Resumption Of DNA Synthesis Research Articles

P53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.

Ectopic p21WAF1 Expression Induces Differentiation-specific Cell Cycle Changes in PC12 Cells Characteristic of Nerve Growth Factor Treatment

Nerve growth factor treatment of PC12 cells results in neuronal differentiation, a process accompanied by induction of the Cdk inhibitor p21(WAF1). To determine the role of p21 in differentiation, PC12 clones containing an inducible p21 construct were utilized to induce growth arrest. Expression of p21 led to accumulation of cyclins D1 and E and to a decrease in cyclins A and B. Levels of Cdc2 and Cdk4 also decreased after p21 induction. Initially, thymidine incorporation into DNA was dramatically inhibited; however, low levels of incorporation were observed during prolonged p21 expression. Fluorescence-activated cell sorter analysis revealed that this low level of DNA synthesis resulted in the generation of polyploid cells. Results from Western blots were consistent with phosphorylation of p21 protein coincident with the resumption of DNA synthesis. Finally, treatment of p21-arrested populations with epidermal growth factor, a known PC12 mitogen, resulted in neurite extension, a key feature of neuronal differentiation. Overall, cell cycle changes following p21 overexpression in PC12 cells closely mimic distinctive events previously shown to occur during differentiation. These results suggest that the mechanism by which nerve growth factor induces the many cellular changes associated with growth arrest during differentiation is through p21(WAF1) induction.

Reexpression of the retinoblastoma protein in tumor cells induces senescence and telomerase inhibition

Normal human diploid cells senesce in vitro and in vivo after a limited number of cell divisions. This process known as cellular senescence is an underlying cause of aging and a critical barrier for development of human cancers. We demonstrate here that reexpression of functional pRB alone in RB/p53-defective tumor cells via a modified tetracycline-regulated gene expression system resulted in a stable growth arrest at the G0/G1 phase of the cell cycle, preventing tumor cells from entering S phase in response to a variety of mitogenic stimuli. These cells displayed multiple morphological changes consistent with cellular senescence and expressed a senescence-associated beta-galactosidase biomarker. Further studies indicated that telomerase activity, which was assumably essential for an extended proliferative life-span of neoplastic cells, was abrogated or repressed in the tumor cell lines after induction of pRB (but not p53) expression. Strikingly, when returned to an non-permissive medium for pRB expression, the pRB-induced senescent tumor cells resumed DNA synthesis, attempted to divide but most died in the process, a phenomenon similar to postsenescent crisis of SV40 T-antigen-transformed human diploid fibroblasts in late passage. These observations provide direct evidence that overexpression of pRB alone in RB/p53-defective tumor cells is sufficient to reverse their immortality and cause a phenotype that is, by all generally accepted criteria, indistinguishable from replicative senescence. The results suggest that pRB may play a causal role in the intrinsic cellular senescence program.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: Effects of aphidicolin

To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomere division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 μM and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos.

Zygotic Transcription Is Required to Block a Maternal Program of Apoptosis inXenopusEmbryos

At the midblastula transition duringXenopusdevelopment, the cell cycle is remodeled, and zygotic transcription is initiated. Additionally, cyclin E1 is degraded at the midblastula transition independently of protein synthesis, the number of cell cycles, and the nuclear-to-cytoplasmic ratio. In the studies reported here, cell cycles were delayed by transient inhibition of protein synthesis with cycloheximide (100 μg/ml) prior to the midblastula transition. Even after reaccumulation of mitotic cyclins and resumption of cell divisions, cycloheximide-treated embryos did not resume DNA synthesis, failed to initiate transcription, and synchronously became apoptotic before the gastrula stage. These results were independent of the stage at which embryos were treated or the duration of treatment. Inhibition of zygotic transcription with α-amanitin also induced apoptosis. These data suggest that a developmental checkpoint at the midblastula transition is maternally regulated and can trigger apoptosis. Apoptosis induced by cycloheximide or α-amanitin was blocked by injection of RNA encodingXenopusBcl-2, suggesting that this maternal program is normally blocked by expression of an apoptotic inhibitor. Embryos pulsed with lower doses of cycloheximide (10 μg/ml) delayed development prior to the midblastula transition but resumed DNA synthesis, initiated transcription, and gastrulated normally. This indicates that the apoptotic response is initiated only when delayed embryos are unable to support initiation of zygotic transcription.

