It is the goal of this project to generate a complete physical map of human chromosome 18 and ultimately, a bank of overlapping precisely mapped cosmids and ordered evenly spaced linking clones covering the chromosome from one end to the other. This resource will facilitate the isolation of genes for human genetic disorders mapped to this chromosome. It will provide material for experiments to further our understanding of the molecular basis of chromosome structure since chromosome 18 has one of the most distinct banding patterns of any human chromosome with very dark and very lightly staining G-bands placed next to each other. The detailed physical map will allow alignment with the genetic linkage map to determine the relationships between physical and genetic distances. This information will be used to determine whether the type of DNA sequences and chromatin structure underlying the chromosome banding patterns can be correlated with the frequency and sites of meiotic crossover events. A multi-faceted approach will be used that integrates high-resolution cytogenetics, chromosome fractionation by X-irradiation of a Chinese hamster x human hybrid cell line with human chromosome 18 as its only human chromosomal material and rescue by fusion to other hamster cell lines under non-selective, and alternatively under specific selective, conditions. In irradiation-rescue hybrids the regions surrounding the human genes for thymidylate synthetase and asparaginyl tRNA synthetase can be selected for and used as focal points for bidirectional mapping. The relative location of 18- specific probes that are available or derived from a flow-sorted library to each other will be determined by probing Southern blots of a large number of the irradiation-rescue hybrids. Physical mapping with respect to chromosome bands and subbands will be carried out. The 18-only hybrid cell line will be used for large scale restriction mapping by pulsed field gel electrophoresis (PFGE), and for the generation of chromosome-18-specific cosmid and linking libraries of inserts containing restriction sites for rare cutting enzymes. By a different cloning strategy chromosome-18- specific single copy sequences closely associated with both telomeres will be isolated that constitute the telomeric ends of the map and can be used as hybridization probes for PFGE mapping of the telomeric sub-bands by partial digestion.
