post-S Phase Research Articles

Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae.

Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3. ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3. While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3Δ but not pif1Δ cells, and was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3. These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.

Hos1 Is a Lysine Deacetylase for the Smc3 Subunit of Cohesin

Chromosome cohesion is a cell-cycle-regulated process in which sister chromatids are held together from the time of replication until the time of separation at the metaphase-to-anaphase transition, ensuring accurate chromosome segregation [1-9]. Chromosome cohesion is established during S phase, and this process requires the four subunits of the cohesin complex (Smc1, Smc3, Mcd1/Scc1, and Irr1/Scc3) and the acetyltransferase Eco1 [10-13]. Acetylation of Smc3 by Eco1 at two evolutionarily conserved lysine residues promotes cohesion establishment during S phase in budding yeast and humans [14-16]. Here we report that Hos1, a member of the evolutionarily conserved class I histone deacetylase family, acts as a deacetylase for Smc3 in S. cerevisiae. We examine the Smc3 acetylation level in nine histone deacetylase deletion strains and find that the acetylation level is increased specifically in a hos1Δ strain post-S phase. Coimmunoprecipitation experiments show that Hos1 interacts with Smc3 and that the interaction is most pronounced as cells reach anaphase. We provide direct evidence that Hos1 can deacetylate Smc3 and retains a soluble pool of deacetylated Smc3. Overexpression of Hos1 results in less acetylation of Smc3 and cohesion defects in both WT and eco1 mutant strains; mutation of the Hos1 active site abolishes the defects. Hos1 may help to maintain a pool of unacetylated Smc3 that can be used for new chromosome cohesion.

Spindle assembly checkpoint gene mdf-1 regulates germ cell proliferation in response to nutrition signals in C. elegans

When newly hatched Caenorhabditis elegans larvae are starved, their primordial germ cells (PGCs) arrest in the post-S phase. This starvation-induced PGC arrest is mediated by the DAF-18/PTEN-AKT-1/PKB nutrient-sensing pathway. Here, we report that the conserved spindle assembly checkpoint (SAC) component MDF-1/MAD1 is required for the PGC arrest. We identified 2 Akt kinase phosphorylation sites on MDF-1. Expression of a non-phosphorylatable mutant MDF-1 partially suppressed the defect in the starvation-induced PGC arrest in L1 larvae lacking DAF-18, suggesting that MDF-1 regulates germ cell proliferation as a downstream target of AKT-1, thereby demonstrating a functional link between cell-cycle regulation by the SAC components and nutrient sensing by DAF-18-AKT-1 during post-embryonic development. The phosphorylation status of MDF-1 affects its binding to another SAC component, MDF-2/MAD2. The loss of MDF-2 or another SAC component also caused inappropriate germ cell proliferation, but the defect was less severe than that caused by mdf-1 hemizygosity, suggesting that MDF-1 causes the PGC arrest by two mechanisms, one involving MDF-2 and another that is independent of other SAC components.

Mitotic control of dTTP pool: a necessity or coincidence?

The fidelity of DNA replication in eukaryotic cells requires a balanced dNTP supply in the S phase. During the cell cycle progression, the production of dTTP is highly regulated to coordinate with DNA replication. Intracellular thymidine is salvaged to dTTP by cytosolic thymidine kinase (TK1) and thymidylate kinase (TMPK), both of which expression increase in the G1/S transition and diminish in the mitotic phase via proteolytic destruction. Anaphase promoting complex/cyclosome (APC/C)-mediated ubiquitination targets TK1 and TMPK to undergo proteasomal degradation in mitosis, by which dTTP pool is minimized in the early G1 phase of the next cell cycle. In this review, we will focus on regulation of TK1 in the post-S phase and the importance of mitotic proteolysis in controlling dNTP balance, replication stress and genomic stability. Finally, we discuss how thymidine pool and oligomeric forms of TK1 can affect mitotic control of dTTP.

Repression of Cell Cycle Progression by Antisense HMG2 RNA

The expression level of the HMG2 gene during the cell cycle of rat fibroblast cell lines was analyzed. Northern analysis demonstrated that the level of HMG2 mRNA was markedly enhanced in the post-S phase and reached a maximum at G2 phase, suggesting that expression of the HMG2 gene is not coupled with DNA synthesis. Progression of the cell cycle of COS-1 cells was repressed during G1 to S phase by expression of the antisense RNA for HMG2, resulting in a decrease of cell growth. These results suggest that the fluctuation of the HMG2 message during the cell cycle is not a consequence of but a prerequisite for cell proliferation.

