-responsive element (tetO). In the absence of tetracycline, the tTA protein will bind to the tetO sequence and activate transcription from an adjacent minimal promoter. The combined effect is that the effector gene is conditionally expressed in essentially the pattern of the promoter driving tTA. As effector gene, Thomas et al. (2000) used a constitutively active version of a regulatory protein (Ras 64BV12, Fortini et al., 1992) while Heinrich and Scott (2000) used the proapoptotic gene head involution defective (hid, Grether et al., 1995). Female-specific lethality can also be achieved with a gene product that is toxic only to females (Thomas et al., 2000). An alternative approach to genetic sexing would be a single transgene system which activates a proinsecticide exclusively in females and causes femalespecific lethality only upon exposure to the pro-insecticide. We call this method Sensitization of Insect Populations to Pro-insecticides (SIPP). In addition to genetic sexing for SIT, SIPP could be used without sterilization of released males. In this approach, released flies would transmit to their progeny gene(s) expressing a harmless pro-insecticide activating enzyme only in female flies. Treatment of the fly population with the appropriate pro-insecticide would then result selectively in death of female progeny, while male progeny will transmit the gene to half of their daughters which will also die, giving a degree of additional effectiveness in later generations. To demonstrate proof of principle, we have developed a model system using transgenic Drosophila melanogaster lines expressing the Escherichia coli enzyme cytosine deaminase (CD; EC 3.5.4.1). CD, which is encoded by the codA gene, catalyzes deamination of the low-toxicity nucleoside analogue 5-Fluorocytosine (5-FC) to the highly toxic metabolite 5-Fluorouracil (5FU) which triggers cell apoptosis due to inhibition of both RNA and DNA synthesis (Polak and Scholer, 1975; Diasio et al., 1978; Vermes et al., 2000). Since
CD is present only in bacteria and fungi, the CD gene provides a useful system for the selective killing of gene-modified cells in higher organisms, for example in enzyme/prodrug gene therapy approaches to cancer treatment (Huber et al., 1991; Mullen et al., 1992; Austin and Huber, 1993; Mullen et al., 1994; Huber et al., 1994). In this paper we report the introduction of the Escherichia coli CD gene, under the control of a Drosophila female-specific promoter, into the Drosophila melanogaster genome, its expression in adult females and the resultant sensitivity of transgenic flies to 5-FC. We demonstrate that 5-FC induces sterility selectively in CD-expressing females. At high 5-FC concentrations, transgene-depedent lethality is also observed. 2. Materials and methods 2.1. Plasmid constructions Transposon pCaSpeRYPCD (Fig. 1) was constructed as follows: The coding sequence of the E. coli CD gene (Austin and Huber, 1993) was obtained by PCR using template DNA from an E. coli XL1 blue strain. Oligonucleotides 50 -CGGGATCCACCATGTCGAATAACGCTTTAC-30 (corresponding to the 50 end of the CD coding sequence but changing the start codon from GTG to ATG, introducing a Kozak consensus sequence (Kozak, 1986) and incorporating a BamHI restriction site upstream of the start codon) and 50 -GCTCTAGATCAACGTTTGTAATCGATGGC-30 (corresponding to the 30 end and inserting an XbaI restriction site)
Fig. 1. Female-specific expression of the E. coli codA gene in D. melanogaster. Top: The pCaSpeRYPCD transposon. Arrows indicate direction of transcription. The bar indicates the probe used in Northern analysis. Bottom: Northern blot analysis. Total RNA from 3-day old male and female flies was separated on a formaldehyde–agarose gel (20–25 lg RNA per lane), blotted onto a nylon membrane filter and hybridized successively with 32P-labeled codA and Drosophila tubulin probes. Non-transformed flies were used as controls.
M. Markaki et al. / Insect Biochemistry and Molecular Biology 34 (2004) 131–137
were used as primers. The 50-ll reactions contained 200 ng of genomic DNA, 50 pM forward and reverse primers, 200 lM dNTPs, 2 mM MgCl2 and 2.5 U Taq DNA polymerase (Promega) in buffer supplied by the v manufacturer. Reactions were heated to 94 C for 5 v min then cycled 35 times (45 s at 94 C; 1 min at 55 v v C; 1 min 30 s at 72 C) with a final extension at 72 C for 10 min in a Perkin-Elmer/Cetus DNA thermo cycler. A 1.3 kb product was purified by agarose gel electrophoresis and subcloned into a pGEM-T Easy Vector (Promega). Cloning of the correct fragment was confirmed by DNA sequencing. The CD coding region was excised as a NotI fragment from the pGEM-T Easy Vector and inserted into the NotI site of the polylinker of pCaSpeR-hs (Thummel and Pirrotta, 1992). An XhoI/BamHI fragment containing the Yp1 gene promoter/enhancer from 362 to +54 (Hung and Wensink, 1981; Hung and Wensink, 1983) was isolated from plasmid pBluescriptYp where it was previously cloned as a DNA fragment obtained by PCR using D. melanogaster DNA as template. This fragment was subsequently cloned into the XhoI/BglII sites of pCaSpeR-hs replacing the hsp70 (87C1) promoter. The final construct pCaSpeRYPCD was used for generating transgenic flies. v
2.2. Fly strains and germ-line transformation For germ-line transformation, the pCaSpeRYPCD transposon DNA (400 lg/ml) and the D2,3 helper plasmid (Laski et al., 1986) (100 lg/ml) were injected into yw pre-blastoderm embryos using standard procedures (Rubin and Spradling, 1982). Adults derived from the injected embryos (G0) were back-crossed to a yw strain and the G1 progeny were screened for the appearance of non-white eye phenotype. Single non-white G1 flies were mated to yw flies and then bred to homozygosity. Seven transformed lines were established of which four were examined for CD expression. Drug-induced lethality was determined by transferring two-day old adult flies into vials containing food supplemented with 5-FC (100 mM) or 5-FU (50 mM) and counting surviving flies daily. Drug-induced sterility was tested by back-crossing transgenic flies to yw flies; yw flies were used as controls. Twenty pairs from each cross were allowed to breed on media containing various concentrations of the drugs or on normal media and viability of the progeny was determined by counting the number of pupae which developed. 2.3. RNA analysis Total RNA was extracted from adult (3-day old) flies homozygous for the introduced gene using the TRIzol reagent (Gibco-BRL, Gaithersburg, MD)
