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Short DNA fragments induce site specific recombination in mammalian cells

Summary

A defective hprt gene was corrected by homologous recombination in a lymphocyte cell line deficient in Hypoxanthine-phosphoribosyl-transferase activity (hprt). In a novel approach, only a fragment of a cDNA clone of the functional hprt gene was used to induce homologous recombination. The mutation that was corrected corresponds to a single base change in exon III of the hprt gene.

Two transfection methods, electroporation and the previously unreported use of polyoma capsids containing only short DNA fragments, were able to induce the recombinational event. After transfection cells with a functional hprt gene were selected and homologous recombination events were identified using polymerase chain reaction.

Double stranded fragments and both coding and non-coding single stranded fragments resulted in conversion to a functional gene.

Analysis of the resulting hprt positive cells revealed that most cells had undergone a simple replacement reaction. Interestingly, however, some cells had lost an intron adjacent to the site of mutation. Potential mechanisms for this phenomenon, including the possible involvement of RNA in DNA repair, are discussed.

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References

  1. 1.

    Radding CM: Homologous pairing and strand exchange in genetic recombination. Ann Rev Genet 16: 405–437, 1982

  2. 2.

    Watt VM, Ingles CJ, Urdea MS, Rutter WJ: Homology requirements for recombination in Escherichia coli. Proc Natl Acad Sci USA 82: 4768–4772, 1985

  3. 3.

    Orr-Weaver TL, Szostak JW, Rothstein RJ: Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78: 6354–6358, 1981

  4. 4.

    Doetschman T, Maeda N, Smithies O: Targeted mutation of the hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci USA 85: 8583–8587, 1988

  5. 5.

    Mansour SL, Thomas KR, Capecchi MR: Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348–352, 1988

  6. 6.

    Singer BS, Gold L, Gauss P, Doherty DH: Determination of the amount of homology required for recombination in bacteriophage T4. Cell 31: 25–33, 1982

  7. 7.

    Shen P, Huang HV: Homologous recombination in Escherichia coli: Dependence on substrate length and homology. Genetics 112: 5350–5357, 1986

  8. 8.

    Waldman AS, Liskay RM: Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology. Mol Cell Biol 8: 5350–5357, 1988

  9. 9.

    Thomas KR, Folger KR, Capecchi MR: High frequency targeting of genes to specific sites in the mammalian genome. Cell 44: 419–428, 1986

  10. 10.

    Liskay RM, Letsou A, Stachelek JL: Homology requirement for efficient gene conversion between duplicated chromosomal sequences in mammalian cells. Genetics 115: 161–167, 1987

  11. 11.

    Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS: Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317: 230–234, 1985

  12. 12.

    Subramani S, Berg P: Homologous and nonhomologous recombination in monkey cells. Mol Cell Biol 3: 1040–1052, 1983

  13. 13.

    Lin F-L, Sperle K, Sternberg N: Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences. Proc Natl Acad Sci USA 82: 1391–1395, 1985

  14. 14.

    Song K-Y, Schwartz F, Maeda N, Smithies O, Kucherlapati RS: Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci USA 84: 6820–6824, 1987

  15. 15.

    Rauth S, Song K-Y, Ayares D, Wallace L, Moore PD, Kucherlapati R: Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts. Proc Natl Acad Sci USA 83: 5587–5591, 1986

  16. 16.

    Simon JR, Moore PD: Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae. Mol Cell Biol 7: 2329–2334, 1987

  17. 17.

    Gellert M, Nash H: Communication between segments of DNA during site-specific recombination. Nature 325: 401–404, 1987

  18. 18.

    Meselson M, Radding C: A general model for genetic recombination. Proc Natl Acad Sci USA 72: 358–361, 1975

  19. 19.

    Roth DB, Wilson JH: Non-homologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol 6: 4295–4304, 1986

  20. 20.

    Anderson RA, Eliason SL: Recombination of homologous DNA fragments into mammalian cells occurs predominantly by terminal pairing. Mol Cell Biol 6: 3246–3252, 1986

  21. 21.

    Lopez B, Rousset S, Coppey J: Homologous recombination intermediates between two duplex DNA catalyzed by human cell extracts. Nucl Acid Res 15: 5643–5655, 1987

  22. 22.

    Hsieh P, Meyn MS, Camerini-Otero RD: Partial purification and characterization of a recombinase from human cells. Cell 44: 885–894, 1986

  23. 23.

    Ganea D, Moore P, Chekuri L, Kucherlapati R: Characterization of an ATP-dependent DNA strand transferase from human cells. Mol Cell Biol 7: 3124–3130, 1987

  24. 24.

    Graham F, Van der Eb AJ: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456–467, 1973

  25. 25.

    McCutchan JH, Pagano JS: Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyldextran. J Natl Cancer Inst 41: 351–356, 1968

  26. 26.

    Neuman E, Schaefer-Ridder M, Wang Y, Hofschneider PH: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1: 841–845, 1982

  27. 27.

    Kelley WN, Rosenbloom FM, Henderson JF, Seegmiller JE: A specific enzyme defect in gout associated with over-production of uric acid. Proc Natl Acad Sci USA 57: 1735–1739, 1967

  28. 28.

    Lesch M, Nyhan WL: A familial disorder of uric acid metablism and central nervous system function. Am J Med 36: 561–570, 1964

  29. 29.

    Wilson JM, Kelley WN: Human hypoxanthine-guanine phosphoribosyltransferase: structural alteration in a disfunctional enzyme variant (hprt-Munich) isolated from a patient with gout. J Biol Chem 259: 27–36, 1984

  30. 30.

    Jolly DJ, Okayama H, Berg P, Esty AC, Filpula D, Bohlen P, Johnson GG, Shively JE, Hunkapillar T, Friedmann R: Isolation and characterization of a full length expressible cDNA for human hypoxanthine phosphoribosyltransferase. Proc Natl Acad Sci USA 80: 477–481, 1983

  31. 31.

    Kim SH, Mooes JC, David D, Respess JG, Jolly DJ, Friedmann T: The organization of the human hprt gene. Nucl Acid Res 14: 3103–3118, 1986

  32. 32.

    Bertling W, Hunger-Bertling K, Cline M: Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells. J Biochem Biophys Meth 14: 223–232, 1987

  33. 33.

    Shibata T, DasGupta C, Cunningham RP, Williams JGK, Osber L, Radding CM: Homologous pairing in genetic recombination. J Biol Chem 256: 7565–7572, 1981

  34. 34.

    Cunningham RP, Shibata T, DasGupta C, Radding CM: Single strands induce recA protein to unwind duplex DNA for homologous pairing. Nature 281: 191–195, 1979

  35. 35.

    Slilaty SN, Aposhian HV: Gene transfer by polyoma like particles assembled in a cell-free system. Science 220: 725–727, 1983

  36. 36.

    Saiki RK, Gelfand DH, Stoffel S, Scharf S, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491, 1988

  37. 37.

    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Ehrlich HA, Arnheim N: Enzymatic amplification of β-globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354, 1985

  38. 38.

    Wilson JM, Baugher BW, Mattes PM, Daddona PE, Kelley WN: Human hypoxanthine-guanine phosphoribosyltransferase: Demonstration of structural variants in lymphoblas toid cells derived from patients with a deficiency of the enzyme. J Clin Invest 69: 706–715, 1981

  39. 39.

    Bertling W: Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation. Biosci Rep 7: 107–112, 1987

  40. 40.

    Maxam AM, Gilbert W: Sequencing end-labeled DNA with base-specific chemical cleavages. Meth Enzymol 65: 499–547, 1980

  41. 41.

    Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13, 1983

  42. 42.

    Register III JC, Griffith J: The direction of recA protein assembly onto single-strand DNA is the same as the direction of strand assimilation during strand exchange. J Biol Chem 260: 12308–12312, 1985

  43. 43.

    Aposhian HV, Thayer RE, Qasba PK: Formation of nucleoprotein complexes between polyoma empty capsids and DNA. J Virol 15: 645–653, 1975

  44. 44.

    Crawford LV: The adsorption of polyoma virus. Virology 18: 177–181, 1962

  45. 45.

    Maniatis T, Fritsch EF, Sambrook J: in Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1982

  46. 46.

    Southern E: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517, 1975

  47. 47.

    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977

  48. 48.

    Chen EY, Seeburg PH: Supercoiled sequencing: Fast and simple method for sequencing plasmid-DNA. DNA 4: 165–172, 1985

  49. 49.

    Miller JH, Lebkowski JS, Greison KS, Calos MP: Specificity of mutations induced in transfected DNA by mammalian cells. EMBO J 3: 3117–3121, 1984

  50. 50.

    Loyter A, Scangos GA, Ruddle FH: Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci USA 79: 422–426, 1982

  51. 51.

    Leavitt AD, Roberts TM, Garcea RL: Polyoma virus major capsid protein, VP1. J Biol Chem 260: 12803–12809, 1985

  52. 52.

    Smith GR: Homologous recombination in procaryotes. Microbiol Rev 52: 1–28, 1988

  53. 53.

    Kucherlapati RS, Spencer J, Moore PD: Homologous recombination catalyzed by human cell extracts. Mol Cell Biol 5: 714–720, 1985

  54. 54.

    Folger KR, Thomas KR, Capecchi MR: Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei. Mol Cell Biol 5: 59–69, 1985

  55. 55.

    Thomas KR, Capecchi MR: Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51: 503–512, 1987

  56. 56.

    Chu M-L, Gargiulo V, Williams CJ, Ramirez F: Multiexon deletion in an osteogenesis imperfecta variant with increased type III collagen mRNA. J Biol Chem 260: 691–694, 1985

  57. 57.

    Barsh GS, Roush CL, Bonadio J, Byers PH, Gelinas RE: Intron mediated recombination may cause a deletion in an α1 type I collagen chain in a lethal form of osteogenesis imperfecta. Proc Natl Acad Sci USA 82: 2870–2874, 1985

  58. 58.

    Resnick MA: The repair double-strand breaks in DNA: a model involving recombination. J Theor Biol 59: 97–106, 1976

  59. 59.

    Szostak JW, Orr-Weaver T, Rothstein RJ, Stahl FW: The double-strand-break repair model for recombination. Cell 33: 25–35, 1983

  60. 60.

    Kucherlapati RS, Eves EM, Song K-Y, Morse BS, Smithies O: Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proc Natl Acad Sci USA 81: 3153–3157, 1984

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Correspondence to Katharina Hunger-Bertling.

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Hunger-Bertling, K., Harrer, P. & Bertling, W. Short DNA fragments induce site specific recombination in mammalian cells. Mol Cell Biochem 92, 107–116 (1990). https://doi.org/10.1007/BF00218128

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Key words

  • homologous recombination
  • polyoma capsids
  • electroporation
  • lymphocytes