S-phase independent silencing establishment in Saccharomyces cerevisiae

The establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using high-resolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that the cell-cycle-regulated step was downstream of Sir protein recruitment. In contrast to prior results, HML and HMR had identical cell-cycle requirements for silencing establishment, with no apparent contribution from a tRNA gene adjacent to HMR. We identified the cause of the S-phase requirement for silencing establishment: removal of transcription-favoring histone modifications deposited by Dot1, Sas2, and Rtt109. These results revealed that silencing establishment was absolutely dependent on the cell-cycle-regulated interplay between euchromatic and heterochromatic histone modifications.

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Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2113-2125.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2113-2125

Author(s):

N Grandin ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Kinase Activity ◽

Mitotic Cell ◽

S Phase ◽

Mitotic Cell Cycle ◽

Meiotic Cell ◽

Cell Cycles ◽

M Phase ◽

Mitotic Cyclin

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.

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Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2113 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2113-2125 ◽

Cited By ~ 101

Author(s):

N Grandin ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Kinase Activity ◽

Mitotic Cell ◽

S Phase ◽

Mitotic Cell Cycle ◽

Meiotic Cell ◽

Cell Cycles ◽

M Phase ◽

Mitotic Cyclin

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.

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SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5829-5842.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5829-5842

Author(s):

P Zheng ◽

D S Fay ◽

J Burton ◽

H Xiao ◽

J L Pinkham ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Dna Synthesis ◽

Genomic Library ◽

Excision Repair ◽

Low Frequency ◽

S Phase ◽

Upstream Region ◽

Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

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Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2794-2802.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2794-2802 ◽

Cited By ~ 13

Author(s):

Neptune Mizrahi ◽

Claire Moore

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Sorting ◽

S Phase ◽

Phase Arrest ◽

Phosphatase Treatment ◽

M Phase ◽

64 Kda Protein ◽

Mitotic Phosphorylation ◽

Cycle Regulation

ABSTRACT The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from α-factor-induced G1 arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G2 and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.

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Histone mRNAs Do Not Accumulate during S Phase of either Mitotic or Endoreduplicative Cycles in the Chordate Oikopleura dioica

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5391-5403.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5391-5403 ◽

Cited By ~ 13

Author(s):

Mariacristina Chioda ◽

Fabio Spada ◽

Ragnhild Eskeland ◽

Eric M. Thompson

Keyword(s):

Cell Cycle ◽

S Phase ◽

Model Organisms ◽

Promoter Elements ◽

Oikopleura Dioica ◽

Stem Loop ◽

Transcript Levels ◽

Cell Cycles ◽

Histone Mrnas ◽

Complete Cell

ABSTRACT Metazoan histones are generally classified as replication-dependent or replacement variants. Replication-dependent histone genes contain cell cycle-responsive promoter elements, their transcripts terminate in an unpolyadenylated conserved stem-loop, and their mRNAs accumulate sharply during S phase. Replacement variant genes lack cell cycle-responsive promoter elements, their polyadenylated transcripts lack the stem-loop, and they are expressed at low levels throughout the cell cycle. During early development of some organisms with rapid cleavage cycles, replication-dependent mRNAs are not fully S phase restricted until complete cell cycle regulation is achieved. The accumulation of polyadenylated transcripts during this period has been considered incompatible with metazoan development. We show here that histone metabolism in the urochordate Oikopleura dioica does not accord with some key tenets of the replication-dependent/replacement variant paradigm. During the premetamorphic mitotic phase of development, expressed variants shared characteristics of replication-dependent histones, including the 3′ stem-loop, but, in contrast, were extensively polyadenylated. After metamorphosis, when cells in many tissues enter endocycles, there was a global downregulation of histone transcript levels, with most variant transcripts processed at the stem-loop. Contrary to the 30-fold S-phase upregulation of histone transcripts described in common metazoan model organisms, we observed essentially constant histone transcript levels throughout both mitotic and endoreduplicative cell cycles.

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Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.85.1.108 ◽

1980 ◽

Vol 85 (1) ◽

pp. 108-115 ◽

Cited By ~ 48

Author(s):

C J Rivin ◽

W L Fangman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cycle Length ◽

Replication Fork ◽

Nitrogen Sources ◽

S Phase ◽

Phase Duration ◽

Yeast Saccharomyces Cerevisiae ◽

Origin Activation ◽

Cell Cycle Length

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

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Identification of a new set of cell cycle-regulatory genes that regulate S-phase transcription of histone genes in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5249-5259.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5249-5259 ◽

Cited By ~ 1

Author(s):

H Xu ◽

U J Kim ◽

T Schuster ◽

M Grunstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Gene Transcription ◽

S Phase ◽

Histone Gene ◽

Mrna Synthesis ◽

Histone Mrna ◽

Complementation Groups ◽

Histone Gene Transcription

Histone mRNA synthesis is tightly regulated to S phase of the yeast Saccharomyces cerevisiae cell cycle as a result of transcriptional and posttranscriptional controls. Moreover, histone gene transcription decreases rapidly if DNA replication is inhibited by hydroxyurea or if cells are arrested in G1 by the mating pheromone alpha-factor. To identify the transcriptional controls responsible for cycle-specific histone mRNA synthesis, we have developed a selection for mutations which disrupt this process. Using this approach, we have isolated five mutants (hpc1, hpc2, hpc3, hpc4, and hpc5) in which cell cycle regulation of histone gene transcription is altered. All of these mutations are recessive and belong to separate complementation groups. Of these, only one (hpc1) falls in one of the three complementation groups identified previously by other means (M. A. Osley and D. Lycan, Mol. Cell. Biol. 7:4204-4210, 1987), indicating that at least seven different genes are involved in the cell cycle-specific regulation of histone gene transcription. hpc4 is unique in that derepression occurs only in the presence of hydroxyurea but not alpha-factor, suggesting that at least one of the regulatory factors is specific to histone gene transcription after DNA replication is blocked. One of the hpc mutations (hpc2) suppresses delta insertion mutations in the HIS4 and LYS2 loci. This effect allowed the cloning and sequence analysis of HPC2, which encodes a 67.5-kDa, highly charged basic protein.

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Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2529 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2529-2531 ◽

Cited By ~ 43

Author(s):

B J Brewer ◽

E Chlebowicz-Sledziewska ◽

W L Fangman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cycle Phase ◽

Cell Cycle Time ◽

Yeast Saccharomyces Cerevisiae ◽

Cell Cycles ◽

Daughter Cells ◽

Mother And Daughter ◽

Cell Cycle Phases ◽

Mother Cells

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

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Cell Cycle Regulation by Light inProchlorococcus Strains

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.782-790.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 782-790 ◽

Cited By ~ 45

Author(s):

Stéphan Jacquet ◽

Frédéric Partensky ◽

Dominique Marie ◽

Raffaella Casotti ◽

Daniel Vaulot

Keyword(s):

Cell Cycle ◽

High Irradiance ◽

S Phase ◽

Low Light ◽

Close Coupling ◽

Cell Cycles ◽

Flow Cytometric ◽

Initiation Of Dna Replication ◽

Cell Cycling ◽

Cycle Regulation

ABSTRACT The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m−2 s−1, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. ShiftingProchlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.

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