Molecular cloning and transcriptional analysis of the start gene CDC25 of Saccharomyces cerevisiae

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Abstract
To isolate the CDC25 gene of Saccharomyces cerevisiae we have transformed a cdc25‐1, trp1 strain with a yeast gene bank constructed in YRp7 vector, selecting trp+ clones able to grow at restrictive temperature. From several independent positive clones we have recovered a plasmid, called pDGEm‐1, that bears a 5‐kb genomic fragment and is able to give a full complementation of the cdc25‐1 mutation. The genomic sequence has been subcloned and a good complementation obtained with a 2‐kb fragment. Several stable integrative trp+, cdc+ transformants have been constructed. Their genetic and molecular analysis indicates that we have cloned the true CDC25 gene. Northern blot hybridization has revealed the presence of a 5‐kb mRNA transcribed by the CDC25 gene. This mRNA is also present in nitrogen‐starved cells and during the re‐enter in cell cycle from starvation, suggesting a constitutive transcription. Transformants bearing the cloned sequence on multicopy plasmid and integrative transformants that bear the CDC25 gene, flanked by plasmid sequences, show an altered control of the cell cycle and fail to arrest in G1 unbudded phase in stationary phase conditions.