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Transformation of Gypsy Moth (Lymantria dispar) Cell Lines by Infection withGlyptapanteles indiensisPolydnavirus
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
225, 764–770 (1996)
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Transformation of Gypsy Moth (Lymantria dispar) Cell Lines by Infection with Glyptapanteles indiensis Polydnavirus Terry A. McKelvey,* Dwight E. Lynn,* Dawn Gundersen-Rindal,* David Guzo,* Donald A. Stoltz,† Kim P. Guthrie,* Philip B. Taylor,‡ and Edward M. Dougherty*,1 *United States Department of Agriculture, ARS, Insect Biocontrol Laboratory, Beltsville, Maryland 20705; †Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada; and ‡United States Department of Agriculture, ARS, Beneficial Insects Introduction Research, Newark, Delaware 19713 Received July 23, 1996 Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells. q 1996 Academic Press, Inc.
Polydnaviruses are complex viruses with genomes consisting of a polydisperse mixture of circular double-stranded DNA molecules (7, 15). These viruses are associated exclusively with certain species of endoparasitic wasps in families Braconidae and Ichneumonidae and replicate only in the reproductive tract of the female wasps (19, 20). During parasitization, female wasps inject parasitoid eggs, venom, and calyx fluid containing polydnavirus into host insects and the virus subsequently infects a variety of host cells and tissues (19, 21). No viral morphogenesis has been observed in infected cells of parasitized hosts (8, 3, 14, 23). Polydnavirus DNA persists, but does not replicate, in host cells parasitized by Campoletis sonorensis (23) or Micropolitis demolitor (22). The nature of polydnaviral persistence in host cells is of considerable interest. Stable transformation of tissue originating from insect pests of agricultural and/or medical importance, especially introduction of DNA into the germ line, has been a seldom achieved goal (1). This event has been achieved thus far only in Drosophila (11) by introduction of the P-element, though other transposable elements have recently been successfully used for transformation of the medfly, Ceratitis capitata (10, 25). Because such transformation events require the injection of embryos, other methods, such as using a viral vector, may be more useful. This is the first report of a polydnavirus associated with the brachonid Glyptapanteles indiensis. In this study, we investigate the persistence of this polydnavirus in several lepidopteran cell lines of somatic origin. We provide evidence that a segment of polydnavirus DNA appears capable of stable integration into the chromosome of lepidopteran cells. MATERIALS AND METHODS Generation of cell lines infected with polydnavirus. Lymantria dispar cell lines IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta were infected with polydnavirus (GiPDV), each from a single female braconid gypsy moth parasitoid
1
To whom correspondence should be addressed. Fax: (301) 504-5104. 764
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G. indiensis. Two ovaries and the venom gland were aseptically dissected from a single female wasp and placed into one well of a 24 well dish containing 40-60% confluent IPLB-Ld652Y, IPLB-LdEp or IPLB-LdEIta cells in 0.5 ml ExCell 400 medium (JRH Biosciences, Lenexa, KS) supplemented with 25 mg per ml gentamicin. The organs were minced with forceps, releasing the calyx fluid and the venom, gently mixed, and incubated with cells at 277C. Infected cells were split normally as determined by the cell density. Analysis of putatively transformed L. dispar cell lines for GiPDV sequences. Total nucleic acids were extracted and analyzed by Southern blotting following digestion with either BamHI, HindIII, or XbaI using digoxigenin-labeled GiPDV genomic DNA as a probe. Total genomic DNA was prepared from GiPDV infected and non-infected cells using standard techniques. Genomic DNA from each sample was digested then electrophoresed through a 0.7% agarose gel and transferred to nylon membrane as described (13). Digoxigenin-labeled total GiPDV DNA (Boehringer Manheim Biochemicals, Indianapolis, IN) was used as a hybridization probe. Annealed DNAs were visualized by fluorography (BMB, Indianapolis, IN). Isolation of GiPDV clones. To isolate clones of viral DNA maintained in GiPDV infected cells, a plasmid library of GiPDV DNA was generated by inserting GiPDV DNA fragments into BamHI restriction site in pGEM7zf(/) vector (Promega Corp., Madison, WI). Individual clones of the GiPDV library were isolated and digested with BamHI. The digested DNAs were electrophoresed through 0.7% agarose gel and compared to the restriction patterns of the DNA from the GiPDV cells after hybridization with labeled GiPDV DNA. Clones which had viral DNA inserts of same size as those of transformed cell digests were labeled and used to probe dot blots of transformed IPLB-LdEp and control IPLB-LdEp genomic DNAs. Isolation of clones from the IPLB-LdEp cellular/GiPDV viral integration border. To isolate DNA clones containing both viral and cellular sequences, a lambda phage library was generated from IPLB-LdEp cells infected with GiPDV DNA as described. MboI-digested DNA fragments were inserted into the BamHI site of the Lambda Dash II vector (Stratagene, Inc., La Jolla, California). Individual clones hybridizing to labeled GiPDV were isolated by plaque lift technique (12). Clones were analyzed by restriction digestion and Southern hybridization (13) using digoxigeninlabeled (BMB, Indianapolis, IN) total GiPDV and IPLB-LdEp DNA as probes.
RESULTS AND DISCUSSION
Infection of L. dispar cells with GiPDV (in the presence of venom) induced distinct, though temporary, changes in host cell morphology and growth rates (Fig. 1, B, D, and F) compared to uninfected cells (Fig. 1, A, C, and E). Infected cells formed aggregates not seen in uninfected cells. The division rate of the cells was greatly slowed compared to uninfected cells. These differences gradually faded during weekly passage of the cell lines. By two months post infection, the growth rates of infected and uninfected cells were similar (data not shown). Infected cells were analyzed for the presence of GiPDV viral sequences. No signal was detected from DNA of uninfected LdEp cells (Fig. 2, lanes D, F, H) when Southern blotted DNAs were probed with labeled total genomic GiPDV DNA. However, labeled GiPDV DNA hybridized to three prominent bands (approximate sizes of 15 kb, 7 kb and 5 kb) in BamHI digests of DNA extracted from infected cells after nine passages (60 days) (Fig. 2, lanes E, G, and I). Comparison of detected bands in this digest to total BamHI digested GiPDV DNA (Fig. 2, lane J) indicated that only part of the total GiPDV genome was present in infected cells. Based on BamHI restriction patterns, the portion of the GiPDV genome present in the infected cells appeared approximately 27 Kb. Restriction patterns of the GiPDV DNAs isolated from the GiPDV infected IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cell lines were identical. Restriction patterns of HindIII digests and XbaI digests of DNA extracted from GiPDV infected cells also showed identical patterns in all three cell lines, and GiPDV present totaled 25 to 27 Kb (data not shown). This suggested that the same part of the GiPDV genome was maintained in each cell line, though it was also possible that the GiPDV DNA was present extrachromosomally. After weekly passage of the transformed cells (not selected) for over 175 passages (two and a half years), no significant change in restriction patterns was observed (data not shown). Thus the GiPDV DNA appeared stably maintained. To determine if the GiPDV DNA was maintained integrated in the insect cell chromosome or present extrachromosomally, a viral clone that hybridized to transformed but not to nontransformed cellular DNA, pTM145 (7.5 kb, one internal BamHI site), was used. A Southern 765
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FIG. 1. L. dispar cell lines IPLB-Ld652Y uninfected (A) and infected (B) with GiPDV, IPLB-LdEp uninfected (C) and infected (D) with GiPDV, and IPLB-LdEIta uninfected (E) and infected (F) with GiPDV, seven days post infection.
