A complete cDNA copy of the preprotoxin (ppTox, M1-P1) gene of type 1 killer M1-dsRNA has recently been constructed and found to express type 1 immunity and toxin at high levels in cells lacking M1-dsRNA. Its expression will be studied at the levels of transcription and toxin precursor processing in vivo. The effects of modification of this gene on expression will be studied in order to understand the requirements (e.g., glycosylation) for efficient secretion, cleavage and function of its component parts (leader Delta, toxin components Alpha and Beta and presumptive glycosylated immunity determinant Gamma in the order DeltaAlphaGammaBeta). Sites for in frame fusion to points just downstream of specific peptide cleavage points in the ppTox sequence (the DeltaAlpha, AlphaGamma and GammaBeta boundaries) are being introduced for insertion of protein sequences to allow individual assay of the cleavage events and testing of the pptox gene fragments as secretion signals for the secretion of foreign proteins in yeast. Comparisons will be made with signals derived from the MFAlpha1 gene for prepro Alpha Factor and the PH05 gene for secreted acid phosphatase. Beta-lactamase will be employed as a robust, easily assayed target protein and chromogenic substrates will be employed to allow screening for hypersecreting S. cerevisiae mutants. Mutants will be analyzed for changes in the efficiency of specific processing events and to identify the stage of secretion enhanced. The gene for SKI5, a minor secreted protease, will be cloned, and its product studied. In collaboration with Dr. Keith Bostian, the effects of TPCK on ppTox processing will be studied, the KEX1 gene cloned, and its product, probably involved in ppTox processing, will be analyzed.
