Abstract
The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of β-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, β-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.
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References
Bates GW, Carle SA, Piastuch WC (1990) Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization. Plant Mot Biol 14:899–908
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336
Bilang R, Iida S, Peterhans A, Potrykus I, Paszowski J (1991) The 3′-terminal region of the hygromycin B resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum. Gene 100:247–250
Carle GF, Frank M, Olson MV (1986) Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 232:65–68
Dhawale SS, Marzluf GA (1985) Transformation of Neurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA. Curr Genet 10:205–212
Farman ML, Oliver RP (1992) Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen. Mol Gen Genet 231:243–247
Folger KR, Wong EA, Wahl G, Capecchi MR (1982) Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol 2:1372–1387
Folger KR, Thomas K, Capecchi MR (1985) Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei. Mol Cell Biol 5:59–69
Förster H, Coffey MD, Elwood H, Sogin ML (1990) Sequence analysis of the small subunit ribosomal RNAs of three zoosporic fungi and implications for fungal evolution. Mycologia 82:306–312
Fotheringham S, Holloman WK (1990) Pathways of transformation in Ustilago maydis determined by DNA conformation. Genetics 124:833–843
Goyon C, Faugeron G (1989) Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences. Mol Cell Biol 9:2818–2827
Hicks JB, Hinnen A, Fink GR (1978) Properties of yeast transformation. Cold Spring Harbor Symp Quant Biol 43:1305–1313
Hoy CA, Fuscoe JC, Thompson LH (1987) Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatic exchange. Mol Cell Biol 7:2007–2011
Judelson HS, Michelmore RW (1991) Transient expression of foreign genes in the oomyeete Phytophthora infestans using Bremia lactucae regulatory sequences. Curr Genet 19:453–459
Judelson HS, Tyler BM, Michelmore RW (1991) Transformation of the oomycete pathogen, Phytophthora infestans. Mol Plant Microbe Interact 4:602–607
Judelson HS, Tyler BM, Michelmore RW (1992) Regulatory sequences for expressing genes in oomycete fungi. Mol Gen Genet 234:138–146
Katz KS, Ratner DI (1988) Homologous recombination and the repair of double-stranded breaks during cotransformation of Dictyostelium discoideum. Mol Cell Biol 8:2779–2786
Landau NR, Schatz DG, Rosa M, Baltimore D (1987) Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol 7:3237–3243
Langin T, Daboussi MJ, Gerlinger C, Brygoo Y (1990) Influence of biological parameters and gene transfer technique on transformation of Fusarium oxysporum. Curr Genet 17:313–319
Maliga P, Svab Z, Harper EC, Jones JDG (1988) Improved expression of streptomycin resistance in plants due to a deletion in the streptomycin phosphotransferase coding sequence. Mol Gen Genet 214:456–459
Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10:3959–3970
Miller CK, Temin HM (1983) High-efficiency ligation and recombination of DNA fragments by vertebrate cells. Science 220:606–609
Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358
Parsons KA, Chumley FG, Valent B (1987) Genetic transformation of the fungal pathogen responsible for rice blast disease. Proc Natl Acad Sci USA 84:4161–4165
Perucho M, Hanahan D, Wigler M (1980) Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22:309–317
Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20
Reid LH, Shesely EG, Kim HS, Smithies O (1991) Cotransformation and gene targeting in mouse embryonic stem cells. Mol Cell Biol 11:2769–2777
Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. Proc Natl Acad Sci USA 83:5602–5606
Roth DB, Wilson JH (1985) Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc Natl Acad Sci USA 82:3355–3359
Thode S, Schäfer A, Pfeiffer P, Vielmetter W (1990) A novel pathway of DNA end-to-end joining. Cell 60:921–928
Tooley PW, Therriën CD (1987) Cytophotometric determination of the nuclear DNA content of 23 Mexican and 18 non-Mexican isolates of Phytophthora infestans. Exp Mycol 11:19–26
Wang J, Holden DW, Leong SA (1988) Gene transfer system for the phytopathogenic fungus Ustilago maydis. Proc Natl Acad Sci USA 85:865–869
Wernars K, Goosen T, Wennekes BMJ, Swart K, van den Hondel CAMJJ, van den Broek HWJ (1987) Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes. Mol Gen Genet 209:71–77
Wigler M, Sweet R, Sim GK, Wold P, Pellicer A, Lacy E, Maniatis T, Silverstein S, Axel R (1979) Transformation of mammalian cells with genes from prokaryotes and eukaryotes. Cell 16:777–785
Wilson JH, Berget PB, Pipas JM (1982) Somatic cells efficiently join unrelated DNA segments end-to-end. Mol Cell Biol 2:1258–1269
Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474
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Communicated by C.A.M.J.J. van den Hondel
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Judelson, H.S. Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans . Molec. Gen. Genet. 239, 241–250 (1993). https://doi.org/10.1007/BF00281624
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DOI: https://doi.org/10.1007/BF00281624