The goal of this project is to produce a high resolution map of the long arm of chromosome 11 using cosmid and yeast artificial chromosome (YAC) cloning techniques. Cosmid libraries will be prepared in vector sCos-1 using DNA from somatic cell hybrid cell lines containing portions of chromosome 11, and DNA prepared from human chromosome 11 purified by flow cytometry. As reference sets, cosmids will be arrayed and archived on nitrocellulose of nylon-backed membranes and identified by grid coordinates. Low level """"""""bottom-up"""""""" contig construction will be carried out using hybridization-based screening in a multiplexed procedure and identifying cosmids mapped to chromosome 11 by high resolution in situ hybridization using non-isotopic labeling and laser scanning confocal microscopy. Using this technique, at least 330 to 350 cosmids will be mapped to chromosome 11q and 170 to 200 to 11p, with a resolution of 1-2 mb. Where cosmids can not be distinguished by order, and thus may be separated by less than 1-2 mb. Where cosmids can not be distinguished by order, and thus may be separated by less than 1-2 mb, analysis of somatic cell hybrids including a set of radiation reduction somatic cell hybrid lines will be carried out. The mapping of this number of cosmids to the 100 mb chromosome 11 long arm will generate a set of mapped markers with an average spacing of about 300 kb. The clones mapping to the short arm will be supplied to Project 2 (S. Sukumar) for further analysis. Identifying DNA sequence information (STS - """"""""Sequence-tagged site"""""""" - information) will be determined for each of the mapped clones. This information will be obtained by direct sequencing using the double-stranded cosmid DNA template and either T3 or T7 polymerase promoter sites, present in the cosmid vector, as templates. This information will allow a concise approach to map closure and provide information for precise quantification of the progress of the project, as well as to allow rapid and inexpensive exchange of probe information with other investigators. This sequence information will be used for the generation of a set of PCR primer oligonucleotide from each of the cosmid landmarks. When a sufficient density of mapped markers is obtained, these PCR identifiers will be used for the multiplex screening of one or more YAC libraries. These libraries will include the 4X human genomic library of Olson (St. Louis) as well as chromosome 11 specific libraries constructed in Project 3. With an average spacing of landmark clones of 300 kb, and an average size of YAC clones of 300 to 500 kb, much of the map will be closed by bridging YAC and cosmid clones. Where gaps are present, one or more steps of """"""""walking"""""""" using probes synthesized from the ends of YAC clones may be able to span the gaps. This project will serve as the basis for the localization of important human disease genes, the production of a chromosome 11 transcript map, and the eventual production of templates for DNA sequencing.
