A versatile and general splitting technology for generating targeted YAC subclones

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 (C(4)A(2))(6) repeats, 400-bp target region, CEN4, H4ARS and Km(r) (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.

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

“…However, target-specific sequences necessary for splitting must be newly cloned into a YAC splitting vector harboring amplification elements, pCGS966, for each splitting event, which is prohibitively time-consuming. In a previous study, we developed an effective PCR-mediated chromosome splitting (PCS) method that can generate any targeted region of a plant chromosome without endogenous ARS elements as a new YAC [4]. The PCS method is a simple, efficient method that splits YAC-harboring plant chromosome regions as well as natural yeast chromosomes [4,15].…”

“…In a previous study, we developed an effective PCR-mediated chromosome splitting (PCS) method that can generate any targeted region of a plant chromosome without endogenous ARS elements as a new YAC [4]. The PCS method is a simple, efficient method that splits YAC-harboring plant chromosome regions as well as natural yeast chromosomes [4,15]. In this study, the Center, Columbus, OH, USA).…”

“…To amplify the split YAC, the amplification medium con- To generate each splitting fragment, pSKCHY (loxP-CgHIS3-loxP) [4] and pBGTK were used as template plasmids for PCR. The pBGTK (Fig.…”

“…A PCR product containing the TK gene with a flanking BamHI-NotI site was generated using pCGS966 [14] as a template and TK-F3 and TK-R3 as forward and reverse primers, respectively, and a PCR product containing GAL1/CEN4 with a flanking KpnI site was generated using pCGS966 [14] as a template and GC4-F and GC4-R as forward and reverse primers, respectively. The oligonucleotides used are listed in Table 1, and some of the primers were [4]. Other methods, i.e., CHEF gel electrophoresis and Southern hybridization were performed as described in Kim et al [3].…”

“…One splitting fragment (2.2 kb), Am-I (Fig. 1B), consisted of the CgHIS3 gene, a yeast transformant selective marker; a telomeric 5'-(C4A2) 6-3' repeat sequence; and a target sequence that corresponded to a sequence from nucleotide position 448,711 to 449,100 of YAC CIC9e2; this fragment was prepared by the same procedure and using the same primers as used for amplification of the 100-I fragment described in a previous report [4]. The other splitting fragment (6.4 kb), Am-II ( Fig.…”