blot of restriction digested transformed and non-transformed IPLB-LdEp cellular DNAs and digested and uncut GiPDV DNA was probed with labeled pTM145 DNA (Fig. 3). The pTM145 clone DNA hybridized to DNA corresponding exactly to the chromosomal DNA from transformed cells (Fig. 3, lane D) but not to DNA from non-transformed cells (Fig. 3, lane C). No bands were detected in transformed cells that corresponded to supercoiled or relaxed circular virus bands observed in undigested GiPDV DNA (Fig. 3, lane E). This suggested that polydnavirus DNA maintained in transformed cells is integrated into the host genome and is not extrachromosomal. Clone pTM145 hybridized to only one segment of the multipartite GiPDV 766
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FIG. 2. Southern blot analysis of L. dispar cell lines transformed by infection with the G. indiensis polydnavirus. L. dispar cell lines, IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta, were infected with polydnavirus. Lanes D, F, and H contain BamHI digested genomic DNA from IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cells, respectively, not infected with GiPDV. Lanes E, G, and I contain BamHI digested genomic DNA from IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cells, respectively, infected with GiPDV. Lane J contains BamHI digested GiPDV DNA. Lanes A and B contain molecular standards indicated by size in kilobase pairs. Lanes C and K are blank.
genome (Fig. 3), indicating that this single circular genome segment represents or contains the integrating DNA. To characterize the GiPDV genome segment involved in transformation of L. dispar cell lines, a lambda phage library containing fragments of putatively transformed cellular (LdEp/ Gi) DNA was constructed. Clones containing GiPDV or LdEp/Gi DNA were isolated by probing the library with labeled GiPDV DNA. Two clones derived from the transformed cell line (E/G 12 and E/G 25) clearly contained sequences from both GiPDV and insect cell genome as shown by reciprocal hybridization of EcoRI digests of clones E/G 12 and E/G 25 separately with labeled total GiPDV and IPLB-LdEp insect cell DNAs (Fig. 4). Each clone hybridized to the same single DNA segment of the multipartite GiPDV genome as clone pTM145 did (data not shown). These two clones did not cross hybridize (data not shown), indicating each was from a different viral insertion boundary within the transformed cell line. This further suggested that polydnaviral DNA maintained in transformed cells exists in an integrated form in the insect cell chromosome. Previous studies have determined that ichneumonid polydnavirus genome segments are integrated into the wasp genome (4, 5, 6, 24) and that parasitoid viral markers routinely 767
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FIG. 3. Southern blot analysis of G. indiensis genomic segment maintained in L. dispar cell lines. Genomic DNA (10mg) from transformed and non-transformed IPLB-LdEp digested with BamHI or XbaI, undigested cellular, and uncut viral DNA was electrophoresed through a 0.7 % agarose gel and transferred to nylon membrane as described (13). Digoxigenin-labeled DNA (BMB, Indianapolis, IN) from a plasmid clone pTM145 (described in the text) was used as a hybridization probe. Lane C contains genomic DNA from uninfected IPLB-LdEp cells. Lane D contains genomic DNA from GiPDV infected IPLB-LdEp cells. Lane E contains uncut GiPDV DNA. Lanes F and H contain genomic DNA from GiPDV infected IPLB-LdEp cells digested with BamHI and XbaI, respectively. Lanes G and I contain GiPDV DNA digested with BamHI and XbaI, respectively. Lane A contains molecular size standard, indicated by size in kilobase pairs.
segregate in Mendelian fashion, suggesting vertical transmission (17). Polydnavirus DNA (but no polydnavirus particles) can be found in males and in female non-ovarian tissues, and males can transmit polydnaviruses to female offspring (18). Although there is strong evidence for the chromosomal transmission of polydnavirus among wasp populations, extrachromosomal polydnavirus in these tissues has been detected (4, 18). The data shown here strongly suggest that part of the polydnavirus genome can integrate into the genome of an infected host. This polydnaviral system, along with that using a densovirus described by Bergoin et al. (2), provide potential for virus-derived transformation vectors. Currently the commonly used baculovirus expression vector system has limitations because protein expression is transient, killing the host cells and necessitating extra bioreactors producing viral inocula and insect cells for further infection. A polydnavirus-based shuttle vector system could be developed to 768
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FIG. 4. Southern blot analysis of DNAs cloned from IPLB-LdEp cells infected with GiPDV, suggesting that GiPDV DNA is integrated in the IPLB-LdEp genome. (Lanes) DNAs isolated from (B) non-transformed IPLB-LdEp cells, (C) transformed IPLB-LdEp cells, (D) GiPDV DNA from female wasps, (E) clone E/G 12, and (F) clone E/G 25 were digested with EcoRI and electrophoresed through a 0.7% agarose gel (panel a). The DNAs were transferred to nylon membrane as described (13). Digoxigenin-labeled total DNA (BMB, Indianapolis, IN) isolated from IPLBLdEp cells (panel b) and GiPDV virus (panel c) were used as hybridization probes. Lane A contains molecular size standard as indicated by size in kilobase pairs.
