Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Sep;16(9):5117–5126. doi: 10.1128/mcb.16.9.5117

Stable episomal maintenance of yeast artificial chromosomes in human cells.

K Simpson 1, A McGuigan 1, C Huxley 1
PMCID: PMC231512  PMID: 8756669

Abstract

Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ambinder R. F., Mullen M. A., Chang Y. N., Hayward G. S., Hayward S. D. Functional domains of Epstein-Barr virus nuclear antigen EBNA-1. J Virol. 1991 Mar;65(3):1466–1478. doi: 10.1128/jvi.65.3.1466-1478.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banerjee S., Livanos E., Vos J. M. Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein-Barr virus. Nat Med. 1995 Dec;1(12):1303–1308. doi: 10.1038/nm1295-1303. [DOI] [PubMed] [Google Scholar]
  3. Beverley S. M. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis. Anal Biochem. 1989 Feb 15;177(1):110–114. doi: 10.1016/0003-2697(89)90023-7. [DOI] [PubMed] [Google Scholar]
  4. Brown K. E., Barnett M. A., Burgtorf C., Shaw P., Buckle V. J., Brown W. R. Dissecting the centromere of the human Y chromosome with cloned telomeric DNA. Hum Mol Genet. 1994 Aug;3(8):1227–1237. doi: 10.1093/hmg/3.8.1227. [DOI] [PubMed] [Google Scholar]
  5. Brown W. R. Molecular cloning of human telomeres in yeast. Nature. 1989 Apr 27;338(6218):774–776. doi: 10.1038/338774a0. [DOI] [PubMed] [Google Scholar]
  6. Burgers P. M., Percival K. J. Transformation of yeast spheroplasts without cell fusion. Anal Biochem. 1987 Jun;163(2):391–397. doi: 10.1016/0003-2697(87)90240-5. [DOI] [PubMed] [Google Scholar]
  7. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cowell J. K. Double minutes and homogeneously staining regions: gene amplification in mammalian cells. Annu Rev Genet. 1982;16:21–59. doi: 10.1146/annurev.ge.16.120182.000321. [DOI] [PubMed] [Google Scholar]
  9. Cross S. H., Allshire R. C., McKay S. J., McGill N. I., Cooke H. J. Cloning of human telomeres by complementation in yeast. Nature. 1989 Apr 27;338(6218):771–774. doi: 10.1038/338771a0. [DOI] [PubMed] [Google Scholar]
  10. Duff K., McGuigan A., Huxley C., Schulz F., Hardy J. Insertion of a pathogenic mutation into a yeast artificial chromosome containing the human amyloid precursor protein gene. Gene Ther. 1994 Jan;1(1):70–75. [PubMed] [Google Scholar]
  11. Featherstone T., Huxley C. Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells. Genomics. 1993 Aug;17(2):267–278. doi: 10.1006/geno.1993.1321. [DOI] [PubMed] [Google Scholar]
  12. Gahn T. A., Schildkraut C. L. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell. 1989 Aug 11;58(3):527–535. doi: 10.1016/0092-8674(89)90433-9. [DOI] [PubMed] [Google Scholar]
  13. Garza D., Ajioka J. W., Burke D. T., Hartl D. L. Mapping the Drosophila genome with yeast artificial chromosomes. Science. 1989 Nov 3;246(4930):641–646. doi: 10.1126/science.2510296. [DOI] [PubMed] [Google Scholar]
  14. Gnirke A., Barnes T. S., Patterson D., Schild D., Featherstone T., Olson M. V. Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes. EMBO J. 1991 Jul;10(7):1629–1634. doi: 10.1002/j.1460-2075.1991.tb07685.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green E. D., Olson M. V. Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping. Science. 1990 Oct 5;250(4977):94–98. doi: 10.1126/science.2218515. [DOI] [PubMed] [Google Scholar]
  16. Gussander E., Adams A. Electron microscopic evidence for replication of circular Epstein-Barr virus genomes in latently infected Raji cells. J Virol. 1984 Nov;52(2):549–556. doi: 10.1128/jvi.52.2.549-556.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haaf T., Warburton P. E., Willard H. F. Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation. Cell. 1992 Aug 21;70(4):681–696. doi: 10.1016/0092-8674(92)90436-g. [DOI] [PubMed] [Google Scholar]
  18. Haase S. B., Calos M. P. Replication control of autonomously replicating human sequences. Nucleic Acids Res. 1991 Sep 25;19(18):5053–5058. doi: 10.1093/nar/19.18.5053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hampar B., Tanaka A., Nonoyama M., Derge J. G. Replication of the resident repressed Epstein-Barr virus genome during the early S phase (S-1 period) of nonproducer Raji cells. Proc Natl Acad Sci U S A. 1974 Mar;71(3):631–633. doi: 10.1073/pnas.71.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harris A., Young B. D., Griffin B. E. Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt's lymphoma-derived cell lines. J Virol. 1985 Oct;56(1):328–332. doi: 10.1128/jvi.56.1.328-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heinzel S. S., Krysan P. J., Tran C. T., Calos M. P. Autonomous DNA replication in human cells is affected by the size and the source of the DNA. Mol Cell Biol. 1991 Apr;11(4):2263–2272. doi: 10.1128/mcb.11.4.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huxley C., Hagino Y., Schlessinger D., Olson M. V. The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion. Genomics. 1991 Apr;9(4):742–750. doi: 10.1016/0888-7543(91)90369-p. [DOI] [PubMed] [Google Scholar]
  23. Huxley C. Transfer of YACs to mammalian cells and transgenic mice. Genet Eng (N Y) 1994;16:65–91. [PubMed] [Google Scholar]
  24. Igarashi T., Ikegami H., Yamazaki H., Minami M. Bam HI restriction fragment length polymorphisms for hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of carriers and controls of HPRT deficiency in Japan. Acta Paediatr Jpn. 1990 Feb;32(1):12–15. doi: 10.1111/j.1442-200x.1990.tb00777.x. [DOI] [PubMed] [Google Scholar]
  25. Igarashi T., Kamoshita S. Carrier detection of partial hypoxanthine-guanine phosphoribosyltransferase deficiency by analysis with BamHI restriction fragment length polymorphisms and oligonucleotide probes. Pediatr Res. 1990 Apr;27(4 Pt 1):417–421. doi: 10.1203/00006450-199004000-00022. [DOI] [PubMed] [Google Scholar]
  26. Imai T., Olson M. V. Second-generation approach to the construction of yeast artificial-chromosome libraries. Genomics. 1990 Oct;8(2):297–303. doi: 10.1016/0888-7543(90)90285-3. [DOI] [PubMed] [Google Scholar]
  27. Jakobovits A., Moore A. L., Green L. L., Vergara G. J., Maynard-Currie C. E., Austin H. A., Klapholz S. Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature. 1993 Mar 18;362(6417):255–258. doi: 10.1038/362255a0. [DOI] [PubMed] [Google Scholar]
  28. Johnson C. V., Singer R. H., Lawrence J. B. Fluorescent detection of nuclear RNA and DNA: implications for genome organization. Methods Cell Biol. 1991;35:73–99. [PubMed] [Google Scholar]
  29. Krysan P. J., Calos M. P. Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol Cell Biol. 1991 Mar;11(3):1464–1472. doi: 10.1128/mcb.11.3.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krysan P. J., Haase S. B., Calos M. P. Isolation of human sequences that replicate autonomously in human cells. Mol Cell Biol. 1989 Mar;9(3):1026–1033. doi: 10.1128/mcb.9.3.1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Larin Z., Fricker M. D., Tyler-Smith C. De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells. Hum Mol Genet. 1994 May;3(5):689–695. doi: 10.1093/hmg/3.5.689. [DOI] [PubMed] [Google Scholar]
  32. Lupton S., Levine A. J. Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. Mol Cell Biol. 1985 Oct;5(10):2533–2542. doi: 10.1128/mcb.5.10.2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Manuelidis L. Chromosomal localization of complex and simple repeated human DNAs. Chromosoma. 1978 Mar 22;66(1):23–32. doi: 10.1007/BF00285813. [DOI] [PubMed] [Google Scholar]
  34. Margolskee R. F., Kavathas P., Berg P. Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. Mol Cell Biol. 1988 Jul;8(7):2837–2847. doi: 10.1128/mcb.8.7.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Markie D., Ragoussis J., Senger G., Rowan A., Sansom D., Trowsdale J., Sheer D., Bodmer W. F. New vector for transfer of yeast artificial chromosomes to mammalian cells. Somat Cell Mol Genet. 1993 Mar;19(2):161–169. doi: 10.1007/BF01233531. [DOI] [PubMed] [Google Scholar]
  36. Masumoto H., Masukata H., Muro Y., Nozaki N., Okazaki T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J Cell Biol. 1989 Nov;109(5):1963–1973. doi: 10.1083/jcb.109.5.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nonet G. H., Wahl G. M. Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously. Somat Cell Mol Genet. 1993 Mar;19(2):171–192. doi: 10.1007/BF01233532. [DOI] [PubMed] [Google Scholar]
