Vladimir Larionov, Natalya Kouprina, Joan Graves, X-N Chen[1], Julie R. Korenberg[1], and Michael A. Resnick
Laboratory of Molecular Genetics, NIEHS, Box 12233, Research Triangle Park, NC 27709.
DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that Transformation-Associated Recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1 selected transformants had YACs containing human DNA inserts that varied in size from 70 kb to more than 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 + M2) vector(s) was comparable suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu were used, the YACs were likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrate that human DNA can be efficiently cloned from mouse cells that contain a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization (FISH). We propose that TAR-cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and it may be useful for isolating families of genes and specific genes from total genome DNA.
Support was provided in part by the US Department of Energy (DE-FG0394ER61402) to J. A. K and by an interagency grant (1-YO2-HG-60021-01) from the NIH Human Genome Center to M. A. R.
[1] Ahmanson Department of Pediatrics, Division of Genetics, Cedars-Sinai Research Institute, UCLA, CA 90048-1869.