FUNCTIONAL COPIES OF A HUMAN GENE CAN BE DIRECTLY ISOLATED BY TRANSFORMATION-ASSOCIATED RECOMBINATION CLONING WITH A SMALL 3'-END TARGET SEQUENCE

Unique, small sequences (sequence tag sites) have been identified at t he 3' ends of most human genes that serve as landmarks in genome mappi ng. We investigated whether a single copy gene could be isolated direc tly from total human DNA by transformation-associated recombination (T AR) cloning in yeast using a short, 3' unique target. A TAR cloning ve ctor was constructed that, when linearized, contained a small amount ( 381 bp) of 3' hypoxanthine phosphoribosyltransferase (HPRT) sequence a t one end and an 189-bp Alu repeat at the other end. Transformation wi th this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3' end sequence to various Alu positions as much as 600 kb u pstream, These YACs were retrofitted with a Neo(R) and a bacterial art ificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most o f the HPRT isolates were functional, demonstrating that TAR cloning re tains the functional integrity of the isolated material. Thus, this mo dified version of TAR cloning, which we refer to as radial TAR cloning , can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.