SpringerProtocols: Abstract: 6. Transformation of Yeast Using Bioactive Beads With Surface-Immobilized Yeast Artificial Chromosomes

6. Transformation of Yeast Using Bioactive Beads With Surface-Immobilized Yeast Artificial Chromosomes

By: Shigeki Kawakami^{2 3}, Satoshi Harashima⁴, Akio Kobayashi⁵, Kiichi Fukui⁶

Abstract

Full Text

Download PDF (267K)

Yeast artificial chromosomes (YACs) are useful cloning vectors with the capacity to carry large DNA inserts. The largest barrier using such large DNA molecules in transformation experiments has been their physical instability in a solution. We developed a new method for transforming yeast with chromosome-sized DNA. The method uses bioactive beads composed of calcium alginate to immobilize yeast chromosomal DNAs. Chromosomal DNA immobilized on bioactive beads is physically stable when compared with naked chromosomal DNAs. The bead-mediated transformation performed well, not only with respect to the transformation frequency, but also in successful transformation using split chromosomal DNA that exceeded 450 kb in size. In this chapter we introduce a new method for transforming yeast using bioactive beads. In conjunction with genomic YAC libraries and the yeast chromosome-splitting method, this technique will pave the way to stable and effective transfer of YACs into yeast cells.

Affiliation(s): (2) Venture Business Laboratory Center for Advanced Science, Osaka, Japan
(3) Innovation Osaka University, Osaka, Japan
(4) Laboratory of Molecular Genetics, Department of Biotechnology, Graduate School of Engineering Osaka University, Suita, Osaka, Japan
(5) Laboratory of Cell Technology, Department of Biotechnology, Graduate School of Engineering Osaka University, Suita, Osaka, Japan
(6) Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering Osaka University, Suita, Osaka, Japan

Book Title: YAC Protocols

Series: Methods in Molecular Biology | Volume: 349 | Pub. Date: Aug-18-2006 | Page Range: 61-65 | DOI: 10.1385/1-59745-158-4:61

Subject: Genetics/Genomics

Key Words: Yeast artificial chromosome (YAC) - transformation - chromosome-sized DNA - bioactive beads - calcium alginate

References

Download References

1.	Beggs, J. D. (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–109.

2.	Hinnen, A., Hicks, J. B., and Fink, G. R. (1978) Transformation of yeast. Biotechnology 24, 337–341.

3.	Hashimoto, H., Morikawa, H., Yamada, K., and Kimura, A. (1985) A novel method fortransformation of intact yeast cells by electroinjection of plasmid DNA. Appl. Microbiol. Biotechnol. 21, 336–339.

4.	Schiestl, R. H. and Gietz, R. D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16, 339–346.

5.	Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806–812.

6.	Vollrath, D., Davis, R. W., Connelly, C., and Hieter, P. (1988) Physical mapping of large DNA by chromosome fragmentation. Proc. Natl. Acad. Sci. USA 85, 6027–6031.

7.	Widianto, D., Mukai, Y., Kim, K., Harashima, S., and Oshima, Y. (1996) One-step splitting of a chromosome in haploid cells of Saccharomyces cerevisiae and its effect on the cell proliferation. J. Ferm. Bioeng. 82, 199–204.

8.	Bauchwitz, R. and Costantini, F. (1998) YAC transgenesis: a study of conditions to protect YAC DNA from breakage and a protocol for transfection. Biochim. Biophys. Acta. 1401, 21–37.

9.	Connelly, C., McCormick, M. K., Shero, J., and Hieter, P. (1991) Polyamines eliminate an extreme size bias against transformation of large yeast artificial chromosome DNA. Genomics 10, 10–16.

10.	Mizukami, A., Nagamori, E., Takakura, Y., et al. (2003) Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA. Biotechniques 35, 734–740.

11.	Liu, H., Kawabe, A., Matsunaga, S., et al. (2004) Obtaining transgenic plants using the bioactive beads method. J. Plant Res. 117, 95–99.

12.	Higashi, T., Nagamori, E., Sone, T., Matsunaga, S., and Fukui, K. (2004) A novel transfection method for mammalian cells using calcium alginate microbeads J. Bioscience Bioengineering 97, 191–195.

13.	Sone, T., Nagamori, E., Ikeuchi, T., et al. (2002) A novel genedelivery system in plants with calcium alginatemicro-beads. J. Biosci. Bioeng. 94, 87–91.

14.	McCormick, M. K., Shero, J., Connelly, C., Antonarakis, S., and Hieter, P. (1990) Methods forcloning large DNA segments as artificial chromosomesin S. cerevisiae. Techniques 2, 65–71.

15.	Burgers, P. M. and Percival, K. J. (1987) Transformation of yeast spheroplasts without cell fusion. Anal. Biochem. 163, 391–397.

Comments (Loading...)

Post comment/View all comments