Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Formation of de novo centromeres and construction of first-generation human artificial microchromosomes

Abstract

We have combined long synthetic arrays of alpha satellite DNA with telomeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6–10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-generation system for the construction of human artificial chromosomes should be suitable for dissecting the sequence requirements of human centromeres, as well as developing constructs useful for therapeutic applications.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

References

  1. Clarke, L. & Carbon, J. Isolation of a yeast centromere and construction of a functional small circular chromosome. Nature 287, 504–509 (1980).

    Article  CAS  Google Scholar 

  2. Murray, A.W. & Szostak, J.W. Construction of artificial chromosomes in yeast. Nature 305, 189–193 (1983).

    Article  CAS  Google Scholar 

  3. Beach, D., Piper, M. & Shall, S. Isolation of chromosomal origins of replication in yeast. Nature 284, 185–187 (1980).

    Article  CAS  Google Scholar 

  4. Bloom, K.S. & Carbon, J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell 29, 305–317 (1982).

    Article  CAS  Google Scholar 

  5. Marahrens, Y. & Stillman, B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255, 817–823 (1992).

    Article  CAS  Google Scholar 

  6. Shampay, J., Szostak, J.W. & Blackburn, E.H. DNA sequences of telomeres maintained in yeast. Nature 310, 154–157 (1984).

    Article  CAS  Google Scholar 

  7. Walmsley, R.W., Chan, C.S., Tye, B.K. & Petes, T.D. Unusual DNA sequences associated with the ends of yeast chromosomes. Nature 310, 157–160 (1984).

    Article  CAS  Google Scholar 

  8. Tyler-Smith, C. & Willard, H.F. Mammalian chromosome structure. Curr. Opin. Genet. Devel. 3, 390–397 (1993).

    Article  CAS  Google Scholar 

  9. Willard, H.F. Chromosome manipulation: a systematic approach toward understanding human chromosome structure and function. Proc. Natl. Acad. Sci. USA 93, 6847–6850 (1996).

    Article  CAS  Google Scholar 

  10. Orkin, S. & Motulsky, A. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. National Institutes of Health(1995).

  11. Fitzgerald-Hayes, M., Clarke, L. & Carbon, J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29, 235–244 (1982).

    Article  CAS  Google Scholar 

  12. Hahnenberger, K.M., Baum, M.P., Polizzi, C.M., Carbon, J. & Clarke, L. Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 86, 577–581 (1989).

    Article  CAS  Google Scholar 

  13. Clarke, C. & Baum, M.P. Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences. Mol. Cell. Biol. 10, 1863–1872 (1990).

    Article  CAS  Google Scholar 

  14. Willard, H.F. & Waye, J.S. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet. 3, 192–198 (1987).

    Article  CAS  Google Scholar 

  15. Bloom, K. The centromere frontier: kinetochore components, microtubule-based motility, and the CEN-value paradox. Cell 73, 621–624 (1993).

    Article  CAS  Google Scholar 

  16. Neil, D., Villasante, A., Fisher, R., Vetrie, D., Cox, B. & Tyler-Smith, C. Structural instability of human tandemly repeated DNA sequences cloned in yeast artifical chromosome vectors. Nucl. Acids Res. 18, 1421–1428 (1990).

    Article  CAS  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 70, 681–696 (1992).

    Article  CAS  Google Scholar 

  18. 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. 3, 689–695 (1994).

    Article  CAS  Google Scholar 

  19. Tyler-Smith, C. et al. Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes. Nature Genet. 5, 368–375 (1993).

    Article  CAS  Google Scholar 

  20. Wevrick, R. & Willard, H.F. Long-range organization of tandem arrays of alpha satellite DNA at the centromeres of human chromosomes: high-frequency array-length polymorphism and meiotic stability. Proc. Natl. Acad. Sci. USA 86, 9394–9398 (1989).

    Article  CAS  Google Scholar 

  21. Brown, K.E. et al. Dissecting the centromere of the human Y chromosome with cloned telomeric DNA. Hum. Mol. Genet. 3, 1227–1237 (1994).

