| RRC ID |
879
|
| 著者 |
Kim Y, Sugiyama M, Yamagishi K, Kaneko Y, Fukui K, Kobayashi A, Harashima S.
|
| タイトル |
A versatile and general splitting technology for generating targeted YAC subclones.
|
| ジャーナル |
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.
|
| 巻・号 |
69(1)
|
| ページ |
65-70
|
| 公開日 |
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
|
| 引用数 |
8
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| リソース情報 |
| 酵母 |
BY6173(SH6173)
BY5679(SH5679) |
