Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis

Gene isolation methods used during positional cloning rely on physical cont igs consisting of bacterial artificial chromosomes, P1, or cosmid clones. H owever, in most instances, the initial framework For physical mapping consi sts of contigs of yeast artificial chromosome (YACs), large vectors that ar e suboptimal substrates For gene isolation. Here we report a strategy to id entify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from t he YAC in mammalian cells. The RDA tester cDNAs were generated from a previ ously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick t ype C (NP-C). The driver cDNAs were generated from a control hamster cell l ine that did not contain the YAC that expressed NPC1. Among the gene fragme nts obtained by RDA, NPC1 was the most abundant product. In addition, two n on-NPC1 fragments were isolated that were mapped to and expressed from 911D 5. One of these RDA gene fragments (7-R) spans more than one exon and has 9 8% sequence identity with a human cDNA clone reported previously as an expr essed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known ma mmalian genes or ESTs, was Further localized to the region of overlap betwe en YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in whi ch stable YAC transfer is followed by cDNA RDA should be a useful adjunct s trategy to expedite the cloning of human genes when a YAC contig is availab le across a candidate interval.