generate transformed insect cells containing foreign genes of interest that could be expressed in an inducible system. If germ line tissue can be transformed as readily as somatic cells, this potential transgenic technology could be used for biocontrol, reducing dependence on chemical pesticides. REFERENCES 1. Ashburner, M. (1995) Science 270, 1941–1942. 2. Bergoin, M., Jousset, F.-X., Jourdan, M., Giraud, C., Rolling, F., Li, Y., Romane, C., Yuan, S., and Bossin, H. (1995) 1st Int. Workshop on Transgen. Invertebrates of Med., Agricult., and Aquacult. Importance, Montpellier, France. [Abstract] 3. Blissard, G. W., Fleming, J. G. W., Vinson, S. B., and Summers, M. D. (1986) J. Insect Physiol. 32, 351–359. 4. Fleming, J. G. W., and Summers, M. D. (1990) in Molecular Insect Science (Hagedorn, H. H., et al., Eds.), pp. 99–105. 5. Fleming, J. G. W. (1991) Biological Control 1, 127–135. 6. Fleming, J. G. W., and Summers, M. D. (1986) J. Virology 57, 552–562. 7. Fleming, J. G. W., and Krell, P. J. (1993) in Parasites and Pathogens of Insects (Beckage, N. E., Thompson, S. N., and Federici, B. A., Eds.), Vol. 1, pp. 189–225. Academic Press, San Diego. 8. Fleming, J. G. W., Blissard, G. W., Summers, M. D., and Vinson, S. B. (1983) J. Virology 48, 74–78. 9. Handler, A. M., and O’Brochta, D. A. (1991) Ann. Rev. Entomol. 36, 159–183. 10. Loukeris, T. G., Livadaras, I., Arca, B., Zabalou, S., and Savakis, C. (1995) Science 270, 2002–2003. 11. Rubin, G. M., and Spradling, A. C. (1982) Science 218, 348–353. 12. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 13. Southern, E. M. (1975) J. Mol. Biol. 98, 503–517. 14. Stoltz, D. B., Guzo, D., Belland, E. R., Lucarotti, C. J., and MacKinnon, E. A. (1988) J. Gen. Virol. 69, 903– 907. 769
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Stoltz, D. B., Krell, P., Summers, M. D., and Vinson, S. B. (1984) Intervirol. 21, 1. Stoltz, D. B., and Xu, D. (1990) Can. J. Microbiol. 36, 538–543. Stoltz, D. B. (1990) J. Gen. Virol. 71, 1051–1056. Stoltz, D. B., Guzo, D., and Cook, D. (1986) Virology 155, 120–131. Stoltz, D. B., and Vinson, S. B. (1979a) Advances in Virus Research 24, 125–171. Stoltz, D. B. (1993) in Parasites and Pathogens of Insects, (Beckage, N. E., Thompson, S. N., and Federici, B. A., Eds.), Vol. 1, pp. 167–187. Academic Press, San Diego. Stoltz, D. B., and Vinson, S. B. (1979b) Can. J. Microbiol. 25, 207–216. Strand, M. R., McKenzie, D. I., Grassl, V., Dover, B. A., and Aiken, J. M. (1992) J. Gen. Virol. 73, 1627–1635. Theilmann, D. A., and Summers, M. D. (1988) Virology 167, 329–341. Xu, D., and Stoltz, D. B. (1991) J. Virology 65, 6693–6704. Zweibel, L. J., Saccone, G., Zacharopoulou, A., Besansky, N. J., Favia, G., Collins, F. H., Louis, C., and Kafatos, F. C. (1995) Science 270, 2005–2008.
