THE REGION SURROUNDING THE PKD1 GENE - A 700-KB P1 CONTIG FROM A YAC-DEFICIENT INTERVAL

As part of an effort to identify the gene responsible for the predomin ant form of polycystic kidney disease (PKDI), we used a gridded human Fl library for contig assembly. The interval of interest, a 700-kb seg ment on chromosome 16p13.3, can be physically delineated by the geneti c markers D16S125 and D16S84 and chromosomally characterized as a GC-r ich isochore enriched for CpG islands, genes, and Alu-like repeats. Ou r attempts to recover CEPH YACs that encode this region of chromosome 16 were unsuccessful. However, we screened an arrayed Pi library using 15 distinct probes from the D16S125-D16S84 interval and identified 56 independent P1 clones. Only one probe from the interval was unsuccess ful in identifying a P1 clone. Forty-four P1 clones were determined to be unique based on restriction enzyme analysis, and 42 of these were found to originate from chromosome 16p13.3, based on FISH to metaphase chromosomes. The 700-kb interval could be defined by a single sequenc e-ready contig comprised of 12 Fl clones and 1 cosmid clone. Our studi es support the use of multiple libraries to generate the requisite phy sical reagents for positional cloning and encourage the use of Escheri chia coli-based large-insert cloning systems to recover clones from YA C-deficient chromosomal intervals.