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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Transformation-associated recombination in yeast can be used to generate large fragment (YAC) genome libraries
V. Larionov, N. Kouprina, J. Graves, and M.A. Resnick
Laboratory of Molecular Genetics, National Institute of Environmental Health Science (NIH), Research Triangle Park, NC 27709 (Fax: 919/541-7593)
A first step in the molecular characterization of eukaryote genomes involves cloning of large chromosomal fragments. This generally requires considerable in vitro DNA manipulation that could result in broken, or nicked molecules, incomplete ligation or ligation between different chromosomal fragments, all of which may contribute to the frequently reported artifacts such as deletions and chimeras.
Previously, we showed that DNA undergoing transformation in yeast is highly recombinogenic, even when diverged (1). We reasoned that this transformation-associated recombination (TAR) could be used as an alternative method for cloning large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation. To test this, human DNA was gently isolated by lysis of cells within agarose plugs. The DNA was transformed directly into yeast along with linearized plasmids that contain a telomere at one end, a centromere, a selectable marker M1, and an Alu or Line sequence at the other end. Nearly all the transformants (selection was for the M1 marker) appeared to be due to recombination between the plasmid - which lacked an ARS - and human DNA based on inter-Alu PCR analysis and the presence of a new band on OFAGE gels. Many of the YACs were greater than 500 kb. Their mitotic segregation was comparable to that for YACs generated by standard in vitro addition of telomeres. Presumably, the ARS-like and telomere-generating sequences contained in human DNAs enabled propagation of these YACs in yeast. Given the high efficiency of TAR between plasmid and human DNA, we investigated the possibility of including another linear plasmid (TEL--Marker M2-ARS--Repeat) in order to generate YACs with two genetically marked ends. Selection for the M1 marker usually led to YACs that also contained the unselected M2 marker.
We conclude that TAR can be considered an efficient in vivo method for generating YAC libraries. Because (i) in vitro manipulation of DNAs is minimized, (ii) the concentrations can be varied, and (iii) there is no enzymatic treatment, we anticipate that the integrity of libraries could be greatly improved over the more traditional approaches. This method may also be used for the generation of libraries from any eukaryote whose chromosomal DNA contains frequent large repeats.
1. Larionov et al., Yeast 10 (1994) 93-104.