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Transformation of Gypsy Moth (Lymantria dispar) Cell Lines by Infection with Glyptapanteles indiensis Polydnavirus Terry A. McKelvey,* Dwight E. Lynn,* Dawn Gundersen-Rindal,* David Guzo,* Donald A. Stoltz,† Kim P. Guthrie,* Philip B. Taylor,‡ and Edward M. Dougherty*,1 *United States Department of Agriculture, ARS, Insect Biocontrol Laboratory, Beltsville, Maryland 20705; †Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada; and ‡United States Department of Agriculture, ARS, Beneficial Insects Introduction Research, Newark, Delaware 19713 Received July 23, 1996 Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells. q 1996 Academic Press, Inc.
Polydnaviruses are complex viruses with genomes consisting of a polydisperse mixture of circular double-stranded DNA molecules (7, 15). These viruses are associated exclusively with certain species of endoparasitic wasps in families Braconidae and Ichneumonidae and replicate only in the reproductive tract of the female wasps (19, 20). During parasitization, female wasps inject parasitoid eggs, venom, and calyx fluid containing polydnavirus into host insects and the virus subsequently infects a variety of host cells and tissues (19, 21). No viral morphogenesis has been observed in infected cells of parasitized hosts (8, 3, 14, 23). Polydnavirus DNA persists, but does not replicate, in host cells parasitized by Campoletis sonorensis (23) or Micropolitis demolitor (22). The nature of polydnaviral persistence in host cells is of considerable interest. Stable transformation of tissue originating from insect pests of agricultural and/or medical importance, especially introduction of DNA into the germ line, has been a seldom achieved goal (1). This event has been achieved thus far only in Drosophila (11) by introduction of the P-element, though other transposable elements have recently been successfully used for transformation of the medfly, Ceratitis capitata (10, 25). Because such transformation events require the injection of embryos, other methods, such as using a viral vector, may be more useful. This is the first report of a polydnavirus associated with the brachonid Glyptapanteles indiensis. In this study, we investigate the persistence of this polydnavirus in several lepidopteran cell lines of somatic origin. We provide evidence that a segment of polydnavirus DNA appears capable of stable integration into the chromosome of lepidopteran cells. MATERIALS AND METHODS Generation of cell lines infected with polydnavirus. Lymantria dispar cell lines IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta were infected with polydnavirus (GiPDV), each from a single female braconid gypsy moth parasitoid
1
To whom correspondence should be addressed. Fax: (301) 504-5104. 764
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G. indiensis. Two ovaries and the venom gland were aseptically dissected from a single female wasp and placed into one well of a 24 well dish containing 40-60% confluent IPLB-Ld652Y, IPLB-LdEp or IPLB-LdEIta cells in 0.5 ml ExCell 400 medium (JRH Biosciences, Lenexa, KS) supplemented with 25 mg per ml gentamicin. The organs were minced with forceps, releasing the calyx fluid and the venom, gently mixed, and incubated with cells at 277C. Infected cells were split normally as determined by the cell density. Analysis of putatively transformed L. dispar cell lines for GiPDV sequences. Total nucleic acids were extracted and analyzed by Southern blotting following digestion with either BamHI, HindIII, or XbaI using digoxigenin-labeled GiPDV genomic DNA as a probe. Total genomic DNA was prepared from GiPDV infected and non-infected cells using standard techniques. Genomic DNA from each sample was digested then electrophoresed through a 0.7% agarose gel and transferred to nylon membrane as described (13). Digoxigenin-labeled total GiPDV DNA (Boehringer Manheim Biochemicals, Indianapolis, IN) was used as a hybridization probe. Annealed DNAs were visualized by fluorography (BMB, Indianapolis, IN). Isolation of GiPDV clones. To isolate clones of viral DNA maintained in GiPDV infected cells, a plasmid library of GiPDV DNA was generated by inserting GiPDV DNA fragments into BamHI restriction site in pGEM7zf(/) vector (Promega Corp., Madison, WI). Individual clones of the GiPDV library were isolated and digested with BamHI. The digested DNAs were electrophoresed through 0.7% agarose gel and compared to the restriction patterns of the DNA from the GiPDV cells after hybridization with labeled GiPDV DNA. Clones which had viral DNA inserts of same size as those of transformed cell digests were labeled and used to probe dot blots of transformed IPLB-LdEp and control IPLB-LdEp genomic DNAs. Isolation of clones from the IPLB-LdEp cellular/GiPDV viral integration border. To isolate DNA clones containing both viral and cellular sequences, a lambda phage library was generated from IPLB-LdEp cells infected with GiPDV DNA as described. MboI-digested DNA fragments were inserted into the BamHI site of the Lambda Dash II vector (Stratagene, Inc., La Jolla, California). Individual clones hybridizing to labeled GiPDV were isolated by plaque lift technique (12). Clones were analyzed by restriction digestion and Southern hybridization (13) using digoxigeninlabeled (BMB, Indianapolis, IN) total GiPDV and IPLB-LdEp DNA as probes.
