Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2

Summary The cell cycle of Schizosaccharomyces pombe in continuous culture is controlled at two steps, one which limits the transition from G₁ to S phase and the other which determines the timing of cell division. We have investigated, by means of flow-cytofluorometry, the cell cycle characteristics of nutritionally starved cells in stationary phase. Cells were shown to become arrested in either G₁ or G₂, in ratios which depended on the composition of the growth medium. G₁ and G₂ stationary phase cells share certain properties. (1) They become relatively resistant to heat shock. (2) They can reenter the cell cycle after subculture into fresh medium. (3) The G₁ and G₂ arrested populations have equal long-term viability in stationary phase. (4) Both populations require the activity of the cdc2 ⁺ gene for reentry into the cell cycle. We suggest that cell cycle arrest in stationary phase is regulated by the activity of the same G₁ and G₂ controls which limit the rate of cell cycle progression in continuous culture. The data demonstrate that in fission yeast the transition from G₁ to S phase does not mark a point of commitment to the completion of the cell cycle.     

Common genes and pathways in the regulation of the mitotic and meiotic cell cycles of Schizosaccharomyces pombe

Summary Cell division cycle mutants defective in G₁, DNA replication or nuclear division were tested for sporulation at semi-restrictive temperatures. In cdc1-7, cdc5-120, cdc17-L16 and cdc18-46 no abnormalities were observed; cdc10-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with a low efficiency; cdc22-M45 was completely defective in meiosis, but could conjugate and formed zygotes with a single nucleus. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc13 were blocked in meiosis II. None of the wee1-50, adh.nim1 ⁺ and win1 ⁺ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis. The meiotic function of cdc13 is TBZ-sensitive and probably exerted downstream of cdc2. Single mutants in cut1 or cut2 did not effect sporulation, whereas the double mutant cut1 cut2 formed two-spored asci. The results demonstrate that the cell division cycle and the meiotic developmental pathway share common genes and regulatory cascades.     

Expression of NPAT, a novel substrate of cyclin E–CDK2, promotes S-phase entry

To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E–CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G₁/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E–CDK2. These results suggest that NPAT is a substrate of cyclin E–CDK2 and plays a role in S-phase entry.     

The Ste16 WD-repeat protein regulates cell-cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe

The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G₁-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G₁ arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16 ⁺ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G₂-phase after the nutritional down-shift. When transferred to fresh growth medium, the G₂-arrested ste16Δ haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Δ, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy. Received: 10 September / 6 October 1997     

Immunochemical detection of cell cycle synchronization in a human erythroid cell line, K562

Cells of the human erythroleukemic line K562 can be manipulated by culture conditions to arrest within the G₁ phase of synchronously after release from G₁. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescence. This protocol offers a method for synchronization of K562 cells at the G₁/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.     

Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G₁ cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G₁ restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G₁/S transition, and suggest that continued phosphorylation of RB beyond G₁/S is required for the completion of DNA replication.     

The induction of class I HLA by interferon-α is independent of the cell cycle,but the expression is enhanced by a G1/S block

The induction of class I HLA expression by interferon-α (IFN-α) was studied in lymphoid cells arrested or traversing different stages of the cell cycle. Exponential cultures of MOLT-4 cells and the MOLT-4 cell variant YHHH were treated with the cell cycle inhibitors aphidicolin and colcemid to obtain cell populations arrested in G₁/S and G₂/M, respectively, and also cells traversing from S to M and vice versa. Cytofluorimetry with the monoclonal antibody YTH/76.3 (which specifically detects those class I molecules which are most susceptible to IFN-α induction) was used to quantitate the class I HLA response to IFN-α. The results showed that the response to IFN-α is not restricted to a given stage of the cell cycle. These studies also revealed that when the cells were arrested at G₁/S, the absolute level of class I HLA expression was enhanced 2–3-fold, both in the presence or absence of either IFN-α or IFN-γ. Therefore, even when absolute levels changed, the ratio of IFN-induced expression to basal expression remained constant at all cell cycle stages. The level of expression of another surface antigen (the CD1 antigen HTA-1) was not affected by the G₁/S block. The results were confirmed by dot blot hybridization of poly (A)⁺ RNA using cDNA-specific probes. These findings suggest that the effect of IFN-α is continuous throughout the cell cycle but that a G₁-dependent event determines the extent of class I HLA expression, and leads to a synergistic superinduction by IFN in G₁/S-arrested cells.     

Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast

The eukaryotic cell cycle is driven by a cascade of cyclins and kinase partners including the G₁ cyclin Cln3p in yeast. As the first step in this cascade, Cln3p is uniquely positioned to determine the critical growth-rate threshold for division. To analyze factors regulating CLN3 expression, we identified a short upstream open reading frame (uORF) in the 5′ leader of CLN3 mRNA as a translational control element. This control element is critical for the growth-dependent regulation of Cln3p synthesis because it specifically represses CLN3 expression during conditions of diminished protein synthesis or slow growth. Inactivation of the uORF accelerates the completion of Start and entry into the cell cycle suggesting that translational regulation of CLN3 provides a mechanism coupling cell growth and division.     

A temperature-sensitive DNA synthesis mutant isolated from the Chinese hamster ovary cell line

A temperature-sensitive DNA synthesis mutant, tsC8, was isolated from mutagenized Chinese hamster ovary cells by the fluorodeoxyuridine suicide technique. The tsC8 cells showed inhibition of DNA synthesis at the nonpermissive temperature (NPT) with little effect on initial levels of RNA and protein synthesis. Temperature-arrested tsC8 cells had G₁ or S DNA content and the temperature-sensitive (ts) period of the tsC8 cell cycle was the interval between the G₁/S border and the middle of the S period. The tsC8 cells were unable to enter the S phase when exposed to the NPT during the G₁ period of the cell cycle. When S phase tsC8 cells were shifted to the NPT, they incorporated [³H]thymidine at rates similar to the parental cell type for only 2 h, indicating ats defect in DNA synthesis. The tsC8 mutation is expressed in a recessive manner and is in a gene distinct from those affected in other DNA synthesis mammalian cell mutants.     

Cyclin-dependent kinase 2 (cdk2) in the murine cdc2 kinaseTS mutant

Kinases of the mammalian cdc2 family including cdk2 (cyclin-dependent kinase 2) are thought to be involved in both the G₂/M transition and DNA replication. To investigate the role of cdc2 kinase and cdk2 in cell cycle progression, murine tsFT210 cells bearing a temperaturesensitive cdc2 mutation were used. These kinases were purified by column chromatography, using a peptide with the consensus phosphorylation site of cdc2 kinase as the substrate. In this mutant, cdc2 kinase activity was temperature sensitive and cdk2 activity was not. At the restrictive temperature, the mutant was only arrested in the G₂ phase and not in the G₂-S phase, suggesting that cdk2 did not compensate for cdc2 kinase at the G₂/M transition but did function at the G₁-S phase. This suggestion was supported by the finding that transfection of cdk2 cDNA did not improve the growth of the mutant cell line at the restrictive temperature, although transfection of cdc2 cDNA did.     

Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G₁. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G₁ results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G₂ leads to cell death in subsequent metaphase–anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G₁. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase.     

The essential Schizosaccharomyces pombe cdc23 DNA replication gene shares structural and functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene

The fission yeast cdc23 gene is required for correct DNA replication: cdc23 mutants show reduced rates of DNA synthesis and become elongated after cell-cycle arrest. We have cloned the Schizosaccharomyces pombe cdc23 gene by complementation of the temperature-sensitive phenotype of cdc23-M36 and confirmed the identity of the gene by integrative mapping. Analysis of the DNA sequence reveals that cdc23 can encode a protein of 593 amino acids (M_r=67 kDa) with 22% overall identity and many structural homologies with the product of the Saccharomyces cerevisiae DNA43 (MCM10) gene which is required for correct initiation of DNA synthesis at chromosomal origins of replication. Construction of a cdc23 null allele has established that the cdc23 gene is essential for viability, with cdc23 deletion mutant spores germinating but undergoing arrest with undivided nuclei in the first or second cell cycle. The S. pombe cdc23 gene on an expression plasmid is able to complement the S. cerevisiae dna43-1 mutant. These structural and functional homologies between two distantly related species suggest that cdc23 and DNA43 may represent genes for a conserved essential eukaryotic DNA replication function. Received: 12 April / 6 July 1998     

S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe

Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest. Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis. Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents. Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G₁ or G₂. Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26. Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response. We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins. This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins.     

Loss of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected synovial sarcomas

Deletions of the short arm of chromosome 9 have been observed in many tumours and cell lines. This chromosomal region is frequently targeted during malignant transformation because it contains at least two known tumour suppressor genes: p16 ^INK4 and p15 ^INK4B . p16 ^INK4A acts as a negative cell cycle regulator by inhibiting G₁ cyclin-dependent kinases that phosphorylate the retinoblastoma protein and therefore block the progression of the cell cycle from G₁ to S phase. The role of p16 ^INK4A in the development of synovial sarcoma has not been comprehensively investigated. Ten samples of synovial sarcomas were examined for allelic imbalance/loss of heterozygosity (AI/LOH) of the 9p region and p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells, amplified by polymerase chain reaction and analysed for AI/LOH by using six microsatellite markers that map to the 9p region. Immunohistochemistry for p16 expression was done. AI/LOH with at least one microsatellite marker on 9p21 was detected in six of ten samples. The most frequent allelic deletions were observed within the coding sequence of p16 ^INK4A . Loss of p16 immunoreactivity was detected in eight samples, six of which showed evidence of alterations at 9p21 region. These findings suggest a possible role of loss of p16 ^INK4A in the development of synovial sarcoma.