Comparison of silver-staining nucleolar organizer region (AgNOR) counts and proliferating cell nuclear antigen (PCNA) expression in reactive mesothelial hyperplasia and malignant mesothelioma

Previous workers have used counts of colloidal silver-stained nucleolar organizer regions (AgNORs) to help distinguish reactive from malignant mesothelial processes. We sought to compare the demanding technique of AgNOR counting with the identification of proliferating cell nuclear antigen (PCNA) in 10 pleural malignant mesotheliomas and 11 reactive mesothelial proliferations. The mean AgNOR count was significantly higher in malignant compared to reactive lesions (5.10 [95% confidence interval, CI 4.35-5.84] vs 3.68 [95% CI 3.17-4.19], p = 0.004), as was the PCNA index (26.9 [95% CI 17.37-36.49] vs 9.67 [95% CI 4.94-14.39], p = 0.004). Less overlap was seen with PCNA indices and no reactive mesothelial proliferations had a PCNA index greater than 20%, suggesting that scores over this level may be a specific indicator of malignancy in this setting. There was only a weak correlation between the 2 proliferation indices suggesting that PCNA and AgNORs are present at somewhat different times of the cell cycle and/or persist in the post-S phase cell for variable lengths of time.

Saccade-related activity in the lateral intraparietal area. I. Temporal properties; comparison with area 7a

1. The cortex of the inferior parietal lobule (IPL) contains neurons whose activity is related to saccadic eye movements. The exact role of the IPL in relation to saccades remains, however, unclear. In this and the companion paper, we approach this problem by quantifying many of the spatial and temporal parameters of the saccade-related (S) activity. These parameters have hitherto been largely unstudied. 2. The activity of single neurons was recorded from Macaca mulatta monkeys while they were performing a delayed-saccade task. The analysis presented here is based on 161 neurons recorded from the lateral intraparietal area (LIP), a recently defined subdivision of the IPL; and 54 neurons recorded from the neighboring part of the IPL, area 7a. Overall, 409 IPL neurons were isolated in this study. 3. The typical activity of IPL neurons during the delayed-saccade task has three basic phases: light sensitive (LS), memory (M), and S. These basic phases are common to neurons of both areas LIP and 7a. In each phase (LS, M, and S), individual neurons may or may not be active. Most LIP neurons, however, are active in more than one phase. 4. To compare the activity levels of different neurons, the actual firing rate was weighted by each neuron's background level, yielding an "activity index" for each neuron, in each phase of the task. We calculated the activity index for the LS and M phases and for three phases related to the saccade: a presaccadic (Pre-S), a saccade-coincident (S-Co), and a postsaccadic (Post-S) phase. For area LIP neurons the median values of the activity index were high for the LS, M, Pre-S, and S-Co activities, and slightly lower in the Post-S period. In area 7a the median values were low for the LS phase and, in particular, for the M and Pre-S phases, somewhat higher coincident with the saccade, and high post-saccadically. 5. In area LIP, in each phase, 49-63% of the neurons had excitatory activity, and 10-17% had inhibitory responses. 6. In contrast, in area 7a excitatory responses were most frequent in the Post-S phase (56%). Excitation was particularly infrequent during M (28%) and Pre-S (22%). The incidence of inhibitory responses varied too (4-18%). The time course of inhibition was roughly opposite that of excitation; the highest frequency of inhibitory responses occurred during the saccade.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell cycle specific effects of selenium on the lens epithelium studied in vivo by the direct chemical approach.

We attempted to separate S phase from post-S phase effects of selenium upon the lens epithelium by exposing the lens in vivo to selenium either during S phase synthesis of DNA or immediately after completion of DNA synthesis. Incorporation of 3H-thymidine into lens epithelial cell DNA is complete within about 3 hours after intraperitoneally injecting this substrate. Young rats were given selenium either 5 hours before injecting 3H-thymidine (selenium present at the time of labeled-DNA synthesis) or 5 hours after 3H-thymidine (selenium present immediately after completion of S phase). By measuring changes in the distribution of 3H-DNA between the epithelium and lens body for 14 days after injection we estimated migration times and rates of differentiation of the labeled cell population. When selenium was present during DNA synthesis, DNA labeling was decreased by 70%, net migration time (movement from the germinative zone to the equator) was markedly prolonged and the rate of differentiation was slightly accelerated. Selenium had little affect on these parameters if first present immediately after S phase. We conclude that selenium insult to the lens epithelium is largely confined to germinative epithelial cells in S or pre-S phases of the cell cycle.

Exotic seismic phases recorded near Mammoth Lakes and their use in the delineation of shallow-crustal (magma?) anomalies

Observations of several hundredexotic seismic phases (herein defined) recorded in and near Long Valley caldera, California, have been cataloged. I discuss here four classes of such seismograms: (1) seismograms with missing S-waves, (2) seismograms with an unusual pre-S phase seen at the single station SLK northwest of the caldera, (3) seismograms with a strong pre-S phase as seen at a number of stations south of the caldera, and (4) a very large, very slow (<2 km/sec) post-S phase seen at the single station Benton. For each of these phenomena, it is not yet possible to pin down an unambiguous and unique theoretical explanation. However, for each, I have presented an explanation, summarizing current thinking, which involves nonplanar reflections/refractions within shallow-crustal anomalous zones which can reasonably be supposed to be magma bodies. If these explanations are even partially pertinent, then the investigation of exotic phases near complex regions like Mammoth Lakes and other volcanic areas is potentially a way to bring precise resolving power on the nature and geometry of local crustal anomalies.

Biogenesis and cell cycle relationship of poly(A) − actin mRNA in mouse ascites cells

A variety of rapidly growing mammalian cells contain a substantial portion of their actin mRNA in a poly(A) − form. We have used DNA-driven hybridization of a cloned actin cDNA-containing plasmid with pulse-labeled RNA from mouse S-180 ascites cells to examine newly synthesized actin mRNA. Our results indicate that the same proportion of newly synthesized and steady-state actin mRNA (approx. 40%) exists in a poly(A)-deficient form. This suggests that the poly(A) − form arises by some process other than slow cytoplasmic de-adenylation of a poly(A) + precursor. We have also examined cell cycle-enriched populations of S-180 ascites cells for the presence of poly(A) − actin mRNA. Results from these experiments indicate that cells in G1 phase of the cell cycle contain predominantly poly(A) + actin mRNA, while the poly(A) − form is restricted to late-S and post-S phase Cells.

Decrease in the levels of specific non-polysomal messenger ribonucleoproteins during the mouse sarcoma-180 ascites cell cycle

A few mRNAs in S-180 ascites cells are found as non-polysomal messenger ribonucleoprotein particles, whose translation is restricted both in , vivo and in , vitro . To determine if these mRNPs remain untranslated throughout the cell cycle we obtained S-180 ascites cells enriched in cell cycle stages by centrifugation through ficoll gradients. The “inactive” mRNA species were clearly present in the pre-polysomal fraction from S and pre-S phase cells, however they were not detected in the mRNP fraction from post S phase cells. Hybridization of a cloned cDNA probe for one of these species (P-40) to polysomal RNA from pre- or post-S phase cells demonstrated it was present in substantially higher concentrations in polysomes from post-S phase cells, consistent with its translation after S phase. We also observed that a poly (A)-minus form of the mRNA coding for actin was restricted to post S phase cells.

Glucocorticoids and lymphocytes. II. Cell cycle-dependent changes in glucocorticoid receptor content.

To study variations in glucocorticoid receptor levels during the cell cycle, we have separated mitogen-stimulated human peripheral lymphocytes and rat lymph node cells by unit gravity sedimentation and measured glucocorticoid binding in the resultant fractions. By morphologic criteria and thymidine incorporation, the fractions were separated into populations of G0 and G1 phase and S and post-S phase cells. A 2- to 3-fold increase in glucocorticoid receptor sites per cell, for cells in the S and post-S phase over those in G0 and G1, was observed with both nonstimulated rat lymph node cell suspensions and concanavalin A-stimulated human peripheral lymphocytes. These observations together with those from other studies indicate that formation of new glucocorticoid receptors near the S phase may be a general phenomenon in proliferating cells. We propose that this increase in glucocorticoid receptors during the cell cycle may explain the increase in glucocorticoid receptors in mitogen-stimulated lymphocytes.

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: Effects of inhibitors of cyclic AMP phosphodiesterase and Na+−K+-ATPase

Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10(-3) M) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7 X 10(-6) M), Persantin (5 X 10(-5) M) or RO-20-1724 (10(-4) M), prevents cell replication induced by PMA or serum. In contrast, ouabain (10(-4) M) and N,N'-dicyclohexylcarbodiimide (10(-5) M), inhibitors of Na+-K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (Go) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane enzymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.