Development of Simultaneous YAC Manipulation-Amplification (SYMA) system by Chromosome Splitting Technique Harboring Copy Number Amplification System

Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation- Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 ㎎/㎖ sulfanilamide and 50 ㎍/㎖ methotrexate (S3/M50) as inducing substances.