Applied Genetics and Molecular Biotechnology

Applied Microbiology and Biotechnology

, Volume 69, Issue 1, pp 65-70

First online:

A versatile and general splitting technology for generating targeted YAC subclones

  • YeonHee KimAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Minetaka SugiyamaAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Kazuo YamagishiAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Yoshinobu KanekoAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Kiichi FukuiAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Akio KobayashiAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University
  • , Satoshi HarashimaAffiliated withDepartment of Biotechnology, Graduate School of Engineering, Osaka University Email author 

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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.