We characterize a component of the E. coli bacterial nucleoid H1a, which accumulates in stationary phase. This protein, identical with the major component of a plasmid-protein complex previously isolated in our laboratory, has a pI close to 7.5. Acrylamide gel electrophoresis and sedimentation in sucrose gradient have shown that the protein H1a induces significant compaction into DNA. This compaction is equivalent to that observed in nucleosome core although it introduces only a slight change in linking number. In addition, the structural change induced in the lactose L8UV5 promoter by H1a results in the decrease in the kinetic of formation of the open complex with RNA polymerase.
In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock. This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription. An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved. These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation. It was also noted that Msn4p is transiently increased at the diauxic transition. Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.
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.
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