Onset Of DNA Synthesis Research Articles

Cell-Cycle Regulated Expression of Rap1 in Regenerating Liver

Rap1 proteins are capable of competing with Ras p21 for binding to effectors, and of antagonizing some Ras-induced effects, but their participation in normal growth regulation has not been established. The level of Rap1 protein and the expression of therap1Agene were examined by immunoblotting and Northern analysis during the regenerative growth response in rat liver following partial hepatectomy. Protein and mRNA were significantly down-regulated prior to and during the onset of DNA synthesis. The timing of this effect is consistent with a model in which expression of Rap1 is turned off or down to allow the initiation of proliferation.

Expression of the Aryl Hydrocarbon Receptor Is Regulated by Serum and Mitogenic Growth Factors in Murine 3T3 Fibroblasts

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological responses to planar aromatic hydrocarbons such as benzo[alpha]pyrene. However, no endogenous physiological ligand for the AhR has been identified. Since the AhR regulates bioactivity to common environmental pollutants, and since it is predicted to play an important physiological function, we have investigated the expression of the AhR during the cell cycle of murine 3T3 fibroblasts. We show here that stimulation of growth-arrested 3T3 cells with serum results in increased expression of AhR protein. Serum-induced expression of AhR in synchronized, serum-stimulated cells occurs at the onset of DNA synthesis (S phase) and is maximal at time points corresponding to late S phase. Transient transfections with an AhR-promoter-luciferase construct demonstrate that reporter gene transcription from the AhR promoter is regulated in a serum-dependent manner. Serum-dependent induction of AhR expression is prevented by an inhibitor of tyrosine kinase activity. Ligand-activated growth factor receptors (platelet-derived growth factor receptor basic fibroblast growth factor receptor) as well as an ectopically expressed tyrosine kinase (the v-Src oncoprotein) induce AhR expression in the absence of serum. Therefore, tyrosine kinase signaling is both necessary and sufficient for induction of AhR expression. Studies with the G1 blocker sodium butyrate show that the signal transduction pathways mediating serum-stimulated progression through the cell cycle are distinct from those that induce AhR expression. These data suggest that transcriptional regulation of the AhR is important in determining cellular sensitivity to the actions of AhR ligand(s) and that the AhR may play a role during the cellular proliferative response.

Expression of PRL-1 nuclear PTPase is associated with proliferation in liver but with differentiation in intestine.

Mechanisms controlling the tyrosine phosphorylation of cellular proteins are important in the regulation of cellular processes including growth and differentiation. It has become clear that a number of protein tyrosine phosphatases (PTPases) that dephosphorylate tyrosyl residues may play a role in the growth response, both in growth-promoting and growth-inhibiting capacities. We identified PRL-1, a unique nuclear PTPase that is an immediate-early gene in liver regeneration and is positively associated with growth, including fetal and neoplastic hepatic growth and anchorage-independent growth after overexpression in fibroblasts. In this study, we show that PRL-1 nuclear protein levels in regenerating liver parallel those of its mRNA, although the peak occurs later, just before the onset of DNA synthesis. We further show that PRL-1 is significantly expressed in intestinal epithelia and that, in contrast to the expression pattern of PRL-1 in liver, its expression is associated with cellular differentiation in intestine. Specifically, PRL-1 is expressed in villus but not crypt enterocytes and in confluent differentiated but not undifferentiated proliferating Caco-2 colon carcinoma cells. The expression of PRL-1 in intestine shows inverse correlation with proliferating cell nuclear antigen expression, a marker for S-phase cells. These results suggest that PRL-1 may play different roles in these two digestive tissues. Such a dichotomy of roles has previously been described for some protein tyrosine kinases and might be due to the availability of alternate substrates in different tissues.

The Cell Cycle in Polyploid Megakaryocytes Is Associated with Reduced Activity of Cyclin B1-dependent Cdc2 Kinase

The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1-dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1.

