No abstract

In budding yeast G1 cells increase in cell mass until they reach a critical cell size, at which point (called Start) they enter S phase, bud and duplicate their spindle pole bodies. Activation of the Cdc28 protein kinase by G1‐specific cyclins Cln1, Cln2 or Cln3 is necessary for all three Start events. Transcriptional activation of CLN1 and CLN2 by SBF and MBF transcription factors also requires an active Cln‐Cdc28 kinase and it has therefore been proposed that the sudden accumulation of CLN1 and CLN2 transcripts during late G1 occurs via a positive feedback loop. We report that whereas Cln1 and Cln2 are required for the punctual execution of most, if not all, other Start‐related events, they are not required for the punctual activation of SBF‐ or MBF‐driven transcription. Cln3, on the other hand, is essential. By turning off cyclin B proteolysis and turning on proteolysis of the cyclin B‐Cdc28 inhibitor p40SIC1, Cln1 and Cln2 kinases activate cyclin B‐Cdc28 kinases and thereby trigger S phase. Thus the accumulation of Cln1 and Cln2 kinases which starts the yeast cell cycle is set in motion by prior activation of SBF‐ and MBF‐mediated transcription by Cln3‐Cdc28 kinase. This dissection of regulatory events during late G1 demands a rethinking of Start as a single process that causes cells to be committed to the mitotic cell cycle.

In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G,) is necessary for subsequent initiation of DNA replication triggered by activation in late GI of Cdc28lCdkl kinases associated with B-type cyclins Clbl-Clb6. The absence of pre-RCs during G, and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G , depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and ClbbICdkl in late GI. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G, and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once ClbICdkl kinases have been activated. We show that Cdc6p associates with ClbICdkl kinases from late G, until late anaphase, which might be important for inhibiting pre-RC assembly during Sf G, , and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.

Both dexamethasone and tadalafil decrease systolic pulmonary artery pressure and may reduce the incidence of HAPE in adults with a history of HAPE. Dexamethasone prophylaxis may also reduce the incidence of AMS in these adults. ClinicalTrials.gov identifier: NCT00274430.