The goal is to characterize available temperature-sensitive mutants of Saccharomyces cerevisiae (amn mutants) which accumulate polyA+ mRNA in the nucleus at 37oC. These mutants fall into eleven complementation groups. After determining the extent of polyA+ RNA accumulation, the reversibility of mRNA accumulation, and the rate of turnover of mRNA at 37o, epistatic relations among three phenotypically-distinguishable classes of amn mutants, the extent of protein import into the nucleus at 37o, and morphological alterations of the nuclear envelope at 37o will be studied. Using wild type and amn cells at 37o, the structure of the 5' and 3' terminae of average mRNA will then be characterized, along with assessment of splicing and the fate of a pre-mRNA which normally is spliced. Moreover, mRNA intranuclear location will be determined and the set of proteins with which intranuclear mRNA is associated will be identified. Further studies will monitor the synthesis and fate of tRNA and rRNAs at 37o. In parallel, the genes responsible for the Ts- lesions will be cloned by complementation and sequenced, the consequences of under and overexpression of AMN genes will be studied, AMN transcripts will be characterized and antisera will be raised against the corresponding gene products. The antisera will be used to localize the AMN gene products and to study their biosynthesis and turnover.
