Reference - Detail
| RRC ID | 879 |
|---|---|
| Author | Kim Y, Sugiyama M, Yamagishi K, Kaneko Y, Fukui K, Kobayashi A, Harashima S. |
| Title | A versatile and general splitting technology for generating targeted YAC subclones. |
| Journal | Appl Microbiol Biotechnol |
| 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. |
| Volume | 69(1) |
| Pages | 65-70 |
| Published | 2005-11-1 |
| DOI | 10.1007/s00253-005-1970-x |
| PMID | 15864580 |
| MeSH | Arabidopsis / genetics Chromosome Mapping Chromosomes / genetics* Chromosomes, Artificial, Yeast / genetics* Cloning, Molecular / methods* DNA Replication / genetics* DNA, Fungal Gene Library Genetic Vectors Polymerase Chain Reaction / methods* Recombination, Genetic Saccharomyces cerevisiae / genetics |
| IF | 3.53 |
| Times Cited | 8 |
| WOS Category | BIOTECHNOLOGY & APPLIED MICROBIOLOGY |
| Altmetric score |
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 1 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Yeast | BY6173(SH6173) BY5679(SH5679) |