NDLI: Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

Content Provider	PubMed Central
Author	Larionov, Vladimir Kouprina, Natalya Graves, Joan Resnick, Michael a.
Copyright Year	1996
Abstract	Transformation-associated recombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the generation of circular YACs by using a single centromere vector containing various human repeats at opposite ends. Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of ≈150 kb. The circular YACs are stable and they can be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette. More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human–rodent hybrid cell lines. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats. The level of enrichment of human DNA was nearly 3000-fold. A comparable level of enrichment was demonstrated with DNA isolated from a radiation hybrid cell line containing only 5 Mb of human DNA. A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human–rodent chimeras were detected among YACs generated by TAR cloning. The results with a circular TAR cloning vector or two vectors differed from results with a single-telomere vector in that the latter often resulted in a series of terminal deletions in linear YACs. This could provide a means for physical mapping of cloned material.
Starting Page	13925
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	24
Volume Number	93
Language	English
Publisher	The National Academy of Sciences of the USA
Publisher Date	1996-11-26
Access Restriction	Open
Rights Holder	The National Academy of Sciences of the USA
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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