Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 May 11;22(9):1651–1654. doi: 10.1093/nar/22.9.1651

Chimeric YACs were generated at unreduced rates in conditions that suppress coligation.

M Wada 1, K Abe 1, K Okumura 1, H Taguchi 1, K Kohno 1, F Imamoto 1, D Schlessinger 1, M Kuwano 1
PMCID: PMC308044  PMID: 8202367

Abstract

Chimerism is a major limitation of current YAC libraries. A method based on partially filled-in ends of restriction fragments was designed to avoid coligation as a possible source of chimeras. Model experiments using plasmid DNA as an insert showed that coligation was clearly avoided by this method. Pilot collections of YACs with an average insert size of 650kb were then constructed with and without the partial fill-in treatment. Starting from a mixture of a equal amounts of human and mouse DNA, none of 108 clones was positive by hybridization with both Alu and B2 probes, again suggesting that coligation was effectively blocked. However, 4 out of 10 clones still hybridized to 2 or more locations by FISH on chromosomes in human metaphase spreads, level similar to that in the clones made without the partial fill-in step. These results strongly suggest that chimeric clones generally arise by a mechanism independent of coligation, presumptively based on recombination.

Full text

PDF
1651

Images in this article

1653Image
on p.1653
1653Image
on p.1653

Selected References

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

  1. Abidi F. E., Wada M., Little R. D., Schlessinger D. Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences. Genomics. 1990 Jul;7(3):363–376. doi: 10.1016/0888-7543(90)90170-y. [DOI] [PubMed] [Google Scholar]
  2. Albertsen H. M., Abderrahim H., Cann H. M., Dausset J., Le Paslier D., Cohen D. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4256–4260. doi: 10.1073/pnas.87.11.4256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett K. L., Hill R. E., Pietras D. F., Woodworth-Gutai M., Kane-Haas C., Houston J. M., Heath J. K., Hastie N. D. Most highly repeated dispersed DNA families in the mouse genome. Mol Cell Biol. 1984 Aug;4(8):1561–1571. doi: 10.1128/mcb.4.8.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  9. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  10. Green E. D., Riethman H. C., Dutchik J. E., Olson M. V. Detection and characterization of chimeric yeast artificial-chromosome clones. Genomics. 1991 Nov;11(3):658–669. doi: 10.1016/0888-7543(91)90073-n. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lichter P., Cremer T., Borden J., Manuelidis L., Ward D. C. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet. 1988 Nov;80(3):224–234. doi: 10.1007/BF01790090. [DOI] [PubMed] [Google Scholar]
  13. Little R. D., Pilia G., Johnson S., D'Urso M., Schlessinger D. Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):177–181. doi: 10.1073/pnas.89.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marchuk D., Collins F. S. pYAC-RC, a yeast artificial chromosome vector for cloning DNA cut with infrequently cutting restriction endonucleases. Nucleic Acids Res. 1988 Aug 11;16(15):7743–7743. doi: 10.1093/nar/16.15.7743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCormick M. K., Campbell E., Deaven L., Moyzis R. Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1063–1067. doi: 10.1073/pnas.90.3.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nakano N. Establishment of cell lines in vitro from a mammary ascites tumor of mouse and biological properties of the established lines in a serum containing medium. Tohoku J Exp Med. 1966 Jan 25;88(1):69–84. doi: 10.1620/tjem.88.69. [DOI] [PubMed] [Google Scholar]
  17. Neil D. L., Villasante A., Fisher R. B., Vetrie D., Cox B., Tyler-Smith C. Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors. Nucleic Acids Res. 1990 Mar 25;18(6):1421–1428. doi: 10.1093/nar/18.6.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schlessinger D., Little R. D., Freije D., Abidi F., Zucchi I., Porta G., Pilia G., Nagaraja R., Johnson S. K., Yoon J. Y. Yeast artificial chromosome-based genome mapping: some lessons from Xq24-q28. Genomics. 1991 Dec;11(4):783–793. doi: 10.1016/0888-7543(91)90001-u. [DOI] [PubMed] [Google Scholar]
  19. Selleri L., Eubanks J. H., Giovannini M., Hermanson G. G., Romo A., Djabali M., Maurer S., McElligott D. L., Smith M. W., Evans G. A. Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization. Genomics. 1992 Oct;14(2):536–541. doi: 10.1016/s0888-7543(05)80263-0. [DOI] [PubMed] [Google Scholar]
  20. Tatsumi K., Toyoda M., Hashimoto T., Furuyama J., Kurihara T., Inoue M., Takebe H. Differential hypersensitivity of xeroderma pigmentosum lymphoblastoid cell lines to ultraviolet light mutagenesis. Carcinogenesis. 1987 Jan;8(1):53–57. doi: 10.1093/carcin/8.1.53. [DOI] [PubMed] [Google Scholar]
  21. Wada M., Little R. D., Abidi F., Porta G., Labella T., Cooper T., Della Valle G., D'Urso M., Schlessinger D. Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes. Am J Hum Genet. 1990 Jan;46(1):95–106. [PMC free article] [PubMed] [Google Scholar]
  22. Zabarovsky E. R., Allikmets R. L. An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends. Gene. 1986;42(1):119–123. doi: 10.1016/0378-1119(86)90158-7. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES