SACCHAROMYCES-CEREVISIAE CDC15 MUTANTS ARRESTED AT A LATE-STAGE IN ANAPHASE ARE RESCUED BY XENOPUS CDNAS ENCODING N-RAS OR A PROTEIN WITH BETA-TRANSDUCIN REPEATS

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitivity defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, betaTrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, betaTrcp is composed of seven beta-transducin repeats. betaTrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.