CONSTRUCTION OF COSMID CONTIGS AND HIGH-RESOLUTION RESTRICTION MAPPING OF THE HUNTINGTON DISEASE REGION OF HUMAN CHROMOSOME-4

The gene responsible for Huntington disease (HD) has been localized to a 2.2 million base pair (Mbp) region between the loci D4S10 and D4S98 on the short arm of human chromosome 4. As part of a strategy origina lly designed to clone the gene based on its chromosomal location, we a nd others previously identified overlapping yeast artificial chromosom e (YAC) clones covering most of this region. While these YAC clones we re useful for initially obtaining long-range clone continuity, a numbe r of features of the YACs indicated that smaller clones are generally more useful in the subsequent steps of the positional cloning strategy . In this paper, we use these YAC clones to generate sets of overlappi ng cosmid clones covering most of the HD region. We isolated a large n umber of cosmids by screening a chromosome 4-specific cosmid library w ith labeled DNA from a minimal overlapping set of YAC clones. These co smid clones were further analyzed by restriction mapping and hybridiza tion experiments, leading to the assembly of 185 cosmids into eleven c ontigs covering more than 1.65 Mbp and to a fine-structure restriction map of the region. Nine of these contigs cover 90 percent of the 1.7 Mbp subregion between loci D4S125 and D4S98 where the HD gene is now k nown to lie. The detailed restriction map and the cosmid clones should facilitate the identification and localization of cDNAs and polymorph ic markers, and they provide reagents for large scale DNA sequencing o f this region of the human genome. Our results suggest that this strat egy should be generally useful for converting YAC clones into cosmid c ontigs and generating high-resolution restriction maps of genomic regi ons of interest.