A Nuclear Protein, Synthesized in Growth-Arrested Human Hepatoblastoma Cells, is a Novel Member of the Short-Chain Alcohol Dehydrogenase Family

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723–729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.

A Nuclear Protein, Synthesized in Growth‐Arrested Human Hepatoblastoma Cells, is a Novel Member of the Short‐Chain Alcohol Dehydrogenase Family

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.

Transforming growth factor-beta stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity.

Macrophage arginine metabolism via nitric oxide (NO) synthase and arginase pathways reduces and enhances tumor cell proliferation, respectively. Transforming growth factor-beta (TGF-beta) has been shown to down-regulate the NO synthase pathway. The present study describes the effect of TGF-beta on the arginase pathway. TGF-beta up-regulated arginase activity in rat peritoneal macrophages as assessed by measuring the generation of [14C]urea from [14C]-L-arginine in the presence of NG-monomethyl-L-arginine (L-NMMA). The stimulation, which reached fivefold after a 48-h exposure of macrophages to 10 ng/ml TGF-beta, was due to reduction in Km value of arginase. TGF-beta-induced up-regulation of arginase activity led to the release of more polyamines, mainly putrescine. The role of this up-regulation on macrophage cytotoxicity toward L-929 tumor cells was analyzed in coculture experiments. Macrophages blunted DNA synthesis by L-929 cells as assessed by measuring the incorporation of [3H]TdR into the cells and the proportion of cells in the G2 phase. Addition of TGF-beta in the presence of L-NMMA permitted L-929 cells cocultured with macrophages to resume DNA synthesis. The mechanism responsible for this restoration was the up-regulation of arginase activity rather than the down-regulation of NO synthase activity since TGF-beta in the presence of L-NMMA failed to further reduce NO synthase activity whereas it still enhanced arginase activity; synthetic putrescine (1-10 microM) also blunted macrophage cytotoxicity toward L-929 cells. This is the first evidence that TGF-beta up-regulates arginase activity in macrophages and, hence, limits macrophage-dependent cytostasis.

Expression and Functional Role of E- and P-Cadherins in Mouse Mammary Ductal Morphogenesis and Growth

Mammary ducts, and the highly mitotic terminal end buds from which they are derived, consist of two layers of ectodermally derived epithelium, forming a tube-within-a-tube structure. We investigated the role of Ca 2+-dependent cell-cell adhesion molecules in maintaining the integrity of these layers. Immunostaining showed abundant E-cadherin on the lateral membranes of end bud body cells and ductal lumenal cells, but no P-cadherin. The basally located cap cells and their differentiated descendants, the ductal myoepithelial cells, displayed only P-cadherin. We investigated the functional significance of this pattern of cadherin expression in situ by surgically implanting small, slow-release plastic implants releasing function-blocking antibodies. End buds exposed to a monoclonal antibody to E-cadherin showed disruption of the body epithelium, with epithelial cells floating freely in the lumen. Epithelial DNA synthesis, which is normally very high in these growth buds, abruptly declined. That this reduction in growth was not due to cell damage was shown by spontaneous reaggregation of the cells into a normal epithelium, with resumption of DNA synthesis, when the blocking antibody was depleted. A monoclonal antibody to P-cadherin had no effect on the lumenal layer but partially disrupted the basally located cap cell layer. These data indicate that spatially selective expression of E- and P-cadherins is required for mammary tissue integrity, which is in turn a prerequisite for normal rates of DNA synthesis.

In vitro inhibition of RecA-mediated homologous pairing by UmuD'C proteins

The process of SOS mutagenesis in Escherichia coli requires: i) the replisome enzymes; ii) RecA protein; and iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. It has recently been shown that the UmuD'C complex, if overproduced, inhibits recombinational repair of a UV-damaged plasmid DNA as well as homologous recombination in an Hfr x F- cross. Since UmuD'C proteins might inhibit an early recombination step by interacting with a RecA nucleo-protein filament, we checked whether UmuD'C proteins will inhibit RecA promoted homologous pairing in vitro. We tested the inhibitory action of UmuD'C proteins in a crude bacterial extract containing possible cofactors such as chaperone proteins that ensure the proper folding of UmuC and the assembly of the UmuD'C complex in vivo. We used a novel recombination assay in which RecA protein promotes the formation of a stable plectonemic joint between a circular single-stranded DNA immobilized onto a membrane and an incoming homologous linear duplex DNA. Under these conditions we show that UmuD'C proteins inhibit the formation of joint molecules.

The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis.

The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.

Alterations in Nuclear Protein Mass and Macromolecular Synthesis Following Heat Shock

Possible correlations between post-heat alterations in nuclear protein mass and the resumption of macromolecular (DNA, RNA, and protein) synthesis were investigated in CHO cells. Nuclear protein content was measured using flow cytometry. Macromolecular synthesis was measured by incorporation of radioactively labeled precursors into TCA-precipitable material of whole cells and isolated nuclei. Following an initial increase which was dependent on the heat dose, nuclear protein mass decreased following a monotonic function which appeared to be multiphasic. The synthesis of DNA, RNA, and protein was inhibited in a manner dependent on the heat dose and remained suppressed for an interval dependent on the heat dose before recovery. The kinetics of resumption of DNA, RNA, and protein synthesis correlated linearly with the nuclear protein mass measured immediately after heating. Also, the time of onset of recovery of RNA and protein syntheses correlated linearly with the time at which nuclear protein mass returned to 125% of control, a level which has been implicated as a possible threshold in previous studies. More significantly, the onset of the resumption of DNA synthesis showed a one-to-one correlation with the time at which the nuclear protein mass returned to 125% of control. These correlations suggest that there may be causal relationships between the resumption of DNA, RNA, and protein synthesis and the reduction of the amount of nuclear protein binding, particularly in the case of DNA synthesis.

Fibroblastic cell cycling in collagen gels.

Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.

Effect of orotic acid on in vivo DNA synthesis in hepatocytes of normal rat liver and in hepatic foci/nodules.

One of the proposed mechanisms by which orotic acid (OA) promotes liver carcinogenesis is by differentially mito-inhibiting the normal hepatocytes while permitting the initiated ones to respond to growth stimuli to form foci/nodules. In an attempt to examine this hypothesis, the present study was designed to determine (i) whether OA inhibits DNA synthesis in normal hepatocytes in vivo, and (ii) whether hepatocytes from hepatic foci/nodules are relatively resistant to the mito-inhibitory effects of OA. The results of this study indicate that OA given i.p. as a tablet of 300 mg at the time of partial hepatectomy (PH) almost completely inhibited liver DNA synthesis. Three days later--a time period by which the implanted tablet disappeared--the hepatocytes resumed DNA synthesis. Exposure to OA results in an accumulation of uridine nucleotides and a decrease in adenosine nucleotides. Creation of such an imbalance in nucleotide pools appears to be important for OA to inhibit DNA synthesis. Adenine (a tablet of 300 mg), an agent that inhibits the metabolism of OA to uridine nucleotides, counteracted the mito-inhibitory effects of OA. To determine whether the hepatocytes in foci/nodules are resistant to the mito-inhibitory effects of OA, rats were initiated with diethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocyte model. Fourteen weeks after the administration of DENA, the rats were subjected to PH in the presence of absence of OA (300 mg tablet). The results indicated that, in contrast to hepatocytes in normal or surrounding non-nodular liver, a subpopulation of hepatocyte foci/nodules appear to be relatively resistant to the mito-inhibitory effects of OA. These findings support the hypothesis that differential mito-inhibition is a possible component in the promoting effect of OA. However, whether this is the mechanism by which OA promotes liver carcinogenesis needs to be further investigated.

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Immunohistochemical observations indicate that human myometrial smooth muscle cells express epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AB and contain EGF and PDGF-beta receptors with no variation in intensity with phases of the menstrual cycle. Furthermore, immunofluorescent microscopic studies revealed that primary myometrial smooth muscle cell cultures also express EGF, PDGF-AB, and contain EGF and PDGF-beta, but not alpha-receptor. Incubation of subconfluent smooth muscle cells in serum-free medium leads to quiescence within 48 h as demonstrated by 3H-thymidine incorporation and labeling index. Exposure of quiescent cells to 10% fetal bovine serum stimulates resumption of DNA synthesis and proliferation in a time-dependent manner with a doubling time of 41.6 h. EGF (1.5-50 ng/ml) and PDGF-AB (1-10 ng/ml) in a dose- and time-dependent manner significantly stimulated 3H-thymidine incorporation by quiescent myometrial smooth muscle cells (P less than 0.05). Combinations of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) significantly increased 3H-thymidine incorporation induced by either growth factor alone (P less than 0.05). PDGF-BB at 10 ng/ml also stimulated 3H-thymidine incorporation and its effect was similar to that induced by PDGF-AB at the same concentration. 17 beta-Estradiol (E2) at 1 microM inhibited 3H-thymidine incorporation by the smooth muscle cells (P less than 0.05). E2 also reduced the stimulatory effect of EGF (15 ng/ml) and PDGF (3 ng/ml). Progesterone at 1 microM either alone or in combination with E2 did not have any effect on 3H-thymidine incorporation or alter the mitogenic action of EGF and PDGF. The effect of EGF and PDGF on cell growth and 3H-thymidine incorporation by myometrial smooth muscle cells was independent of phases of the menstrual cycle. In summary, the results of present studies indicate that human myometrial tissue and myometrial smooth muscle cells in primary culture locally produce EGF and PDGF-AB and contain EGF and PDGF-beta, but not alpha-receptors. Moreover, the myometrial smooth muscle cells in culture respond to the mitogenic action of EGF and PDGF.

Involvement of Fis protein in replication of the Escherichia coli chromosome

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 °C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 × 10 3 base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.

Identification of a novel nuclear protein synthesized in growth‐arrested human hepatoblastoma HepG2 cells

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein.

Post-initiation control of chromosomal replication in Bacillus subtilis: a mechanism for limiting over-replication or for duplicating key growth and sporulation genes?

We used the Bacillus subtilis dnaB37 mutant, which is defective in initiation, to synchronize DNA replication in order to identify the first fragments to be replicated following initiation and to study the control of this process under various conditions. We show by DNA/DNA hybridization analysis that, after returning the mutant from 45°C to the permissive temperature (30°C), the origin region relative to other sequences is over-replicated (approximately 2-fold) during the first round. This was confirmed by autoradiographic analysis. The over-replicated region is however limited to about 190 kb on the left and right arms. Replication apparently resumes from these positions during the following round of replication. We propose that, in B. subtilis, in addition to the first level of control at the origin there is a second level or post-initiation control downstream of the origin which limits DNA replication resulting from premature initiation. We believe that these two levels of control are tightly coupled under conditions of balanced growth. Using the same system, we have now shown that DNA replication is subject to “stringent control”, an important regulatory network in bacteria. These studies demonstrate that the inhibition of replication induced during the “stringent response” does not occur at the primary origin. In fact, by DNA/DNA hybridization, replication forks were found to be blocked at similar positions to the post-initiation control sites described above. Moreover, replication appears to resume from regions close to the stalled replisomes upon removal of the stringent response. Resumption of DNA synthesis, in this case, does not require protein or RNA synthesis or the initiation protein DnaB. We propose that this second level of control, or post-initiation control, is exerted through the reversible inhibition of replisome movement at specific regions on either side of the origin. This may be mediated through the cellular concentration of ppGpp. We have also examined the effect of the stringent response on the initiation of replication in E. coli. Interestingly, in contrast to B. subtilis, inhibition of DNA replication occurs at oriC itself. This difference between the two organisms raises questions over the possible physiological implications. In B. subtilis, many key genes involved in DNA replication and sporulation, together with 7 out of 10 ribosomal operons, are located within the over-replicated region. Differential expression of this important group of duplicated genes under starvation conditions could be crucial in determining the final decision between continued vegetative growth or commitment to sporulation.