RESULTS AND DISCUSSION
Infection of L. dispar cells with GiPDV (in the presence of venom) induced distinct, though temporary, changes in host cell morphology and growth rates (Fig. 1, B, D, and F) compared to uninfected cells (Fig. 1, A, C, and E). Infected cells formed aggregates not seen in uninfected cells. The division rate of the cells was greatly slowed compared to uninfected cells. These differences gradually faded during weekly passage of the cell lines. By two months post infection, the growth rates of infected and uninfected cells were similar (data not shown). Infected cells were analyzed for the presence of GiPDV viral sequences. No signal was detected from DNA of uninfected LdEp cells (Fig. 2, lanes D, F, H) when Southern blotted DNAs were probed with labeled total genomic GiPDV DNA. However, labeled GiPDV DNA hybridized to three prominent bands (approximate sizes of 15 kb, 7 kb and 5 kb) in BamHI digests of DNA extracted from infected cells after nine passages (60 days) (Fig. 2, lanes E, G, and I). Comparison of detected bands in this digest to total BamHI digested GiPDV DNA (Fig. 2, lane J) indicated that only part of the total GiPDV genome was present in infected cells. Based on BamHI restriction patterns, the portion of the GiPDV genome present in the infected cells appeared approximately 27 Kb. Restriction patterns of the GiPDV DNAs isolated from the GiPDV infected IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cell lines were identical. Restriction patterns of HindIII digests and XbaI digests of DNA extracted from GiPDV infected cells also showed identical patterns in all three cell lines, and GiPDV present totaled 25 to 27 Kb (data not shown). This suggested that the same part of the GiPDV genome was maintained in each cell line, though it was also possible that the GiPDV DNA was present extrachromosomally. After weekly passage of the transformed cells (not selected) for over 175 passages (two and a half years), no significant change in restriction patterns was observed (data not shown). Thus the GiPDV DNA appeared stably maintained. To determine if the GiPDV DNA was maintained integrated in the insect cell chromosome or present extrachromosomally, a viral clone that hybridized to transformed but not to nontransformed cellular DNA, pTM145 (7.5 kb, one internal BamHI site), was used. A Southern 765
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FIG. 1. L. dispar cell lines IPLB-Ld652Y uninfected (A) and infected (B) with GiPDV, IPLB-LdEp uninfected (C) and infected (D) with GiPDV, and IPLB-LdEIta uninfected (E) and infected (F) with GiPDV, seven days post infection.
