Transformation of yeast using bioactive beads with surface-immobilized yeast artificial chromosomes.
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.
Kawakami
Shigeki
S
Venture Business Laboratory Center for Advanced Science and Innovation, Osaka University, Japan.
Harashima
Satoshi
S
Kobayashi
Akio
A
Fukui
Kiichi
K
D000464:Alginates / Q000737:chemistry*; D018244:Chromosomes, Artificial, Yeast; D015825:Chromosomes, Fungal; D004271:DNA, Fungal; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013499:Surface Properties; D014170:Transformation, Genetic
D000464:Alginates; D004271:DNA, Fungal; D006603:Hexuronic Acids; D020723:Glucuronic Acid