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according to the manufacturer’s instructions. RNA (20–25 lg per lane) was run on 1.2% agarose/formaldehyde gel and transferred to GeneScreen Plus membrane (NEN Life Science Products, Inc). For hybridizations, the CD probe was prepared by PCR and the Drosophila a-tubulin probe was a 650-bp fragment (a gift of Z. Veneti and K. Bourtzis, IMBBFORTH) purified by a Qiaquick gel extraction protocol (QIAGEN). Hybridization with 32P radiolabeled probes was carried out according to standard protocols (Sambrook et al., 1989). Autoradiographic exposure was 48 and 12 h for the CD and the tubulin probe, respectively. 3. Results 3.1. Expression of the CD gene in transformed flies We used pCaSpeRYPCD, a P element transposon to generate transgenic fly lines expressing CD. The transposon contains the E. coli CD coding sequence flanked by the Drosophila Yolk Protein 1 gene (Yp1) femaleand fat-body-specific promoter/enhancer (Garabedian et al., 1986) and the Drosophila Hsp70 terminator sequences (Fig. 1, top). Total RNA isolated from 3-day old flies of four independently transformed lines was examined for CD expression by RNA blot analysis using the CD coding sequence as a probe. Accumulation of CD transcripts was detected in transgenic females but not in transgenic males or non-transgenic flies (Fig. 1, bottom). To demonstrate that RNA was present in lanes lacking a hybridization signal, the CD probe was removed and the same blot was then rehybridized to a Drosophila a-tubulin gene probe. As it can be seen in the lower panel of Fig. 1, all lanes contain approximately the same amount of RNA. 3.2. Sensitivity of transgenic lines to 5-FC and 5-FU Toxicity of 5-FC and 5-FU was tested in 2-day old flies from four transgenic lines and the control parental strain yw, which was originally used to produce the transformants. Fig. 2 shows the effects of 5-FC and 5FU on transgenic as well as non-transgenic flies. Expression of the CD gene had no obvious effects on viability of flies in the absence of 5-FC (Fig. 2A). Selective elimination of transgenic females was observed in the presence of high (100 mM) 5-FC concentrations 13 days after treatment (Fig. 2B). Transgenic flies showed sensitivity to 5-FU similar to that of the non-transgenic controls (Fig. 2C) indicating that conversion of 5-FC to 5-FU rather than differential sensitivity to the toxic end-product (5-FU) is the cause of lethality in CDexpressing flies. Under identical conditions, transgenic and non-transgenic males showed comparable sensitivity to 5-FC.
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Fig. 2. Sensitivity of CD-transformed Drosophila lines to 5-FC and 5-FU. Transgenic and yw flies were collected within a few hours after eclosion and maintained on normal medium. Two day-old flies were then transferred either to fresh normal medium (A) or to medium containing 100 mM 5-FC (B) or 50 mM 5-FU (C). The number of viable flies was determined daily. t yw non-transgenic controls; v transgenic line 37.3.1; 4 line 32.2.2; & line 15.1.2. Each data point represents the mean of three separate experiments. Standard deviation was <10%.
Although the effect of 5-FU and 5-FC on fly viability was delayed, a relatively fast effect was observed on fly fertility (Table 1). When flies homozygous for stable insertions of Yp1-CD were mated to yw flies on either
normal medium or on medium supplemented with 5FC, a strong transgene- and sex-dependent sterility effect of 5-FC was detected. Overall fertility of the non-transgenic controls was reduced by the drug in a
M. Markaki et al. / Insect Biochemistry and Molecular Biology 34 (2004) 131–137
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Table 1 Female sterility in homozygous CD-transformed D. melanogaster caused by 5-Fluorocytosine. Transgenic flies were backcrossed to the recipient yw strain as indicated (f ¼ females, m ¼ males); the yw strain was used as control Concentration (mM)
0 1 10
Number of progency yw f yw m
12:1 f yw m
yw f 12:1 m
15:1 f yw m
yw f 15:1m
24:1 f yw m
yw f 24:1 m
32:2 f yw m
yw f 32:2 m
1050 780 390
950 550 0
1140 770 670
940 627 0
1022 855 850
1083 216 0
1100 788 589
1030 191 0
1003 1020 670
dose-dependent way. However, a strong sex-specific effect was observed when homozygous transgenic flies were backcrossed to non-transgenic flies; the progeny of transgenic females were markedly reduced at 1 mM 5-FC and eliminated at 10 mM 5-FC. This effect was caused by reduced fertility rather than larval lethality, as very few or no eggs were layed by transgenic females in food containing 10 mM pro-drug, a concentration of drug which had no effect on viability of transgenic or non-transgenic flies. Fertility of transgenic males was not affected by 5-FC, compared to the non-transgenic controls. No progeny was recovered from control or transgenic flies bred on food containing lower concentrations (0.5 mM) of 5-FU (data not shown). 4. Discussion We have described proof of principle for a system which is potentially useful for targeted control of populations of insect pests. The system, SIPP, is based on expression of a harmless pro-drug converting enzyme in females. The SIPP approach is simple, relying on a single transgene, as opposed to two transgenes that are required for similar conditional lethal systems developed previously (Thomas et al., 2000; Heinrich and Scott, 2000). As a model we have used the bacterial CD gene expressed in D. melanogaster flies under the control of a Yp1 female-specific promoter/enhancer, which has been shown previously to drive expression of a heterologous gene in the fat body of adult females. We have shown that, as expected, transformed Drosophila lines carrying the YP1-CD construct express CD in adult females but not in males. We have demonstrated that transgenic female flies show decreased viability upon treatment with 5-FC, compared to non-transgenic flies. We have also shown female sterility in transgenic flies bred on food containing 5-FC. Fly lethality was first detectable on day 6 after exposure to either 5-FU or 5-FC and at relatively high drug and pro-drug concentrations (50 mM and 100 mM, respectively). Inefficient uptake of the analogues by the insect gut may be the cause of the apparent delayed toxicity. Delayed action could also be attributed to the fact that adult flies consist mainly of postmitotic, non-growing cells, which are expected to be
less sensitive to the inhibition of DNA and RNA synthesis caused by 5-FU. Compared to 5-FU, the effect of 5-FC on viability of transgenic females shows an additional delay, with 100% lethality observed 14 days after the beginning of 5-FC treatment, compared to 8 days with 5-FU. This could be due to different rates of uptake of 5-FC and 5-FU, or to slow accumulation of 5-FU by enzymatic deamination of 5-FC in transgenic flies. However, the observation that relatively low concentrations of 5-FC (10 mM) can induce complete sterility in newly eclosed female transgenic flies suggests that CD produced in the fat body can readily deaminate 5-FC into 5-FU in amounts sufficient to kill dividing cells upon diffusion. Unexpectedly, transgenic and non-transgenic males were equally sensitive to 5-FC, indicating conversion to 5-FU at higher rates in males relative to females in the D. melanogaster strain used. Although this particular enzyme-prodrug system provides proof of principle for SIPP, delayed action and high cost of 5-FC, combined with observed sensitivity of non-transgenic males to 5-FC (Fig. 2), would make it unsuitable for large-scale applications. Ideally, for efficient genetic sexing schemes the bioactivation process should take place early in the development of the insect so that doomed females do not take up space in the mass-rearing facility. Unfortunately, the Yp1 promoter/ enhancer drives expression of a gene only in the fat body of adult females. In addition, yolk protein gene promoter regulation in some insect species is diet-dependent. Use of suitable regulatory elements to drive female-specific expression of the converting enzyme during early development would lead to reduced rearing costs. The SIPP approach to population management has a number of attractive applications when used in combination with insect mass rearing technology. First, the development of improved SIT control through genetic sexing, i.e. the elimination of female flies by the application of pro-insecticides during mass rearing, prior to the release of sterile males into the environment. Second, the development of strategies dependent on massive release of fertile transgenic male insects into the environment early in the growing season, followed by the application of pro-insecticide after germline transmission has occurred, to eliminate the female pro-
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geny in the target population. In this work, the CD/5FC combination was used as a proof of principle model system. Although this system is clearly less effective for controlling female viability compared to the tetracycline-repressible expression system used by Heinrich and Scott (2000) and Thomas et al. (2000) it can be improved by using other enzyme/pro-insecticide combinations. We plan to develop SIPP systems for pest population control relying on bioactivation of pro-insecticides into active insecticides in transgenic insects expressing an appropriate converting enzyme. There are a number of approved pro-insecticides whose selective activity in one species as opposed to another is dependent on the presence or absence of converting enzymes. Examples include N-Me-imidaclopid, a pro-insecticide that is converted to the insecticide imidaclopid by a mixed function oxidase system (Yamamoto et al., 1998) and acephate, a low toxicity phosphoramidate insecticide which is converted to the highly toxic acetylcholinesterase inhibitor methamidophos by specific amidases (Kao and Fukuto, 1977). The isolation and species-specific expression of genes encoding such enzymes should permit the application of SIPP strategies using existing pro-insecticides. In addition, the availability of expression systems for pro-insecticide activating enzymes will enable design of novel pro-insecticides from existing insecticides. These applications should lead to improved management of insect pests and disease vectors that can be mass-reared and released, such as the agricultural pests medfly, melon fly, Mexican fruit fly, pink bollworm, boll weevil, corn earworm, gypsy moth, tobacco hornworm, and the disease vectors tsetse fly, house fly and mosquito species. Compared to conventional methods, which rely mostly on release of non-selective highly toxic insecticides, SIPP strategies should provide a selective and environmentally sustainable alternative for insect population management. Acknowledgements We thank Ioannis Livadaras for expert technical assistance and Stefan Oehler for critically reading the manuscript. References Austin, E.A., Huber, B.E., 1993. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol. Pharmacol. 43, 380–387. Bull, D.L., 1979. Fate and efficacy of acephate after application to plants and insects. J. Agric. Food Chem. 27, 268–272. Diasio, R.B., Bennett, J.E., Myers, C.E., 1978. Mode of action of 5fluorocytosine. Biochem. Pharmacol. Biochem. Pharmacol. 27, 703–707.
Fortini, M., Simon, M., Rubin, G., 1992. Signalling by the sevenless protein tyrosine kinase is mimicked by Ras 1 activation. Nature 355, 559–561. Garabedian, M.J., Shepherd, B.M., Wensink, P.C., 1986. A tissuespecific transcription enhancer from Drosophila yolk protein 1 gene. Cell 45, 859–867. Grether, M., Abrams, J., Agapite, J., White, K., Steller, H., 1995. The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Gene Dev. 9, 1694–1708. Heinrich, J.C., Scott, M.J., 2000. A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc. Natl. Acad. Sci. USA 97, 8229– 8232. Hendrichs, J., Franz, G., Rendon, P., 1995. Increased effectiveness and applicability of the sterile insect technique through male-only releases for control of Mediterranean fruit flies during fruiting seasons. J. Appl. Entomol. 119, 371–377. Huber, B.E., Richards, C.A., Krenitsky, T., 1991. Retroviralmediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. Proc. Natl. Acad. Sci. USA 88, 8039–8043. Huber, B.E., Austin, E.A., Richards, C.A., Davis, S.T., Good, S.S., 1994. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: Significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc. Natl. Acad. Sci. USA 91, 8302–8306. Hung, M.-C., Wensink, P.C., 1981. The sequence of Drosophila melanogaster gene for yolk protein 1. Nucleic Acids Res. 9, 6407–6419. Hung, M.-C., Wensink, P.C., 1983. Sequence and structure conservation in yolk proteins and their genes. J. Mol. Biol. 164, 481– 492. Kao, T.-S., Fukuto, T.R., 1977. Metabolism of O, S-dimethyl propionyl and hexanoyphosphoramidothioate in the house fly and white mouse. Pestic. Biochem. Physiol. 7, 83–95. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. Laski, F.A., Rio, D.C., Rubin, G.M., 1986. Identification and immunochemical analysis of biologically active Drosophila P element transposase. Cell 44, 7–19. Magee, P.S., 1982. Structure–activity relationships in phosphoramidates. In: Coats, J.R. (Ed.), Insecticide Mode of Action. Academic Press, Inc, New York, pp. 101–161. Mahajna, M., Quistab, G.B., Casida, J.E., 1997. Acephate insecticide toxicity: safety conferred by inhibition of the bioactivating carboxyamidase by the metabolite methamidophos. Chem. Res. Toxicol. 10, 64–69. Mahajna, M., Casida, J.E., 1998. Oxidative bioactivation of methamidophos insecticide: synthesis of N- hydromethamidophos (a candidate metabolite) and its proposed alternative reactions involving N-O rearrangement or fragmentation through a metaphosphate analogue. Chem. Res. Toxicol. 11, 26–34. Mullen, C.A., Kilstrup, M., Blaese, R.M., 1992. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytocine: A negative selection system. Proc. Natl. Acad. Sci. USA 89, 33–37. Mullen, C.A., Coale, M.M., Lowe, R., Blaese, R.M., 1994. Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. Cancer Res. 54, 1503–1506. Polak, A., Scholer, H.J., 1975. Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 21, 113–130. Riva, M.E., 1987. The use of allyl alcohol as discriminating agentfor genetic sexing in the Mediterranean fruit fly Ceratitis capitata. In: Economopoulos, A.P. (Ed.), Fruit flies. In: Proceedings of the Second International Symposium. Elsevier, Amsterdam, pp. 227–234.
M. Markaki et al. / Insect Biochemistry and Molecular Biology 34 (2004) 131–137 Robinson, A.S., Riva, M.E., Zapater, M., 1986. Genetic sexing in the Mediterranean fruit fly, Ceratitis capitata, using the Adh locus. Theor. Appl. Genet. 72, 455–457. Robinson, A.S., Franz, G., Fisher, K., 1999. Genetic sexing strains in the medfly, Ceratitis capitata: Development, mass rearing and field application. Trends Entomol. 2, 81–104. Rubin, G.M., Spradling, A.C., 1982. Genetic transformation of Drosophila with transposase element vectors. Science 218, 348–352. Sambrook, J., Fritsch, E., Maniatis, F., 1989. Molecular Cloning: A Laboratory Manual. . Cold Spring Harbor Lab. Press, Plainview, NY. Seawright, J.A., Kaiser, P.E., Dame, D.A., Lofgren, C.S., 1978. Genetic method for the preferential elimination of females of Anopheles albimanus. Science 200, 1303–1304. Thomas, D.D., Donnelly, C.A., Wood, R.J., Alphey, L.S., 2000. Insect population control using a dominant, repressible, lethal genetic system. Science 287, 2474–2476.
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Thummel, C.S., Pirrotta, V., 1992. New pCaSpeR P element vectors Drosophila Inf. Service 71, 150. Vermes, A., Guchelaar, H.J., Dankert, J., 2000. Fluorocytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J. Antimicrob. Chemother. 46, 171–179. Vreysen, M.J., 2001. Principles of area-wide integrated tsetse fly control using the sterile insect technique. Med Trop. 61, 397–411. Wyss, J.H., 2000. Screwworm eradication in the Americas. Ann N Y Acad Sci. 91, 186–193. Yamamoto, I., Tomizawa, M., Saito, T., Miyamoto, T., Walcott, E.C., Sumikawa, K., 1998. Structural factors contributing to insecticidal and selective actions of neonicotinoids. Arch. Insect Biochem. Physiol. 37, 24–32.
Insect population control using female specific pro-drug activation Maria Markaki a, Roger K. Craig b, Charalambos Savakis a,b,c, a
c
Institute of Molecular Biology and Biotechnology, FoRTH, Heraklion, Crete, Greece b Minos BioSystems Ltd, Jubilee House Farm, Sandbach, CW11 2XB, UK Division of Medical Sciences, Medical School, University of Crete, Heraklion, Crete, Greece Received 22 December 2002; accepted 21 March 2003
Abstract A system for population control of insects is proposed. It is based on transgenic insects expressing an enzyme which converts an inactive pro-drug into an active, toxic form. A model system is presented which relies on transposon-mediated integration of a bacterial cytosine deaminase (CD) gene into the genome of Drosophila melanogaster. We demonstrate female-specific sterility and transgene-dependent lethality when flies carrying the CD gene under a Drosophila female-specific promoter/enhancer are treated with 5-Fluorocytosine, a low-toxicity nucleoside analogue which is converted to toxic 5-Fluorouracil by the enzyme. The approach can be used with existing pro-insecticides and appropriate converting enzymes in combination with established mass rearing technology, for targeted, environmentally acceptable control of insects of economic and public health importance. # 2003 Elsevier Ltd. All rights reserved. Keywords: Pro-insecticide activation; Transgenic insect; Drosophila; Cytosine deaminase; Genetic sexing
1. Introduction The control of insects which damage crops or act as vectors for human and animal diseases depends on the widespread application of often toxic insecticides. A strategy to minimize undesirable toxicity of insecticides to non-target organisms is to use pro-insecticides, i.e. compounds which are inactive unless converted into an active form by enzymes in the target species (Magee, 1982; Yamamoto et al., 1998). Insects or other organisms exposed to a pro-insecticide and lacking an enzyme capable of metabolizing the pro-drug into its active toxic form are not affected. A small number of pro-insecticides are currently in use and it has been demonstrated that toxicity can depend on bioactivation by the target species; as a result, selective toxicity has been documented in some cases (Kao and Fukuto, 1977; Bull, 1979; Mahajna et al., 1997; Mahajna and Casida, 1998). In principle, it should be possible to sensitize any refractory insect species to any given proinsecticide by integrating into the genome a gene Corresponding author. IMBB-FoRTH, P.O. Box 1527, Heraklion, Greece. Tel.: +30-810-391114; fax: +30-81-391950. E-mail address: [email protected] (C. Savakis).
0965-1748/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2003.03.001
expressing an appropriate converting enzyme, using standard genetic engineering methodologies. Introduction of the gene into a population would then render this population sensitive to the pro-insecticide. This approach can be used to improve the Sterile Insect Technique (SIT), a pest management method that involves mass rearing, sterilization and release of large numbers of insects into the field to compete with wild males for mating, thus drastically reducing the size of pest populations. Conventional SIT is generally carried out by releasing both sexes, and even though the sterilized females play no part in population suppression, they cause fruit damage. In addition, mass releases of females may present a serious risk since, in many cases, diseases vectored by insects are transmitted by the females (Robinson et al., 1999). Successful SIT programs for the eradication of the tsetse fly, Glossina spp., (Vreysen, 2001), the screwworm fly, Cochliomyia hominivorax (Wyss, 2000) and the Mediterranean fruit fly (Medfly), Ceratitis capitata, (Hendrichs et al., 1995) have been carried out. Use of genetic sexing strains, i.e. strains that produce predominantly males, results in a major improvement of SIT. Genetic sexing has obvious economic advantages, reducing the cost of SIT and enhancing the effectiveness of released
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sterile males, which disperse more widely in search of females and thus compete more intensely with wild males. Genetic sexing methods currently rely on translocations of selectable dominant genes to the maledetermining Y chromosome complementing an X-linked or autosomal recessive trait such as pupal color, temperature-sensitive lethality or insecticide resistance (Seawright et al., 1978; Robinson et al., 1986; Riva, 1987; Robinson et al., 1999). Efficient genetic sexing strains based on the use of a two component tetracycline-repressible expression system have independently developed in Drosophila. (Thomas et al., 2000; Heinrich and Scott, 2000). One component of the system is the tetracycline-dependent transactivator gene (tTA) controlled by a female-specific enhancer of a yolk protein gene. The other component consists of a cell-lethal effector gene under the control of a tetracycline-responsive element (tetO). In the absence of tetracycline, the tTA protein will bind to the tetO sequence and activate transcription from an adjacent minimal promoter. The combined effect is that the effector gene is conditionally expressed in essentially the pattern of the promoter driving tTA. As effector gene, Thomas et al. (2000) used a constitutively active version of a regulatory protein (Ras 64BV12, Fortini et al., 1992) while Heinrich and Scott (2000) used the proapoptotic gene head involution defective (hid, Grether et al., 1995). Female-specific lethality can also be achieved with a gene product that is toxic only to females (Thomas et al., 2000). An alternative approach to genetic sexing would be a single transgene system which activates a proinsecticide exclusively in females and causes femalespecific lethality only upon exposure to the pro-insecticide. We call this method Sensitization of Insect Populations to Pro-insecticides (SIPP). In addition to genetic sexing for SIT, SIPP could be used without sterilization of released males. In this approach, released flies would transmit to their progeny gene(s) expressing a harmless pro-insecticide activating enzyme only in female flies. Treatment of the fly population with the appropriate pro-insecticide would then result selectively in death of female progeny, while male progeny will transmit the gene to half of their daughters which will also die, giving a degree of additional effectiveness in later generations. To demonstrate proof of principle, we have developed a model system using transgenic Drosophila melanogaster lines expressing the Escherichia coli enzyme cytosine deaminase (CD; EC 3.5.4.1). CD, which is encoded by the codA gene, catalyzes deamination of the low-toxicity nucleoside analogue 5-Fluorocytosine (5-FC) to the highly toxic metabolite 5-Fluorouracil (5FU) which triggers cell apoptosis due to inhibition of both RNA and DNA synthesis (Polak and Scholer, 1975; Diasio et al., 1978; Vermes et al., 2000). Since
CD is present only in bacteria and fungi, the CD gene provides a useful system for the selective killing of gene-modified cells in higher organisms, for example in enzyme/prodrug gene therapy approaches to cancer treatment (Huber et al., 1991; Mullen et al., 1992; Austin and Huber, 1993; Mullen et al., 1994; Huber et al., 1994). In this paper we report the introduction of the Escherichia coli CD gene, under the control of a Drosophila female-specific promoter, into the Drosophila melanogaster genome, its expression in adult females and the resultant sensitivity of transgenic flies to 5-FC. We demonstrate that 5-FC induces sterility selectively in CD-expressing females. At high 5-FC concentrations, transgene-depedent lethality is also observed. 2. Materials and methods 2.1. Plasmid constructions Transposon pCaSpeRYPCD (Fig. 1) was constructed as follows: The coding sequence of the E. coli CD gene (Austin and Huber, 1993) was obtained by PCR using template DNA from an E. coli XL1 blue strain. Oligonucleotides 50 -CGGGATCCACCATGTCGAATAACGCTTTAC-30 (corresponding to the 50 end of the CD coding sequence but changing the start codon from GTG to ATG, introducing a Kozak consensus sequence (Kozak, 1986) and incorporating a BamHI restriction site upstream of the start codon) and 50 -GCTCTAGATCAACGTTTGTAATCGATGGC-30 (corresponding to the 30 end and inserting an XbaI restriction site)
Fig. 1. Female-specific expression of the E. coli codA gene in D. melanogaster. Top: The pCaSpeRYPCD transposon. Arrows indicate direction of transcription. The bar indicates the probe used in Northern analysis. Bottom: Northern blot analysis. Total RNA from 3-day old male and female flies was separated on a formaldehyde–agarose gel (20–25 lg RNA per lane), blotted onto a nylon membrane filter and hybridized successively with 32P-labeled codA and Drosophila tubulin probes. Non-transformed flies were used as controls.
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were used as primers. The 50-ll reactions contained 200 ng of genomic DNA, 50 pM forward and reverse primers, 200 lM dNTPs, 2 mM MgCl2 and 2.5 U Taq DNA polymerase (Promega) in buffer supplied by the v manufacturer. Reactions were heated to 94 C for 5 v min then cycled 35 times (45 s at 94 C; 1 min at 55 v v C; 1 min 30 s at 72 C) with a final extension at 72 C for 10 min in a Perkin-Elmer/Cetus DNA thermo cycler. A 1.3 kb product was purified by agarose gel electrophoresis and subcloned into a pGEM-T Easy Vector (Promega). Cloning of the correct fragment was confirmed by DNA sequencing. The CD coding region was excised as a NotI fragment from the pGEM-T Easy Vector and inserted into the NotI site of the polylinker of pCaSpeR-hs (Thummel and Pirrotta, 1992). An XhoI/BamHI fragment containing the Yp1 gene promoter/enhancer from 362 to +54 (Hung and Wensink, 1981; Hung and Wensink, 1983) was isolated from plasmid pBluescriptYp where it was previously cloned as a DNA fragment obtained by PCR using D. melanogaster DNA as template. This fragment was subsequently cloned into the XhoI/BglII sites of pCaSpeR-hs replacing the hsp70 (87C1) promoter. The final construct pCaSpeRYPCD was used for generating transgenic flies. v
2.2. Fly strains and germ-line transformation For germ-line transformation, the pCaSpeRYPCD transposon DNA (400 lg/ml) and the D2,3 helper plasmid (Laski et al., 1986) (100 lg/ml) were injected into yw pre-blastoderm embryos using standard procedures (Rubin and Spradling, 1982). Adults derived from the injected embryos (G0) were back-crossed to a yw strain and the G1 progeny were screened for the appearance of non-white eye phenotype. Single non-white G1 flies were mated to yw flies and then bred to homozygosity. Seven transformed lines were established of which four were examined for CD expression. Drug-induced lethality was determined by transferring two-day old adult flies into vials containing food supplemented with 5-FC (100 mM) or 5-FU (50 mM) and counting surviving flies daily. Drug-induced sterility was tested by back-crossing transgenic flies to yw flies; yw flies were used as controls. Twenty pairs from each cross were allowed to breed on media containing various concentrations of the drugs or on normal media and viability of the progeny was determined by counting the number of pupae which developed. 2.3. RNA analysis Total RNA was extracted from adult (3-day old) flies homozygous for the introduced gene using the TRIzol reagent (Gibco-BRL, Gaithersburg, MD)
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according to the manufacturer’s instructions. RNA (20–25 lg per lane) was run on 1.2% agarose/formaldehyde gel and transferred to GeneScreen Plus membrane (NEN Life Science Products, Inc). For hybridizations, the CD probe was prepared by PCR and the Drosophila a-tubulin probe was a 650-bp fragment (a gift of Z. Veneti and K. Bourtzis, IMBBFORTH) purified by a Qiaquick gel extraction protocol (QIAGEN). Hybridization with 32P radiolabeled probes was carried out according to standard protocols (Sambrook et al., 1989). Autoradiographic exposure was 48 and 12 h for the CD and the tubulin probe, respectively. 3. Results 3.1. Expression of the CD gene in transformed flies We used pCaSpeRYPCD, a P element transposon to generate transgenic fly lines expressing CD. The transposon contains the E. coli CD coding sequence flanked by the Drosophila Yolk Protein 1 gene (Yp1) femaleand fat-body-specific promoter/enhancer (Garabedian et al., 1986) and the Drosophila Hsp70 terminator sequences (Fig. 1, top). Total RNA isolated from 3-day old flies of four independently transformed lines was examined for CD expression by RNA blot analysis using the CD coding sequence as a probe. Accumulation of CD transcripts was detected in transgenic females but not in transgenic males or non-transgenic flies (Fig. 1, bottom). To demonstrate that RNA was present in lanes lacking a hybridization signal, the CD probe was removed and the same blot was then rehybridized to a Drosophila a-tubulin gene probe. As it can be seen in the lower panel of Fig. 1, all lanes contain approximately the same amount of RNA. 3.2. Sensitivity of transgenic lines to 5-FC and 5-FU Toxicity of 5-FC and 5-FU was tested in 2-day old flies from four transgenic lines and the control parental strain yw, which was originally used to produce the transformants. Fig. 2 shows the effects of 5-FC and 5FU on transgenic as well as non-transgenic flies. Expression of the CD gene had no obvious effects on viability of flies in the absence of 5-FC (Fig. 2A). Selective elimination of transgenic females was observed in the presence of high (100 mM) 5-FC concentrations 13 days after treatment (Fig. 2B). Transgenic flies showed sensitivity to 5-FU similar to that of the non-transgenic controls (Fig. 2C) indicating that conversion of 5-FC to 5-FU rather than differential sensitivity to the toxic end-product (5-FU) is the cause of lethality in CDexpressing flies. Under identical conditions, transgenic and non-transgenic males showed comparable sensitivity to 5-FC.
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Fig. 2. Sensitivity of CD-transformed Drosophila lines to 5-FC and 5-FU. Transgenic and yw flies were collected within a few hours after eclosion and maintained on normal medium. Two day-old flies were then transferred either to fresh normal medium (A) or to medium containing 100 mM 5-FC (B) or 50 mM 5-FU (C). The number of viable flies was determined daily. t yw non-transgenic controls; v transgenic line 37.3.1; 4 line 32.2.2; & line 15.1.2. Each data point represents the mean of three separate experiments. Standard deviation was <10%.
Although the effect of 5-FU and 5-FC on fly viability was delayed, a relatively fast effect was observed on fly fertility (Table 1). When flies homozygous for stable insertions of Yp1-CD were mated to yw flies on either
normal medium or on medium supplemented with 5FC, a strong transgene- and sex-dependent sterility effect of 5-FC was detected. Overall fertility of the non-transgenic controls was reduced by the drug in a
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Table 1 Female sterility in homozygous CD-transformed D. melanogaster caused by 5-Fluorocytosine. Transgenic flies were backcrossed to the recipient yw strain as indicated (f ¼ females, m ¼ males); the yw strain was used as control Concentration (mM)
0 1 10
Number of progency yw f yw m
12:1 f yw m
yw f 12:1 m
15:1 f yw m
yw f 15:1m
24:1 f yw m
yw f 24:1 m
32:2 f yw m
yw f 32:2 m
1050 780 390
950 550 0
1140 770 670
940 627 0
1022 855 850
1083 216 0
1100 788 589
1030 191 0
1003 1020 670
dose-dependent way. However, a strong sex-specific effect was observed when homozygous transgenic flies were backcrossed to non-transgenic flies; the progeny of transgenic females were markedly reduced at 1 mM 5-FC and eliminated at 10 mM 5-FC. This effect was caused by reduced fertility rather than larval lethality, as very few or no eggs were layed by transgenic females in food containing 10 mM pro-drug, a concentration of drug which had no effect on viability of transgenic or non-transgenic flies. Fertility of transgenic males was not affected by 5-FC, compared to the non-transgenic controls. No progeny was recovered from control or transgenic flies bred on food containing lower concentrations (0.5 mM) of 5-FU (data not shown). 4. Discussion We have described proof of principle for a system which is potentially useful for targeted control of populations of insect pests. The system, SIPP, is based on expression of a harmless pro-drug converting enzyme in females. The SIPP approach is simple, relying on a single transgene, as opposed to two transgenes that are required for similar conditional lethal systems developed previously (Thomas et al., 2000; Heinrich and Scott, 2000). As a model we have used the bacterial CD gene expressed in D. melanogaster flies under the control of a Yp1 female-specific promoter/enhancer, which has been shown previously to drive expression of a heterologous gene in the fat body of adult females. We have shown that, as expected, transformed Drosophila lines carrying the YP1-CD construct express CD in adult females but not in males. We have demonstrated that transgenic female flies show decreased viability upon treatment with 5-FC, compared to non-transgenic flies. We have also shown female sterility in transgenic flies bred on food containing 5-FC. Fly lethality was first detectable on day 6 after exposure to either 5-FU or 5-FC and at relatively high drug and pro-drug concentrations (50 mM and 100 mM, respectively). Inefficient uptake of the analogues by the insect gut may be the cause of the apparent delayed toxicity. Delayed action could also be attributed to the fact that adult flies consist mainly of postmitotic, non-growing cells, which are expected to be
less sensitive to the inhibition of DNA and RNA synthesis caused by 5-FU. Compared to 5-FU, the effect of 5-FC on viability of transgenic females shows an additional delay, with 100% lethality observed 14 days after the beginning of 5-FC treatment, compared to 8 days with 5-FU. This could be due to different rates of uptake of 5-FC and 5-FU, or to slow accumulation of 5-FU by enzymatic deamination of 5-FC in transgenic flies. However, the observation that relatively low concentrations of 5-FC (10 mM) can induce complete sterility in newly eclosed female transgenic flies suggests that CD produced in the fat body can readily deaminate 5-FC into 5-FU in amounts sufficient to kill dividing cells upon diffusion. Unexpectedly, transgenic and non-transgenic males were equally sensitive to 5-FC, indicating conversion to 5-FU at higher rates in males relative to females in the D. melanogaster strain used. Although this particular enzyme-prodrug system provides proof of principle for SIPP, delayed action and high cost of 5-FC, combined with observed sensitivity of non-transgenic males to 5-FC (Fig. 2), would make it unsuitable for large-scale applications. Ideally, for efficient genetic sexing schemes the bioactivation process should take place early in the development of the insect so that doomed females do not take up space in the mass-rearing facility. Unfortunately, the Yp1 promoter/ enhancer drives expression of a gene only in the fat body of adult females. In addition, yolk protein gene promoter regulation in some insect species is diet-dependent. Use of suitable regulatory elements to drive female-specific expression of the converting enzyme during early development would lead to reduced rearing costs. The SIPP approach to population management has a number of attractive applications when used in combination with insect mass rearing technology. First, the development of improved SIT control through genetic sexing, i.e. the elimination of female flies by the application of pro-insecticides during mass rearing, prior to the release of sterile males into the environment. Second, the development of strategies dependent on massive release of fertile transgenic male insects into the environment early in the growing season, followed by the application of pro-insecticide after germline transmission has occurred, to eliminate the female pro-
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geny in the target population. In this work, the CD/5FC combination was used as a proof of principle model system. Although this system is clearly less effective for controlling female viability compared to the tetracycline-repressible expression system used by Heinrich and Scott (2000) and Thomas et al. (2000) it can be improved by using other enzyme/pro-insecticide combinations. We plan to develop SIPP systems for pest population control relying on bioactivation of pro-insecticides into active insecticides in transgenic insects expressing an appropriate converting enzyme. There are a number of approved pro-insecticides whose selective activity in one species as opposed to another is dependent on the presence or absence of converting enzymes. Examples include N-Me-imidaclopid, a pro-insecticide that is converted to the insecticide imidaclopid by a mixed function oxidase system (Yamamoto et al., 1998) and acephate, a low toxicity phosphoramidate insecticide which is converted to the highly toxic acetylcholinesterase inhibitor methamidophos by specific amidases (Kao and Fukuto, 1977). The isolation and species-specific expression of genes encoding such enzymes should permit the application of SIPP strategies using existing pro-insecticides. In addition, the availability of expression systems for pro-insecticide activating enzymes will enable design of novel pro-insecticides from existing insecticides. These applications should lead to improved management of insect pests and disease vectors that can be mass-reared and released, such as the agricultural pests medfly, melon fly, Mexican fruit fly, pink bollworm, boll weevil, corn earworm, gypsy moth, tobacco hornworm, and the disease vectors tsetse fly, house fly and mosquito species. Compared to conventional methods, which rely mostly on release of non-selective highly toxic insecticides, SIPP strategies should provide a selective and environmentally sustainable alternative for insect population management. Acknowledgements We thank Ioannis Livadaras for expert technical assistance and Stefan Oehler for critically reading the manuscript. References Austin, E.A., Huber, B.E., 1993. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol. Pharmacol. 43, 380–387. Bull, D.L., 1979. Fate and efficacy of acephate after application to plants and insects. J. Agric. Food Chem. 27, 268–272. Diasio, R.B., Bennett, J.E., Myers, C.E., 1978. Mode of action of 5fluorocytosine. Biochem. Pharmacol. Biochem. Pharmacol. 27, 703–707.
