Applied Microbiology and Biotechnology

, Volume 69, Issue 1, pp 65–70 | Cite as

A versatile and general splitting technology for generating targeted YAC subclones

  • YeonHee Kim
  • Minetaka Sugiyama
  • Kazuo Yamagishi
  • Yoshinobu Kaneko
  • Kiichi Fukui
  • Akio Kobayashi
  • Satoshi Harashima
Applied Genetics and Molecular Biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C4A2)6 repeats, 400-bp target region, CEN4, H4ARS and Kmr (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu+ transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.

Keywords

Yeast Artificial Chromosome Template Plasmid Autonomously Replicate Sequence Eukaryotic Chromosome Strain SH5679 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Notes

Acknowledgements

We thank the Arabidopsis Biological Resource Center for providing YAC clone CIC9e2. This research was supported by grants from Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • YeonHee Kim
    • 1
  • Minetaka Sugiyama
    • 1
  • Kazuo Yamagishi
    • 1
  • Yoshinobu Kaneko
    • 1
  • Kiichi Fukui
    • 1
  • Akio Kobayashi
    • 1
  • Satoshi Harashima
    • 1
  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversityOsakaJapan

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