    Article  CAS  Google Scholar 

  22. Heller, R., Brown, K.E., Burgtorf, C. & Brown, W.R. Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage. Proc Natl. Acad. Sci. USA 93, 7125–7130 (1996).

    Article  CAS  Google Scholar 

  23. Hofer, B. Construction and stability of a sixfold repeated artificial gene. Eur. J. Biochem. 67, 307–313 (1987).

    Article  Google Scholar 

  24. Sadler, J.R., Tecklenburg, M. & Betz, J.L. Plasmids containing many tandem copies of a synthetic lactose operator. Gene 8, 279–300 (1980).

    Article  CAS  Google Scholar 

  25. Wilson, J.H., Berget, P.B. & Pipas, J.M. Somatic cells efficiently join unrelated DNA segments end-to-end. Mol. Cell. Biol. 2, 1258–1269 (1982).

    Article  CAS  Google Scholar 

  26. Munz, P.L. & Young, C.S. End-joining of DNA fragments in adenovirus transfection of human cells. Virology 183, 160–169 (1991).

    Article  CAS  Google Scholar 

  27. MacGregor, G.R., Zambrowicz, B.P. & Soriano, P. Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 121, 1487–1496 (1995).

    PubMed  CAS  Google Scholar 

  28. Barnett, M.A. et al. Telomere directed fragmentation of mammalian chromosomes. Nucl. Acids Res. 21, 27–36 (1993).

    Article  CAS  Google Scholar 

  29. Farr, C.J. et al. Generation of a human X-derived minichromosome using telomere-associated chromosome fragmentation. EMBO J. 14, 5444–5454 (1995).

    Article  CAS  Google Scholar 

  30. Taylor, S., Larin, Z. & Tyler-Smith, C. Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells. Chromosoma 105, 70–81 (1996).

    Article  CAS  Google Scholar 

  31. Greig, G.M. & Willard, H.F. Beta satellite DNA: characterization and localization of two subfamilies from the distal and proximal short arms of the human acrocentric chromosomes. Genomics 12, 573–580 (1992).

    Article  CAS  Google Scholar 

  32. Pluta, A.F., Mackay, A.M., Ainsztein, A.M., Goldberg, I.G. & Earnshaw, W.C. The centromere: hub of chromosomal activities. Science 270, 1591–1594 (1995).

    Article  CAS  Google Scholar 

  33. Earnshaw, W.C., Ratrie, H. & Stetten, G. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma 98, 1–12 (1989).

    Article  CAS  Google Scholar 

  34. Sullivan, B. & Schwartz, S. Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum. Mol. Genet. 4, 2189–2197 (1995).

    Article  CAS  Google Scholar 

  35. Szostak, J.W. & Blackburn, E.H. Cloning yeast telomeres on linear plasmid vectors. Cell 29, 245–255 (1982).

    Article  CAS  Google Scholar 

  36. Murphy, T.D. & Karpen, G.H. Localization of centromere function in a Drosophila minichromosome. Cell 82, 599–609 (1995).

    Article  CAS  Google Scholar 

  37. Mann, C. & Davis, R. Instability of dicentric plasmids in yeast. Proc. Natl. Acad. Sci. USA 80, 228–232 (1983).

    Article  CAS  Google Scholar 

  38. Koshland, D., Rutledge, L., Fitzgerald-Hayes, M. & Hartwell, L.H. A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae. Cell 48, 801–812 (1987).

    Article  CAS  Google Scholar 

  39. Featherstone, T. & Huxley, C. Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells. Genomics 7, 267–278 (1993).

    Article  Google Scholar 

  40. Sullivan, B.A. & Willard, H.F. Functional status of centromeres in dicentric X chromosomes: evidence for the distance-dependence of centromere/kinetechore assembly and correlation with malsegregation in anaphase. Am. J. Hum. Genet. 59, suppl.A14 (1996).