blot of restriction digested transformed and non-transformed IPLB-LdEp cellular DNAs and digested and uncut GiPDV DNA was probed with labeled pTM145 DNA (Fig. 3). The pTM145 clone DNA hybridized to DNA corresponding exactly to the chromosomal DNA from transformed cells (Fig. 3, lane D) but not to DNA from non-transformed cells (Fig. 3, lane C). No bands were detected in transformed cells that corresponded to supercoiled or relaxed circular virus bands observed in undigested GiPDV DNA (Fig. 3, lane E). This suggested that polydnavirus DNA maintained in transformed cells is integrated into the host genome and is not extrachromosomal. Clone pTM145 hybridized to only one segment of the multipartite GiPDV 766
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FIG. 2. Southern blot analysis of L. dispar cell lines transformed by infection with the G. indiensis polydnavirus. L. dispar cell lines, IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta, were infected with polydnavirus. Lanes D, F, and H contain BamHI digested genomic DNA from IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cells, respectively, not infected with GiPDV. Lanes E, G, and I contain BamHI digested genomic DNA from IPLB-Ld652Y, IPLB-LdEp, and IPLB-LdEIta cells, respectively, infected with GiPDV. Lane J contains BamHI digested GiPDV DNA. Lanes A and B contain molecular standards indicated by size in kilobase pairs. Lanes C and K are blank.
genome (Fig. 3), indicating that this single circular genome segment represents or contains the integrating DNA. To characterize the GiPDV genome segment involved in transformation of L. dispar cell lines, a lambda phage library containing fragments of putatively transformed cellular (LdEp/ Gi) DNA was constructed. Clones containing GiPDV or LdEp/Gi DNA were isolated by probing the library with labeled GiPDV DNA. Two clones derived from the transformed cell line (E/G 12 and E/G 25) clearly contained sequences from both GiPDV and insect cell genome as shown by reciprocal hybridization of EcoRI digests of clones E/G 12 and E/G 25 separately with labeled total GiPDV and IPLB-LdEp insect cell DNAs (Fig. 4). Each clone hybridized to the same single DNA segment of the multipartite GiPDV genome as clone pTM145 did (data not shown). These two clones did not cross hybridize (data not shown), indicating each was from a different viral insertion boundary within the transformed cell line. This further suggested that polydnaviral DNA maintained in transformed cells exists in an integrated form in the insect cell chromosome. Previous studies have determined that ichneumonid polydnavirus genome segments are integrated into the wasp genome (4, 5, 6, 24) and that parasitoid viral markers routinely 767
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FIG. 3. Southern blot analysis of G. indiensis genomic segment maintained in L. dispar cell lines. Genomic DNA (10mg) from transformed and non-transformed IPLB-LdEp digested with BamHI or XbaI, undigested cellular, and uncut viral DNA was electrophoresed through a 0.7 % agarose gel and transferred to nylon membrane as described (13). Digoxigenin-labeled DNA (BMB, Indianapolis, IN) from a plasmid clone pTM145 (described in the text) was used as a hybridization probe. Lane C contains genomic DNA from uninfected IPLB-LdEp cells. Lane D contains genomic DNA from GiPDV infected IPLB-LdEp cells. Lane E contains uncut GiPDV DNA. Lanes F and H contain genomic DNA from GiPDV infected IPLB-LdEp cells digested with BamHI and XbaI, respectively. Lanes G and I contain GiPDV DNA digested with BamHI and XbaI, respectively. Lane A contains molecular size standard, indicated by size in kilobase pairs.
segregate in Mendelian fashion, suggesting vertical transmission (17). Polydnavirus DNA (but no polydnavirus particles) can be found in males and in female non-ovarian tissues, and males can transmit polydnaviruses to female offspring (18). Although there is strong evidence for the chromosomal transmission of polydnavirus among wasp populations, extrachromosomal polydnavirus in these tissues has been detected (4, 18). The data shown here strongly suggest that part of the polydnavirus genome can integrate into the genome of an infected host. This polydnaviral system, along with that using a densovirus described by Bergoin et al. (2), provide potential for virus-derived transformation vectors. Currently the commonly used baculovirus expression vector system has limitations because protein expression is transient, killing the host cells and necessitating extra bioreactors producing viral inocula and insect cells for further infection. A polydnavirus-based shuttle vector system could be developed to 768
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FIG. 4. Southern blot analysis of DNAs cloned from IPLB-LdEp cells infected with GiPDV, suggesting that GiPDV DNA is integrated in the IPLB-LdEp genome. (Lanes) DNAs isolated from (B) non-transformed IPLB-LdEp cells, (C) transformed IPLB-LdEp cells, (D) GiPDV DNA from female wasps, (E) clone E/G 12, and (F) clone E/G 25 were digested with EcoRI and electrophoresed through a 0.7% agarose gel (panel a). The DNAs were transferred to nylon membrane as described (13). Digoxigenin-labeled total DNA (BMB, Indianapolis, IN) isolated from IPLBLdEp cells (panel b) and GiPDV virus (panel c) were used as hybridization probes. Lane A contains molecular size standard as indicated by size in kilobase pairs.
