Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae.

Abstract

The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called ‘START’. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site‐specific gene re‐integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200‐nucleotides mRNA. We have determined the nucleotide sequence of a 5548‐bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587‐amino acid polypeptide chain. Only the C‐terminal part of the ORF appears to be essential for the complementation of the cdc25‐5 allele, suggesting a multidomain protein.

In Saccharomyces cerevisiae an unbalanced level of tyrosine phosphorylation down-regulates the Ras/PKA pathway

“…The Ras-PKA pathway plays a crucial role in the control of metabolism, stress resistance and proliferation, in connection with the availability of nutrients. The Ras-GTP/Ras-GDP ratio is controlled by the balance between the activities of guanine nucleotide exchange factors, Cdc25 (Camonis et al, 1986) and Sdc25 (Damak, Boy-Marcotte, Le-Roscouet, Guilbaud, & Jacquet, 1991), and GTPase Activating Proteins (GAPs) which stimulate Ras-GTPase activity and are encoded by the IRA1,2 genes (Tanaka et al, 1990). In turn, GTP-bound Ras proteins stimulate adenylyl cyclase to yield an increase in intracellular cAMP level that is responsible for the activation of PKA (Toda et al, 1985).…”

Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells.

“…Such mutants are blocked in the traverse of specific phases of the cell cycle at the nonpermissive temperature (reviewed in references 1, 2, and 29). This approach has been used successfully with yeasts, in which several critical cell cycle functions have been identified (37) and their genes cloned (3,(5)(6)(7)31). A number of cell cycle mutants have also been isolated in the higher eucaryotes.…”

The glucose‐induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25‐RAS mediated signal transduction pathway

“…On the other hand the data obtained on temperaturesensitive mutants suggest that the CDC25 gene product that is postulated to be a mediator of glucose response for adenylate cyclase activation [2,3,7], is not involved in glucose-mediated PI stimulation, and also that the stimulus appears to be RAS-independent, as indicated by the data observed with rasl, ras2-ts strain. The two cdc25 alleles used show a difference for cAMP metabolism at restrictive temperature.…”

“…The two cdc25 alleles used show a difference for cAMP metabolism at restrictive temperature. In fact while the cdc25-1 mutant still responds to glucose addition with an increase of intracellular cAMP ( [14], Thevelein, personal communication), the cdc25-5 mutants do not accumulate cAMP [7], in addition the level of intracellular cAMP remains high in cdc25-1 [15], while it rapidly drops in cdc25-5 [7]. Since the stimulation of PI turnover at restrictive temperature is similar in strains bearing the two alleles, we can conclude that this stimulation is clearly not dependent on the transient increase of cAMP that occurs in wild type strains.…”

“…[6]. OL86 was obtained from M. Jacquet [7], TS1-6 was obtained from O. Fasano [8]. Strain X4004[plND25-2] is a transformant of X4004-3A (MATch, lys5, met2, ura3, trpl) bearing a multicopy plasmid plND25-2 that contains the whole ORF of CDC25 gene cloned under the control of an inducible GAL promoter [9,10].…”