SPECIFIC CLONING OF HUMAN DNA AS YEAST ARTIFICIAL CHROMOSOMES BY TRANSFORMATION-ASSOCIATED RECOMBINATION

DNA molecules undergoing transformation into yeast are highly recombin ogenic, 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) linea rized vectors that contained a human Alu sequence at one end and a tel omere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL), Nearl y all the M1-selected transformants had yeast artificial chromosomes ( YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb, Approximately half of these had also acquired the unselected M 2 marker. The mitotic segregational stability of YACs generated from o ne (M1) or two (M1 and M2) vector(s) was comparable, suggesting de nov o generation of telomeric ends, Since no YACs were isolated when roden t DNAs or a vector lacking an Alu sequence was used, the YACs were mos t likely the consequence of TAR between the repeat elements on the vec tor(s) and the human DNA, Using the BLUR13 Alu-containing vector, we d emonstrated that human DNA could be efficiently cloned from mouse cell s that contained a single human chromosome 16, The distribution of clo ned DNAs on chromosome 16 was determined by fluorescence in situ hybri dization, We propose that TAR cloning can provide an efficient means f or generating YACs from specific chromosomes and subchromosome fragmen ts and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.