generate transformed insect cells containing foreign genes of interest that could be expressed in an inducible system. If germ line tissue can be transformed as readily as somatic cells, this potential transgenic technology could be used for biocontrol, reducing dependence on chemical pesticides. REFERENCES 1. Ashburner, M. (1995) Science 270, 1941–1942. 2. Bergoin, M., Jousset, F.-X., Jourdan, M., Giraud, C., Rolling, F., Li, Y., Romane, C., Yuan, S., and Bossin, H. (1995) 1st Int. Workshop on Transgen. Invertebrates of Med., Agricult., and Aquacult. Importance, Montpellier, France. [Abstract] 3. Blissard, G. W., Fleming, J. G. W., Vinson, S. B., and Summers, M. D. (1986) J. Insect Physiol. 32, 351–359. 4. Fleming, J. G. W., and Summers, M. D. (1990) in Molecular Insect Science (Hagedorn, H. H., et al., Eds.), pp. 99–105. 5. Fleming, J. G. W. (1991) Biological Control 1, 127–135. 6. Fleming, J. G. W., and Summers, M. D. (1986) J. Virology 57, 552–562. 7. Fleming, J. G. W., and Krell, P. J. (1993) in Parasites and Pathogens of Insects (Beckage, N. E., Thompson, S. N., and Federici, B. A., Eds.), Vol. 1, pp. 189–225. Academic Press, San Diego. 8. Fleming, J. G. W., Blissard, G. W., Summers, M. D., and Vinson, S. B. (1983) J. Virology 48, 74–78. 9. Handler, A. M., and O’Brochta, D. A. (1991) Ann. Rev. Entomol. 36, 159–183. 10. Loukeris, T. G., Livadaras, I., Arca, B., Zabalou, S., and Savakis, C. (1995) Science 270, 2002–2003. 11. Rubin, G. M., and Spradling, A. C. (1982) Science 218, 348–353. 12. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 13. Southern, E. M. (1975) J. Mol. Biol. 98, 503–517. 14. Stoltz, D. B., Guzo, D., Belland, E. R., Lucarotti, C. J., and MacKinnon, E. A. (1988) J. Gen. Virol. 69, 903– 907. 769
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Stoltz, D. B., Krell, P., Summers, M. D., and Vinson, S. B. (1984) Intervirol. 21, 1. Stoltz, D. B., and Xu, D. (1990) Can. J. Microbiol. 36, 538–543. Stoltz, D. B. (1990) J. Gen. Virol. 71, 1051–1056. Stoltz, D. B., Guzo, D., and Cook, D. (1986) Virology 155, 120–131. Stoltz, D. B., and Vinson, S. B. (1979a) Advances in Virus Research 24, 125–171. Stoltz, D. B. (1993) in Parasites and Pathogens of Insects, (Beckage, N. E., Thompson, S. N., and Federici, B. A., Eds.), Vol. 1, pp. 167–187. Academic Press, San Diego. Stoltz, D. B., and Vinson, S. B. (1979b) Can. J. Microbiol. 25, 207–216. Strand, M. R., McKenzie, D. I., Grassl, V., Dover, B. A., and Aiken, J. M. (1992) J. Gen. Virol. 73, 1627–1635. Theilmann, D. A., and Summers, M. D. (1988) Virology 167, 329–341. Xu, D., and Stoltz, D. B. (1991) J. Virology 65, 6693–6704. Zweibel, L. J., Saccone, G., Zacharopoulou, A., Besansky, N. J., Favia, G., Collins, F. H., Louis, C., and Kafatos, F. C. (1995) Science 270, 2005–2008.
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