Inositol lipid cycle and autonomous nuclear signalling

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observations have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC β 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC β 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC α translocation to the nucleus, (iii) the chromosome mapping of PLC β 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC β 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC β 1 construct, the knock-out of the PLC β 1 gene induces both a loss of PLC β 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC β 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC β 1 gene to the band q35–36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC β 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Coordinated effects of insulin-like growth factor I on inhibitory pathways of cell cycle progression in cultured cardiac muscle cells.

Insulin-like growth factor I (IGF I) plays a key role in the regulation of cell proliferation. Progression of the cell cycle is regulated by stimulatory and inhibitory pathways. In order to understand the mechanisms through which IGF I regulates cardiac muscle growth, we have studied the effects of IGF I on inhibitory pathways involving p53 and WAF1 in cultured cardiac muscle cell line H9C2. The onset of DNA synthesis in response to IGF I stimulation was preceded by activation of p53 expression. In addition, IGF I increased p53-dependent and p53-independent induction of WAF1 in H9C2 cells. Dose-response studies showed that IGF I effects on p53-dependent and p53-independent induction of WAF1 occur at physiological concentrations of IGF I. These data indicate that IGF I coordinately regulates inhibitory pathways of cell cycle progression, and that p53-dependent and p53-independent induction of WAF1 may provide negative control mechanisms to regulate stimulatory pathways of cell cycle progression activated by IGF I.

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.

Ectromelia virus RING finger protein is localized in virus factories and is required for virus replication in macrophages.

We have previously described a gene of ectromelia virus (EV) that codes for a 28-kDa RING zinc finger-containing protein (p28) that is nonessential for virus growth in cell culture but is critical for EV pathogenicity in mice (T. G. Senkevich, E. V. Koonin, and R. M. L. Buller, Virology 198:118-128; 1994). Here, we show that, unlike all tested cell cultures, the expression of p28 is required for in vitro replication of EV in murine resident peritoneal macrophages. In macrophages infected with the p28- mutant, viral DNA replication was not detected, whereas the synthesis of at least two early proteins was observed. Immunofluorescence and biochemical analyses showed that in EV-infected macrophages or BSC-1 cells, p28 is associated with virus factories. By use of a vaccinia virus expression system to examine different truncated versions of p28, it was shown that the disruption of the specific structure of the RING domain had no influence on the intracellular localization of this protein. When viral DNA replication was inhibited with cytosine arabinoside, p28 was found in distinct, focal structures that may be precursors to the factories. We hypothesize that in macrophages, which are highly specialized, nondividing cells, p28 substitutes for an unknown cellular factor(s) that may be required for viral DNA replication or a stage of virus reproduction between the expression of early genes and the onset of DNA synthesis. In the absence of p28, the attenuation of EV pathogenicity can be explained by a failure of the virus to replicate in macrophage lineage cells at all successive steps in the spread of virus from the skin to its target organ, the liver.

CycMs3, a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle.

Cyclins are key regulators of the cell cycle in all eukaryotes. We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes. Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast. The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins. In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells. When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis. In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively. In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively. These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2. We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.

Identification of a gibberellin-induced gene in deepwater rice using differential display of mRNA.

Differential display of mRNA was employed to identify gibberellin (GA)-regulated genes in deepwater rice. One of the first differentially displayed products identified was shown to be ten-fold induced after start of GA treatment. The sequence of the clone shows complete amino acid identity with histone H3, and its increased mRNA level correlates with the onset of DNA synthesis. We also identified a gene whose expression pattern did not change over the course of treatment with GA and can be used as standard to correct for loading differences on northern blots.

Inhibition of DNA synthesis in primary cultures of adult rat hepatocytes by benzo[ a]pyrene and related aromatic hydrocarbons: Role of Ah receptor-dependent events

Studies were conducted to examine the effects of benzo[ a]pyrene (BaP) and related-aromatic hydrocarbons (AHs) on the DNA synthetic profiles of adult rat hepatocytes in primary culture. Scheduled DNA synthesis in control cultures peaked at 64 h and was negligible by 72 h after initial seeding of freshly isolated hepatocytes. A concentration-dependent inhibition of DNA synthesis was observed in 1-day old hepatocyte cultures treated with BaP (0.3–30 μM) for up to 28 h. Comparable inhibitory responses were observed in cultures treated for 24 h with 2,3,7,8-tetrachlorodi-benzo- p-dioxin (TCDD, 0.01–1 nM) or 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.01–1 nM), but not in cultures treated with perylene (0.01–100 nM) or benzo[ e])pyrene (1–1000 nM). Ethoxyresorufin- O-deethylase (EROD) activity was highly inducible in hepatocytes challenged for 24 h with BaP (0.3–3 μM) or TCDD (0.1–100 nM) with peak induction observed at 12 or 36 h after chemical challenge, respectively. To determine if DNA synthesis inhibition by these agents involved aryl hydrocarbon receptor (AhR)-related events, subsequent experiments were conducted to examine the interactions of α-naphthoflavone (α-NF) and ellipticine (ET) with BaP and TCDD in this cell system. Pretreatment with α-NF (10 nM) for 24 h prevented the inhibitory effects of both BaP (3 μM) and TCDD (1 nM), while ET (0.01 nM) pretreatment selectively antagonized the effects of BaP (3 μM). Pretreatment of hepatocytes with TCDD or TCDF (1 nM) for 24 h before the onset of DNA synthesis followed by challenge with BaP (3 μM) partially antagonized the inhibitory response to BaP. These data implicate AhR-related signal transduction in the inhibition of hepatocyte DNA synthesis by BaP and related AHs and suggest that in the case of BaP, metabolism by cytochrome P450 to toxic intermediates contributes to the inhibitory response.

Testosterone Treatment Results in Quiescent Satellite Cells Being Activated and Recruited into Cell Cycle in Rat Levator Ani Muscle

At puberty, the male levator ani (LA) muscle exhibits muscle fiber hypertrophy. This fiber enlargement can be provoked in the female LA muscle by testosterone treatment. In both cases the hypertrophic process is accompanied by an increase in the number of satellite cells and myonuclei. The present ultrastructural autoradiographic study was undertaken in order to investigate (1) whether satellite cells when stimulated by testosterone can undergo DNA synthesis by incorporating [ 3H]thymidine ([ 3H]Tdr); (2) whether the whole satellite cell population is committed in the cell cycle; and (3) whether the new myonuclei originate from fusion of the satellite cells with the preexistant myofibers. Thirty-day-old female rats treated with a single testosterone injection received a single injection of [ 3H]Tdr at 24, 28, 32, to 34 hr after testosterone treatment. LA muscles were removed 2 hr after [ 3H]Tdr injection. This first series of experiments allowed us to determine that onset of DNA synthesis in satellite cells occurs within the 34th and the 36th hour after testosterone treatment. To obtain a more precise timing, 30-day-old female rats treated with a single testosterone injection received multiple injections every 30 min either from Hour 32 to 33.5 or from Hour 34 to 35.5. LA muscles were removed 30 min after the last [ 3H]Tdr injection. This showed that the onset of satellite cell replication occurred between the 32nd and the 34th hour after testosterone treatment. However, only 30% of the satellite cell population was affected by this proliferative process regardless of the experimental protocol used. To confirm that the increase in myonuclei number results from incorporation of satellite cells into mature myofibers, 30-day-old female rats treated with a single injection of testosterone received a single injection of [ 3H]Tdr on the 60th hour after testosterone treatment. LA muscles were removed at 63, 84, to 108 hr after [ 3H]Tdr injection. We conclude that testosterone induces satellite cell proliferation at around the 33rd hour and that the increased number of myonuclei reported in our previous study is due to fusion of labeled satellite cells with myofibers.

Ascorbate free radical and its role in growth control

Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.

The coding sequences of mouse H2A and H3 histone genes contains a conserved seven nucleotide element that interacts with nuclear factors and is necessary for normal expression.

Expression of replication-dependent histone genes of all classes is up-regulated coordinately at the onset of DNA synthesis. The cellular signals involved in coordinate regulation of these genes are not known. Here we report identification of an alpha element, present within the mouse histone coding region activating sequence (CRAS). We show evidence that this element is present in histone genes from two classes, H2a and H3, in the mouse. This element has two biological functions in histone gene expression, i.e. the element interacts with nuclear proteins in regulation of gene expression, as well as encoding the amino acids of the histone proteins. We present both in vivo and in vitro evidence that interaction of nuclear proteins with this element is required for normal expression. The binding site for nuclear protein(s) has been precisely defined by means of synthetic oligonucleotides, as well as DNase I protection and methylation interference. It is interesting to note that the histone CRAS alpha element is mutated in a replication-independent H3.3 gene; 5 of 7 nt in the CRAS alpha box are changed in this gene.

Onset of DNA synthesis in experimentally activated ascidian eggs

AbstractDNA synthesis was studied autoradiographically in unfertilized ascidian eggs (Ascidia malaca and Ciona intestinalis) that had been artificially activated by a K+‐free external medium or a Ca‐ionophore. Naked eggs of A. malaca were incubated in K+‐free seawater that contained [3H]‐thymidine for 30 min, and naked eggs of C. intestinalis were incubated in seawater supplemented with a Ca‐ionophore and [3H]‐thymidine for 15 min. The observations revealed limited activation in eggs treated with K+‐free seawater. Activation consisted of surface modifications and the onset of DNA synthesis. Incorporation of [3H]‐thymidine was detected in female nuclei of treated eggs which had not ejected their polar bodies. In eggs treated with the Ca‐ionophore, a known activating agent, the rate of incorporation of [3H]‐thymidine was higher and nuclear duplication was also observed. Comparison of activation under the two different sets of artificial conditions indicated that, in K+‐free medium, the early calcium‐dependent events, such as deformation of egg shape, extrusion of the polar bodies, and ooplasmic segregation, do not occur, while DNA synthesis is initiated. The results are discussed on the basis of the hypothesis that, in Ascidians, egg‐activation events are only partly regulated by an intracellular increase in levels of calcium ions. DNA synthesis appears to be an independent process that is initiated by changes in the egg plasma membrane. © 1994 Wiley‐Liss, Inc.

Post-transcriptional repression of thymidine kinase expression during cell cycle and growth stimulation

In vertebrates, endogenous thymidine kinase (TK) gene expression is strictly growth-dependent. Here we report that in continuously cycling Ltk − mouse fibroblasts, stably transfected with a vector expressing human TK cDNA from a constitutive promoter, enzyme activity rises 8-fold at the G 1/S phase transition and declines again in G 2. The mechanism did not involve changes in protein stability. When hTK was put under the control of a hormone-inducible promoter, production of high mRNA levels following addition of dexamethasone did not result in any enzyme activity in resting NIH-3T3tk − cells. After growth stimulation with serum, TK activity rose together with the onset of DNA synthesis only in the simultaneous presence of the hormone.

Growth factor modulated calmodulin-binding protein stimulates nuclear DNA synthesis in hemopoietic progenitor cells.

A specific calmodulin-binding protein of 68 kDa (CaM-BP68) is modulated in response to growth factors that induce proliferative stimulation in a variety of hemopoietic progenitor cells. The nuclear localization of the CaM-BP68 coincided temporally with interleukin 3 (IL-3)-dependent progression of synchronized FDC-P1 cells from G1 to S phase [Reddy et al. (1992) Blood 79, 1946-1956]. To delineate the role of the CaM-BP68 in the onset of DNA synthesis (S phase), this protein was purified to an apparent homogeneity from FDC-P1 cells and its effects on DNA replication in permeabilized FDC-P1 cells were examined. Purified CaM-BP exhibited a single silver-stained protein band of 68 kDa on SDS-polyacrylamide gels. This purified protein, when incubated with permeabilized log-growing FDC-P1 cells, caused a 3-4-fold increase in the rate of [3H]dTTP incorporation into DNA as compared to the controls. There was a direct correlation between the increase in the rate of [3H]dTTP incorporation into DNA and the concentration of the added CaM-BP68 in the incubation mixture. These observations suggest that the CaM-BP68, whose nuclear localization is associated with growth factor dependent proliferative stimulation of myeloid progenitor cells, is involved in the regulation of nuclear DNA synthesis.

Expression of a growth arrest specific gene (gas-6) during liver regeneration: molecular mechanisms and signalling pathways.

A set of growth arrest-specific (gas) genes negatively regulated by serum has been identified. To define the role of gas genes in a model of cell proliferation in vivo we analyzed the expression of one of these genes (gas-6) during liver regeneration after partial hepatectomy (PH). We found that gas-6 mRNA was down-regulated 4 hours after PH, within the G0 to G1 transition. Later on, gas-6 mRNA increased over the level found in normal liver with a peak at 16 hours, before the onset of DNA synthesis. This surge was probably triggered by an inflammatory response caused by the surgical trauma, because an increase of similar extent occurring with the same time course was present in livers of sham-operated and turpentine-treated rats. Comparison of mRNA steady state levels with nuclear transcription rates indicated that gas-6 expression is post-transcriptionally regulated. As we found that down-regulation of gas-6 expression was prevented by treatment with Actinomycin D, a labile protein might be involved in the determination of gas-6 mRNA stability. To investigate the mitogenic signals controlling gas-6 expression during liver regeneration we treated hepatectomized rats with a specific alpha-1-adrenoceptor blocker (prazosin) as well as with drugs which modify intracellular calcium levels. The decrease of gas-6 mRNA 4 hours after PH was prevented by prazosin and by neomycin, an inhibitor of calcium release from endogenous stores. These findings suggest that down-regulation of gas-6 expression during hepatic regeneration is triggered by catecholamines interaction with alpha-1-adrenergic receptors and by subsequent calcium release. In addition we found that the rise of gas-6 gene expression occurring at 16 hours after PH was not affected by prazosin but was inhibited by trifluoperazine. Therefore, we suggest that up-regulation of gas-6 gene expression is mediated by the interaction of calcium with calmodulin, independently of catecholamines.

Antibacterial effects of esters of guanidino- and amidino-acids trypsin inhibitors.

Inhibitory effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid and the 4-tert- butylphenyl esters of amidinopiperidine-4-alkanoic, trans-4- amidinocyclohexanealkanoic, trans-4-guanidinoethylcyclohexanecarboxylic and trans-4-guanidinocyclohexanealkanoic acids, all trypsin inhibitors, on the growth of E. coli, B. subtilis, S. aureus and S. epidermidis were examined. 4-tert-Butylphenyl esters strongly inhibited the growth of E. coli and the order of the inhibitory effects correlated with that for the inhibitory effects on proteinase In, which appears immediately before initiation of DNA synthesis in E. coli and closely correlates with the onset of DNA synthesis. No correlation was observed between the inhibitory effects and Ki values for trypsin. The 4-tert-butylphenyl esters also strongly inhibited B. subtilis, S. aureus and S. epidermidis, and the order, of the inhibitory effects on these bacteria roughly coincided with that on E. coli. The order of the inhibitory effects of each ester, on these bacteria was S. epidermidis > S. aureus > B. subtilis > E. coli. Among the esters examined, the biphenyl ester of trans-4-guanidinoethylcyclohexanecarboxylic acid was the most inhibitory on these four bacteria and proteinase In. Hydrolysis of tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide, which is a substrate for proteinase In, in crude extracts of E. coli, B. subtilis and S. epidermidis was examined. The order of this activity in these bacteria was E. coli > B. subtilis > S. epidermidis.

Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.