Genes which specify cytoskeletal proteins as well as genes involved specifically in the progress of the cell division cycle will be studied in the budding yeast Saccharomyces cerevisiae. Mutations in cytoskeletal proteins (actin and tubulins) will be made in vitro starting with the cloned structural genes. The genes will be mutagenized by highly specific segment-directed mutagenesis methods and the mutagenized gene used to replace the normal gene in a yeast chromosome. Pseudoreversion methods will be used to find genes whose products interact with known cytoskeletal or cell-division-cycle genes. The mutations obtained will be used to characterize the mutant phenotypes by a variety of physiological, morphological (including immunofluorescence and electron microscopy), and biochemical methods. The results of these characterizations can be used to deduce the function of these genes in vivo. Monoclonal antibodies will be produced against two purified proteins: bacterial beta-lactamase from E. coli and yeast hexokinase B. Differences in affinities to the several monoclonals exhibited by mutations in the structural genes for the proteins will be correlated with map position of the mutations, in an attempt to probe domain structure without direct crystallography. Results of these investigations will be applied, if warranted, to studies of the yeast cytoskeletal proteins.
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