Abstract
We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.