  41. Murphy, T.D. & Karpen, G.H. Interactions between the nod+ kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome. Cell 81, 139–148 (1995).

    Article  CAS  Google Scholar 

  42. Afshar, K., Barton, N.R., Hawley, R.S. & Goldstein, L.S. DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell 81, 129–138 (1995).

    Article  CAS  Google Scholar 

  43. Hahn, P.J. et al. Double-minute chromosomes as megabase cloning vehicles. Genet. Anal. Tech. Appl. 9, 17–25 (1992).

    Article  CAS  Google Scholar 

  44. Sun, T.Q., Fenstermacher, D.A. & Vos, J.-M. Human artificial episomal chromosomes for cloning large DNA fragments in human cells. Nature Genet. 8, 33–41 (1994).

    Article  CAS  Google Scholar 

  45. McGuigan, A. & Huxley, C. Replication of yeast DNA and novel chromosome formation in mouse cells. Nucl. Acids Res. 24, 2271–2280 (1996).

    Article  CAS  Google Scholar 

  46. Friedmann, T. The promise and overpromise of human gene therapy. Gene Ther. 1, 217–218 (1994).

    PubMed  CAS  Google Scholar 

  47. Strauss, M. Liver-directed gene therapy: prospects and problems. Gene Ther. 1, 156–164 (1994).

    PubMed  CAS  Google Scholar 

  48. Nabel, G.J. et al. Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans. Proc. Natl. Acad. Sci. USA 90, 11307–11311 (1993).

    Article  CAS  Google Scholar 

  49. Perales, J.C., Ferkol, T., Molas, M. & Hanson, R.W. An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. Eur. J. Biochem. 226, 255–66 (1994).

    Article  CAS  Google Scholar 

  50. Waye, J.S. & Willard, H.F., Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol. Cell. Biol. 6, 3156–65 (1986).

    Article  CAS  Google Scholar 

  51. Warburton, P.E., Greig, G.M., Haaf, T. & Willard, H.F. PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 11, 324–333 (1991).

    Article  CAS  Google Scholar 

  52. Shizuya, H. et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794–8797 (1992).

    Article  CAS  Google Scholar 

  53. Seed, B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329, 840–842 (1987).

    Article  CAS  Google Scholar 

  54. Seed, B. & Aruffo, A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA 84, 3365–3369 (1987).

    Article  CAS  Google Scholar 

  55. Heard, J.M. et al. Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system. Mol. Cell. Biol. 7, 2425–2434 (1987).

    Article  CAS  Google Scholar 

  56. McGeady, M.L., Wood, T.G., Maizel, J.V. & Vande, W.G. Sequences upstream from the mouse c-mos oncogene may function as a transcription termination signal. DNA 5, 289–298 (1986).

    Article  CAS  Google Scholar 

  57. Salier, J.P. & Kurachi, K. A CAT expression vector with virtually no background: pUMSVOCAT. Biotechniques 7, 30–31 (1989).

    PubMed  CAS  Google Scholar 

  58. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

    Google Scholar 

  59. Ijdo, J.W., Wells, R.A., Baldini, A. & Reeders, S.T. Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucl. Acids Res. 19, 4780 (1991).

    Article  CAS  Google Scholar 

  60. Earnshaw, W.C. et al. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. Cell Biol. 104, 8177ndash;829 (1987).

    Article  CAS  Google Scholar 

  61. Harrington, J.J. & Lieber, M.R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 8, 1344–1355 (1994).

    Article  CAS  Google Scholar 

  62. Saitoh, H. et al. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70, 115–125 (1992).

    Article  CAS  Google Scholar 

  63. Yen, T.J., Li, G., Schaar, B.T., Szilak, I. & Cleveland, D.W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature 359, 536–539 (1992).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Huntington F. Willard.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Harrington, J., Bokkelen, G., Mays, R. et al. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat Genet 15, 345–355 (1997). https://doi.org/10.1038/ng0497-345

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng0497-345

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing