TOOLS FOR GENETIC MAPPING IN YEAST

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Dr. David Stillman proposed to develop a portable, rapid mapping system for the yeast Saccharomyces cerevisiae. The primary use for this system will be map mutations that have proved difficult to clone by complementation; it would provide the information necessary for positional mapping. The development of the system will proceed in two phases. The first phase involves the construction of sixteen plasmids each of which will be targeted to integrate near the centromere of one of the yeast chromosomes. These plasmids will carry a GAL1 promoter that when activated would transcribe through the centromere, inactivating it and causing chromosome loss. The plasmids also will contain a dominant marker that would allow easy scoring for such a loss. Using these plasmids, a researcher could construct sixteen strains with a specific genetic background, each carrying one of these plasmids in a different chromosome. Each strain could then be mated with the mutant strain, and the loss of the plasmid containing chromosome in each diploid would be induced. In one of the diploid strains, the recessive mutant phenotype would be uncovered when the chromosome carrying the wildtype allele was lost, thus mapping the mutation to that chromosome. The second phase of development involves the construction of a set of 35 chromosome fragmentation vectors. These plasmids will allow a researcher to divide the larger chromosomes into two or three fragments to map the mutation of interest to a subchromosomal segment. Each plasmids will carry a centromere joined to a GAL1 promoter, suitable selectable markers, and a region to which integration is targeted, all contained between telomeric sequences. The restriction enzyme SceI flanked by sites for this enzyme will be inserted between the telomeric sequences opposite the rest of the vector. The integration of this vector at the targeted site will generate an unstable dicentromeric chromosome. The instability will be resolved through cleavage by SceI (which does not cleave nuclear DNA), cutting the chromosome in two between the centromeres. The presence of the telomeric sequences will generate proper ends. Thus, the targeted chromosome will be cut in two at the chosen site. One of the new chromosomes will contain the centromere that can be inactivated when the GAL1 promoter is induced, causing its loss and exposing the recessive mutation if the wildtype allele were contained within that chromosomal segment. Thus, once a mutation is mapped to a particular chromosome, that chromosome could be fragmented to determine which fragment contains the mutation. Once a gene is mapped to a region of a chromosome, complementation could be tested using clones from that region.