REFINEMENT OF THE YEAST PHYSICAL MAP

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The physical map of the laboratory yeast Saccharomyces cerevisiae will be refined by carrying the analysis to higher resolution, providing more extensive cross correlation with the genetic map, improving the quality of the supporting collection of overlapping lambda clones, and characterizing the reasons for residual uncloned sequences. In a separately supported project, work is well advanced on the establishment of a continuous physical map of the 15 Mb (megabase pair) yeast genome with a resolution of 3 kb (kilobase pairs). On the basis of work already completed in this long-term project, the opportunity is nov available to refine the completed portions of the mar at the same time that its final connectivity is being established. By using indirect-end-label, partial-digestion techniques, facilitated by the high resolution achievable using field-inversion gel electrophoresis, the mapping will be extended from its current dependence on a single restriction-enzyme digestion, to mapping with several enzymes that cleave at frequencies ranging from once per 3 kb to once per 30 kb. The extent of cross-correlation with the genetic map, which is presently based on 50 genetic loci, will be extended to include approximately 200 loci. Furthermore, a general method of carrying out functional "chromosome walks," based on direct complementation between the lambda clones and yeast mutations, will also be developed. By enhancing the extent to which the physical and genetic maps are cross-correlated, it will be possible to test the hypothesis that recombinational hot and cold spots make only a small contribution to the overall frequency of meiotic recombination in yeast. To facilitate the refinement of the map and make the supporting clone collection readily accessible to all investigators, a subset of approximately 1000 lambda clones :ha: span the yeast genome will be selected from the overall collection of 5500 clones. The clones in this subset will be regrown, re- analyzed, and stored at high titer; on the basis of past experience, these steps are deemed necessary to assure the permanent availability of a master clone collection of high quality. Finally, the major reasons for residual gaps in the initial clone collection will be established by analyzing representative cases using direct genomic analysis and alternative cloning techniques.