Fortini, M., Simon, M., Rubin, G., 1992. Signalling by the sevenless protein tyrosine kinase is mimicked by Ras 1 activation. Nature 355, 559–561. Garabedian, M.J., Shepherd, B.M., Wensink, P.C., 1986. A tissuespecific transcription enhancer from Drosophila yolk protein 1 gene. Cell 45, 859–867. Grether, M., Abrams, J., Agapite, J., White, K., Steller, H., 1995. The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Gene Dev. 9, 1694–1708. Heinrich, J.C., Scott, M.J., 2000. A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program. Proc. Natl. Acad. Sci. USA 97, 8229– 8232. Hendrichs, J., Franz, G., Rendon, P., 1995. Increased effectiveness and applicability of the sterile insect technique through male-only releases for control of Mediterranean fruit flies during fruiting seasons. J. Appl. Entomol. 119, 371–377. Huber, B.E., Richards, C.A., Krenitsky, T., 1991. Retroviralmediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. Proc. Natl. Acad. Sci. USA 88, 8039–8043. Huber, B.E., Austin, E.A., Richards, C.A., Davis, S.T., Good, S.S., 1994. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: Significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc. Natl. Acad. Sci. USA 91, 8302–8306. Hung, M.-C., Wensink, P.C., 1981. The sequence of Drosophila melanogaster gene for yolk protein 1. Nucleic Acids Res. 9, 6407–6419. Hung, M.-C., Wensink, P.C., 1983. Sequence and structure conservation in yolk proteins and their genes. J. Mol. Biol. 164, 481– 492. Kao, T.-S., Fukuto, T.R., 1977. Metabolism of O, S-dimethyl propionyl and hexanoyphosphoramidothioate in the house fly and white mouse. Pestic. Biochem. Physiol. 7, 83–95. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. Laski, F.A., Rio, D.C., Rubin, G.M., 1986. Identification and immunochemical analysis of biologically active Drosophila P element transposase. Cell 44, 7–19. Magee, P.S., 1982. Structure–activity relationships in phosphoramidates. In: Coats, J.R. (Ed.), Insecticide Mode of Action. Academic Press, Inc, New York, pp. 101–161. Mahajna, M., Quistab, G.B., Casida, J.E., 1997. Acephate insecticide toxicity: safety conferred by inhibition of the bioactivating carboxyamidase by the metabolite methamidophos. Chem. Res. Toxicol. 10, 64–69. Mahajna, M., Casida, J.E., 1998. Oxidative bioactivation of methamidophos insecticide: synthesis of N- hydromethamidophos (a candidate metabolite) and its proposed alternative reactions involving N-O rearrangement or fragmentation through a metaphosphate analogue. Chem. Res. Toxicol. 11, 26–34. Mullen, C.A., Kilstrup, M., Blaese, R.M., 1992. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytocine: A negative selection system. Proc. Natl. Acad. Sci. USA 89, 33–37. Mullen, C.A., Coale, M.M., Lowe, R., Blaese, R.M., 1994. Tumors expressing the cytosine deaminase suicide gene can be eliminated in vivo with 5-fluorocytosine and induce protective immunity to wild type tumor. Cancer Res. 54, 1503–1506. Polak, A., Scholer, H.J., 1975. Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 21, 113–130. Riva, M.E., 1987. The use of allyl alcohol as discriminating agentfor genetic sexing in the Mediterranean fruit fly Ceratitis capitata. In: Economopoulos, A.P. (Ed.), Fruit flies. In: Proceedings of the Second International Symposium. Elsevier, Amsterdam, pp. 227–234.
M. Markaki et al. / Insect Biochemistry and Molecular Biology 34 (2004) 131–137 Robinson, A.S., Riva, M.E., Zapater, M., 1986. Genetic sexing in the Mediterranean fruit fly, Ceratitis capitata, using the Adh locus. Theor. Appl. Genet. 72, 455–457. Robinson, A.S., Franz, G., Fisher, K., 1999. Genetic sexing strains in the medfly, Ceratitis capitata: Development, mass rearing and field application. Trends Entomol. 2, 81–104. Rubin, G.M., Spradling, A.C., 1982. Genetic transformation of Drosophila with transposase element vectors. Science 218, 348–352. Sambrook, J., Fritsch, E., Maniatis, F., 1989. Molecular Cloning: A Laboratory Manual. . Cold Spring Harbor Lab. Press, Plainview, NY. Seawright, J.A., Kaiser, P.E., Dame, D.A., Lofgren, C.S., 1978. Genetic method for the preferential elimination of females of Anopheles albimanus. Science 200, 1303–1304. Thomas, D.D., Donnelly, C.A., Wood, R.J., Alphey, L.S., 2000. Insect population control using a dominant, repressible, lethal genetic system. Science 287, 2474–2476.
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Thummel, C.S., Pirrotta, V., 1992. New pCaSpeR P element vectors Drosophila Inf. Service 71, 150. Vermes, A., Guchelaar, H.J., Dankert, J., 2000. Fluorocytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J. Antimicrob. Chemother. 46, 171–179. Vreysen, M.J., 2001. Principles of area-wide integrated tsetse fly control using the sterile insect technique. Med Trop. 61, 397–411. Wyss, J.H., 2000. Screwworm eradication in the Americas. Ann N Y Acad Sci. 91, 186–193. Yamamoto, I., Tomizawa, M., Saito, T., Miyamoto, T., Walcott, E.C., Sumikawa, K., 1998. Structural factors contributing to insecticidal and selective actions of neonicotinoids. Arch. Insect Biochem. Physiol. 37, 24–32.