Yeast mutants with transposable element (Ty) insertion mutations in the promoter region of the HIS4 gene have a His phenotype. His revertants of these mutations fall into several categories, including mutations in genes unlinked to HIS4, called SPM genes. Seven SPM genes have been identified and all share the phenotypes of suppression of some and destabilization of other insertion mutations at HIS4. This proposal has two aims: further study of SPM3 mutations which affect supercoiling of plasmids in yeast and the cloning and preliminary analysis of the SPM4-SPM7 genes, which have so far been identified by mutation. Study of SPM3 will include several approaches: analysis of the supercoiling effect on plasmids using chloroquine gels which resolve tsopoisomers, verification that the supercoiling effect correlates to a change in chromatin structure of genomic DNA, isolation of pseudo-revertants of spm3 mutations, study of regulation of the SPM3 gene by Northern hybridization and by construction of SPM3lacZ fusions, analysis of the effect of spm3 mutations on the expression of several other genes and identification and localization studies on the SPM3 gene product after making antibody to an SPM3-lacZ fusion protein. Study of SPM4-SPM7 genes will involve cloning the genes by complementation in yeast, construction of deletion and frameshift mutation in these cloned genes in vitro, transplacement of these mutations back into yeast and mutant analysis of strains with these in vitro constructed mutations which will confer the full mutant phenotype.
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