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

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 approximately 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.

Figure 1

Isolation of human DNAs as circular YACs by using the TAR cloning method. (A) TAR cloning vectors. Presented are pVC39-AAH4, -AAT3, -AAH2, -MAH8, -MAH10, and -LAH2 vectors containing Alu, LINE, or MER sequences. CEN6, centromere; Amp^R, ampicillin-resistance gene. (B) The TAR cloning scheme. Yeast spheroplasts are transformed with human DNA along with a TAR cloning vector. Recombination between repeats in the vector and human DNA leads to the establishment of a circular YAC. The filled-in blocks in human DNA and vectors identify repeated Alu, LINE, or MER sequences.

Figure 2

Physical characterization of five randomly selected circular human YACs developed by TAR cloning from total human DNA. Chromosomal-size DNA was separated by TAFE in a gel and blot-hybridized with a human DNA probe. Strong signals located at the positions of the starting wells correspond to circular YACs (lanes 2, 4, 6, 8, and 10). A band corresponding to linear molecules was also detected. Irradiation of the plugs with human YACs resulted in appearance of the bands corresponding to linear forms of the YACs (lanes 3, 5, 7, 9, and 11).

Figure 3

Size distribution of circular YACs generated by the TAR vector pVC39-AAH2 from a human hybrid cell line containing chromosome 16. Presented are the results for 110 YACs.

Figure 4

Alu profiles of 20 YACs generated by the TAR cloning method from hamster radiation hybrid cells containing a 5-Mb human DNA fragment. The profiles were produced by hybridization of an 82-bp Alu probe with TaqI-digested DNA isolated from the transformants. Size markers are kb.