  38. Nonoyama M., Pagano J. S. Homology between Epstein-Barr virus DNA and viral DNA from Burkitt's lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Nature. 1973 Mar 2;242(5392):44–47. doi: 10.1038/242044a0. [DOI] [PubMed] [Google Scholar]
  39. Nussbaum R. L., Crowder W. E., Nyhan W. L., Caskey C. T. A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4035–4039. doi: 10.1073/pnas.80.13.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pachnis V., Pevny L., Rothstein R., Costantini F. Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5109–5113. doi: 10.1073/pnas.87.13.5109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peterson C., Legerski R. High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector. Gene. 1991 Nov 15;107(2):279–284. doi: 10.1016/0378-1119(91)90328-9. [DOI] [PubMed] [Google Scholar]
  42. Rawlins D. R., Milman G., Hayward S. D., Hayward G. S. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell. 1985 Oct;42(3):859–868. doi: 10.1016/0092-8674(85)90282-x. [DOI] [PubMed] [Google Scholar]
  43. Reeves R. H., Pavan W. J., Hieter P. Modification and manipulation of mammalian DNA cloned as YACs. Genet Anal Tech Appl. 1990 Sep;7(5):107–113. doi: 10.1016/0735-0651(90)90015-8. [DOI] [PubMed] [Google Scholar]
  44. Reisman D., Sugden B. trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. Mol Cell Biol. 1986 Nov;6(11):3838–3846. doi: 10.1128/mcb.6.11.3838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Reisman D., Yates J., Sugden B. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol. 1985 Aug;5(8):1822–1832. doi: 10.1128/mcb.5.8.1822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Saemundsen A. K., Perlmann C., Klein G. Intracellular Epstein-Barr virus DNA is methylated in and around the EcoRI-J fragment in both producer and nonproducer cell lines. Virology. 1983 Apr 30;126(2):701–706. doi: 10.1016/s0042-6822(83)80026-9. [DOI] [PubMed] [Google Scholar]
  47. Shalit P., Loughney K., Olson M. V., Hall B. D. Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae. Mol Cell Biol. 1981 Mar;1(3):228–236. doi: 10.1128/mcb.1.3.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Southern E. M., Anand R., Brown W. R., Fletcher D. S. A model for the separation of large DNA molecules by crossed field gel electrophoresis. Nucleic Acids Res. 1987 Aug 11;15(15):5925–5943. doi: 10.1093/nar/15.15.5925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sugden B., Marsh K., Yates J. A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985 Feb;5(2):410–413. doi: 10.1128/mcb.5.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sun T. Q., Fenstermacher D. A., Vos J. M. Human artificial episomal chromosomes for cloning large DNA fragments in human cells. Nat Genet. 1994 Sep;8(1):33–41. doi: 10.1038/ng0994-33. [DOI] [PubMed] [Google Scholar]
  51. Taylor S. S., Larin Z., Smith C. T. Addition of functional human telomeres to YACs. Hum Mol Genet. 1994 Aug;3(8):1383–1386. doi: 10.1093/hmg/3.8.1383. [DOI] [PubMed] [Google Scholar]
  52. Tyler-Smith C., Oakey R. J., Larin Z., Fisher R. B., Crocker M., Affara N. A., Ferguson-Smith M. A., Muenke M., Zuffardi O., Jobling M. A. Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes. Nat Genet. 1993 Dec;5(4):368–375. doi: 10.1038/ng1293-368. [DOI] [PubMed] [Google Scholar]
  53. Willard H. F. Chromosome-specific organization of human alpha satellite DNA. Am J Hum Genet. 1985 May;37(3):524–532. [PMC free article] [PubMed] [Google Scholar]
  54. Yates J. L., Guan N. Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. J Virol. 1991 Jan;65(1):483–488. doi: 10.1128/jvi.65.1.483-488.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yates J. L., Warren N., Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. 1985 Feb 28-Mar 6Nature. 313(6005):812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]
  56. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES