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

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

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709

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.

Footnotes

↵ * On leave from: Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia.
↵ † To whom reprint requests should be addressed. e-mail: larionov{at}niehs.nih.gov.
Ronald W. Davis, Stanford University Medical Center, Stanford, CA
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Abbreviations: YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; TAR, transformation-associated recombination; EtdBr, ethidium bromide; TAFE, transverse alternating field electrophoresis.