Abstract
To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.
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- Allshire R. C., Cranston G., Gosden J. R., Maule J. C., Hastie N. D., Fantes P. A. A fission yeast chromosome can replicate autonomously in mouse cells. Cell. 1987 Jul 31;50(3):391–403. doi: 10.1016/0092-8674(87)90493-4. [DOI] [PubMed] [Google Scholar]
- 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]
- Brownstein B. H., Silverman G. A., Little R. D., Burke D. T., Korsmeyer S. J., Schlessinger D., Olson M. V. Isolation of single-copy human genes from a library of yeast artificial chromosome clones. Science. 1989 Jun 16;244(4910):1348–1351. doi: 10.1126/science.2544027. [DOI] [PubMed] [Google Scholar]
- Burhans W. C., Vassilev L. T., Caddle M. S., Heintz N. H., DePamphilis M. L. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell. 1990 Sep 7;62(5):955–965. doi: 10.1016/0092-8674(90)90270-o. [DOI] [PubMed] [Google Scholar]
- Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
- Caddle M. S., Calos M. P. Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication. Nucleic Acids Res. 1992 Nov 25;20(22):5971–5978. doi: 10.1093/nar/20.22.5971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Coverley D., Laskey R. A. Regulation of eukaryotic DNA replication. Annu Rev Biochem. 1994;63:745–776. doi: 10.1146/annurev.bi.63.070194.003525. [DOI] [PubMed] [Google Scholar]
- 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]
- Davies N. P., Rosewell I. R., Brüggemann M. Targeted alterations in yeast artificial chromosomes for inter-species gene transfer. Nucleic Acids Res. 1992 Jun 11;20(11):2693–2698. doi: 10.1093/nar/20.11.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DePamphilis M. L. Eukaryotic DNA replication: anatomy of an origin. Annu Rev Biochem. 1993;62:29–63. doi: 10.1146/annurev.bi.62.070193.000333. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Hamlin J. L. Mammalian origins of replication. Bioessays. 1992 Oct;14(10):651–659. doi: 10.1002/bies.950141002. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- McManus J., Perry P., Sumner A. T., Wright D. M., Thomson E. J., Allshire R. C., Hastie N. D., Bickmore W. A. Unusual chromosome structure of fission yeast DNA in mouse cells. J Cell Sci. 1994 Mar;107(Pt 3):469–486. doi: 10.1242/jcs.107.3.469. [DOI] [PubMed] [Google Scholar]
- Méchali M., Kearsey S. Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast. Cell. 1984 Aug;38(1):55–64. doi: 10.1016/0092-8674(84)90526-9. [DOI] [PubMed] [Google Scholar]
- Newlon C. S. Yeast chromosome replication and segregation. Microbiol Rev. 1988 Dec;52(4):568–601. doi: 10.1128/mr.52.4.568-601.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Praznovszky T., Keresö J., Tubak V., Cserpán I., Fátyol K., Hadlaczky G. De novo chromosome formation in rodent cells. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11042–11046. doi: 10.1073/pnas.88.24.11042. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riley J. H., Morten J. E., Anand R. Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells. Nucleic Acids Res. 1992 Jun 25;20(12):2971–2976. doi: 10.1093/nar/20.12.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Thomas K. R., Capecchi M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
- Tran C. T., Caddle M. S., Calos M. P. The replication behavior of Saccharomyces cerevisiae DNA in human cells. Chromosoma. 1993 Jan;102(2):129–136. doi: 10.1007/BF00356030. [DOI] [PubMed] [Google Scholar]
- 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]