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A versatile and general splitting technology for generating targeted YAC subclones

  • Applied Genetics and Molecular Biotechnology
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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.

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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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Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka, 565-0871, Japan

    YeonHee Kim, Minetaka Sugiyama, Kazuo Yamagishi, Yoshinobu Kaneko, Kiichi Fukui, Akio Kobayashi & Satoshi Harashima

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  1. YeonHee Kim
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  2. Minetaka Sugiyama
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  3. Kazuo Yamagishi
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  4. Yoshinobu Kaneko
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  5. Kiichi Fukui
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  6. Akio Kobayashi
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  7. Satoshi Harashima
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Correspondence to Satoshi Harashima.

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Kim, Y., Sugiyama, M., Yamagishi, K. et al. A versatile and general splitting technology for generating targeted YAC subclones. Appl Microbiol Biotechnol 69, 65–70 (2005). https://doi.org/10.1007/s00253-005-1970-x

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  • DOI: https://doi.org/10.1007/s00253-005-1970-x

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