A versatile and general splitting technology for generating targeted YAC subclones.
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.
Kim
Yeonhee
Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
Sugiyama
Minetaka
M
Yamagishi
Kazuo
K
Kaneko
Yoshinobu
Y
Fukui
Kiichi
K
Kobayashi
Akio
A
Harashima
Satoshi
S
D017360:Arabidopsis / Q000235:genetics; D002874:Chromosome Mapping; D002875:Chromosomes / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal; D015723:Gene Library; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal