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.
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Clarke, L. & Carbon, J. Isolation of a yeast centromere and construction of a functional small circular chromosome. Nature 287, 504–509 (1980).
Murray, A.W. & Szostak, J.W. Construction of artificial chromosomes in yeast. Nature 305, 189–193 (1983).
Beach, D., Piper, M. & Shall, S. Isolation of chromosomal origins of replication in yeast. Nature 284, 185–187 (1980).
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).
Marahrens, Y. & Stillman, B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255, 817–823 (1992).
Shampay, J., Szostak, J.W. & Blackburn, E.H. DNA sequences of telomeres maintained in yeast. Nature 310, 154–157 (1984).
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).
Tyler-Smith, C. & Willard, H.F. Mammalian chromosome structure. Curr. Opin. Genet. Devel. 3, 390–397 (1993).
Willard, H.F. Chromosome manipulation: a systematic approach toward understanding human chromosome structure and function. Proc. Natl. Acad. Sci. USA 93, 6847–6850 (1996).
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).
Fitzgerald-Hayes, M., Clarke, L. & Carbon, J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29, 235–244 (1982).
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).
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).
Willard, H.F. & Waye, J.S. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet. 3, 192–198 (1987).
Bloom, K. The centromere frontier: kinetochore components, microtubule-based motility, and the CEN-value paradox. Cell 73, 621–624 (1993).
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).
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).
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).
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).
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).
Brown, K.E. et al. Dissecting the centromere of the human Y chromosome with cloned telomeric DNA. Hum. Mol. Genet. 3, 1227–1237 (1994).
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).
Hofer, B. Construction and stability of a sixfold repeated artificial gene. Eur. J. Biochem. 67, 307–313 (1987).
Sadler, J.R., Tecklenburg, M. & Betz, J.L. Plasmids containing many tandem copies of a synthetic lactose operator. Gene 8, 279–300 (1980).
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).
Munz, P.L. & Young, C.S. End-joining of DNA fragments in adenovirus transfection of human cells. Virology 183, 160–169 (1991).
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).
Barnett, M.A. et al. Telomere directed fragmentation of mammalian chromosomes. Nucl. Acids Res. 21, 27–36 (1993).
Farr, C.J. et al. Generation of a human X-derived minichromosome using telomere-associated chromosome fragmentation. EMBO J. 14, 5444–5454 (1995).
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).
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).
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).
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).
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).
Szostak, J.W. & Blackburn, E.H. Cloning yeast telomeres on linear plasmid vectors. Cell 29, 245–255 (1982).
Murphy, T.D. & Karpen, G.H. Localization of centromere function in a Drosophila minichromosome. Cell 82, 599–609 (1995).
Mann, C. & Davis, R. Instability of dicentric plasmids in yeast. Proc. Natl. Acad. Sci. USA 80, 228–232 (1983).
Koshland, D., Rutledge, L., Fitzgerald-Hayes, M. & Hartwell, L.H. A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae. Cell 48, 801–812 (1987).
Featherstone, T. & Huxley, C. Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells. Genomics 7, 267–278 (1993).
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).
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).
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).
Hahn, P.J. et al. Double-minute chromosomes as megabase cloning vehicles. Genet. Anal. Tech. Appl. 9, 17–25 (1992).
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).
McGuigan, A. & Huxley, C. Replication of yeast DNA and novel chromosome formation in mouse cells. Nucl. Acids Res. 24, 2271–2280 (1996).
Friedmann, T. The promise and overpromise of human gene therapy. Gene Ther. 1, 217–218 (1994).
Strauss, M. Liver-directed gene therapy: prospects and problems. Gene Ther. 1, 156–164 (1994).
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).
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).
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).
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).
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).
Seed, B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329, 840–842 (1987).
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).
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).
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).
Salier, J.P. & Kurachi, K. A CAT expression vector with virtually no background: pUMSVOCAT. Biotechniques 7, 30–31 (1989).
Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).
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).
Earnshaw, W.C. et al. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. Cell Biol. 104, 8177ndash;829 (1987).
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).
Saitoh, H. et al. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70, 115–125 (1992).
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).
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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
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