Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA.

In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the beta subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42 degrees C)-sensitive dnaN59 mutant are inhibited at 34-35 degrees C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the beta subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the beta subunit is abundant (present at a level of about 5000 dimers per cell) at 34 degrees C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34 degrees C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean.

The nature and enzymic properties of starch-branching enzyme (SBE) are two of the dominant factors influencing the fine structure of starch. To understand the role of this enzyme's activity in the formation of starch in kidney bean (Phaseolus vulgaris L.), a study was undertaken to identify the major SBE sequences expressed during seed development and to characterize the enzymic properties of the coded recombinant enzymes. Two SBE cDNA species (designated pvsbe2 and pvsbe1) that displayed significant similarity (more than 70%) to other family A and B SBEs respectively were isolated. Northern blot analysis revealed that pvsbe1 and pvsbe2 were differentially expressed during seed development. pvsbe2 showed maximum steady-state transcript levels at the mid-stage of seed maturation, whereas pvsbe1 reached peak levels at a later stage. Western blot analysis with antisera raised against both recombinant proteins (rPvSBE1 and rPvSBE2) showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean. PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with the starch granule fraction. The differences in location suggest that these two SBE isoenzymes have different roles in amylopectin synthesis in kidney bean seeds. rPvSBE1 and rPvSBE2 were purified from Escherichia coli and their kinetic properties were determined. The affinity of rPvSBE2 for amylose (K(m) 1.27 mg/ml) was lower than that of rPvSBE1 (0.46 mg/ml). The activity of rPvSBE2 was stimulated more than 3-fold in the presence of 0.3 M citrate, whereas rPvSBE1 activity was not affected. The implications of the enzymic properties and the distribution of SBEs and amylopectin structure are discussed.

Different localization of SeqA-bound nascent DNA clusters and MukF-MukE-MukB complex in Escherichia coli cells.

MukF, MukE and MukB proteins form a complex that may participate in the organization of folded sister chromosomes in Escherichia coli. We have found that a MukB-GFPuv4 fusion protein is observed as discrete fluorescent foci, which are localized within cellular spaces occupied by nucleoids, but not at the constriction site of cell division in living cells. In contrast, MukB-GFPuv4 is distributed throughout the whole cell when either MukF or MukE is absent. Statistical analysis revealed that most newborn cells have two foci of mukB-gfpUV4 at one-quarter and three-quarter positions in the cell length and one focus of SeqA-bound nascent DNA at or near the middle of the cell. Subsequently, the single SeqA focus divides into two foci, and then these migrate to the one-quarter and three-quarter positions. Before cell division, most long cells have two SeqA foci and four MukB-GFPuv4 foci. In early stationary phase, SeqA foci disappear, but one or two foci of MukB-GFPuv4 remain. We discuss the reorganization and proper arrangement of folded sister chromosome in the cell quarter positions, which are performed after release from the long-time cohesion of sister chromosomes.

A large family of class III plant peroxidases.

Class III plant peroxidase (POX), a plant-specific oxidoreductase, is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms. POXs exist as isoenzymes in individual plant species, and each isoenzyme has variable amino acid sequences and shows diverse expression profiles, suggesting their involvement in various physiological processes. Indeed, studies have provided evidence that POXs participate in lignification, suberization, auxin catabolism, wound healing and defense against pathogen infection. Little, however, is known about the signal transduction for inducing expression of the pox genes. Recent studies have provided information on the regulatory mechanisms of wound- and pathogen-induced expression of some pox genes. These studies suggest that pox genes are induced via different signal transduction pathways from those of other known defense-related genes.

Sister chromosome cohesion of Escherichia coli.

We analysed Escherichia coli cells synchronized for initiation of chromosomal DNA replication by fluorescence in situ hybridization (FISH) using fluorescent DNA probes corresponding to various chromosomal regions. Sister copies of regions in an approximately oriC-proximal half of the chromosome are cohesive with each other after replication until the late period of chromosome replication. Sister copies of regions relatively close to the terminus are also separated from each other in the same late period of replication. It is important that sister copies in all the tested regions are thus separated from each other nearly all at once in the late period of chromosome replication. These results are consistent with results obtained by FISH in randomly growing cultures. Cohesion of sister copies in an oriC-close region is observed in a dam null mutant lacking DNA adenine methyltransferase the same as in the parental isogenic dam+ strain, indicating that the cohesion is independent of DNA adenine methyltransferase. This further implies that hemimethylated DNA-binding proteins, such as SeqA, are not involved in the cohesion. On the other hand, the cohesion of sister copies of the oriC-close region was not observed in mukB null mutant cells, suggesting that MukB might be involved in the chromosome cohesion.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

BACKGROUND: Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. RESULTS: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. CONCLUSIONS: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Dynamic localization of bacterial and plasmid chromosomes.

Plasmid-encoded partition genes determine the dynamic localization of plasmid molecules from the mid-cell position to the 1/4 and 3/4 positions. Similarly, bacterial homologs of the plasmid genes participate in controlling the bidirectional migration of the replication origin (oriC) regions during sporulation and vegetative growth in Bacillus subtilis, but not in Escherichia coli. In E. coli, but not B. subtilis, the chromosomal DNA is fully methylated by DNA adenine methyltransferase. The E. coli SeqA protein, which binds preferentially to hemimethylated nascent DNA strands, exists as discrete foci in vivo. A single SeqA focus, which is a SeqA-hemimethylated DNA cluster, splits into two foci that then abruptly migrate bidirectionally to the 1/4 and 3/4 positions during replication. Replicated oriC copies are linked to each other for a substantial period of generation time, before separating from each other and migrating in opposite directions. The MukFEB complex of E. coli and Smc of B. subtilis appear to participate in the reorganization of bacterial sister chromosomes.

Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants.

Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.

Two types of localization of the DNA-binding proteins within the Escherichia coli nucleoid.

BACKGROUND: The genome DNA of Escherichia coli is folded into the nucleosome-like structure, often called a nucleoid, by the binding of several DNA-binding proteins. We previously determined the specificity and affinity of DNA-binding for 12 species of the E. coli DNA-binding protein, and their intracellular concentrations at various growth phases. The intracellular localization of these proteins in E. coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA-binding proteins. RESULTS: The intracellular localization in Escherichia coli of 10 species of the nucleoid-associated protein, three components of the transcripton apparatus, and three components of the translation machinery was investigated by indirect immuno-fluorescence microscopy. The DNA-binding proteins could be classified into two groups. The group-I proteins, including the major nucleoid-structural proteins, H-NS, HU, IHF, StpA and Dps, are distributed uniformly within the entire nucleoid. In contrast, the group-II proteins, which are presumed to possess regulatory activities of DNA functions accumulate at specific loci within the nucleoid, forming 2 (SeqA), 3-4 (CbpA and CbpB) and 6-10 (Fis and IciA) immuno-stained dots. Each immuno-stained dot may represent either the association of a hundred to one thousand molecules of each DNA-binding protein at a specific locus of the genome DNA or the assembly of protein-associated DNA segments from different domains of the folded genome. Both the RNA polymerase core enzyme and the sigma70 subunit are mainly associated with the nucleoid, but the anti-sigma70 factor (Rsd) appears to be accumulated at the boundary between the nucleoid and the cytosol in the stationary-phase cells. Here we show that the majority of Hfq is present in cytoplasm together with ribosomal proteins L7/L12 and RMF. CONCLUSION: The DNA-binding proteins of E. coli could be classified into two groups. One group proteins was distributed uniformly within the nucleoid, but the other group of proteins showed an irregular distribution, forming immuno-stained spots or clumps.

Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli.

BACKGROUND: We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions. RESULTS: The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 degrees C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division. CONCLUSIONS: A single cluster of SeqA-bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine.

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.

Diverse expression profiles of 21 rice peroxidase genes.

Secretory class III plant peroxidases (POXs) catalyze the oxidation of various reductants, and are encoded by a large multigene family. In rice, 42 independent expressed sequence tags for POXs have been identified. By RNA gel blot analysis using specific probes, we show here that 21 rice POX genes are unique in their developmental, organ specific and external stimuli-responsive expression. This would suggest that encoded POX isoenzymes are involved in a broad range of physiological processes in rice plants, individually.

Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants.

A peroxidase gene, poxA, was isolated from a rice (Oryza sativa L.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots, Plant Cell Rep. 13 (1994) 361-366]. The temporal and spatial expression properties of the poxA gene promoter as well as that from a second related peroxidase gene, poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive cis-elements were located within 144 bp from the translational start codon of the poxA gene. The poxN promoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath.

Modulation of collagen synthesis by tumor necrosis factor alpha in cultured vascular smooth muscle cells.

Collagen synthesis in vascular smooth muscle cells (SMCs) after exposure to tumor necrosis factor alpha (TNF-alpha) was investigated using a culture system. The synthesis of collagenase-digestible proteins (CDP) and noncollagenous proteins (NCP) was evaluated by the [3H]proline incorporation. It was shown that TNF-alpha markedly suppresses the incorporation of [3H]proline into both CDP and NCP in confluent cultures of SMCs but not in sparse cultures of the cells. Such a marked suppression by TNF-alpha was not observed in confluent bovine aortic endothelial cells and human fibroblastic IMR-90 cells. In confluent SMCs, the synthesis of CDP was more strongly inhibited by TNF-alpha than that of NCP. When the CDP synthesis was stimulated by transforming growth factor beta, TNF-alpha suppressed the stimulation in both confluent and sparse SMCs. Human SMCs synthesized types I, III, IV and V collagens; TNF-alpha markedly decreased the relative proportion of types IV and V. It was therefore suggested that TNF-alpha modulates the collagen synthesis by SMCs depending on their cell density and modifies the formation of atherosclerotic lesions.

Analysis of urea nitrogen and creatinine kinetics in hemodialysis: comparison of a variable-volume two-compartment model with a regional blood flow model and investigation of an appropriate solute kinetics model for clinical application.

To investigate an appropriate solute kinetics model for clinical application, we analyzed urea nitrogen (UN) and creatinine (Cr) kinetics by a variable-volume two-compartmental model (2CM) and a regional blood flow model (RBF) in 44 hemodialysis patients with varying proportions of first compartmental volume and regional volume (p(1)). Solute kinetics could not be solved in some of the patients with higher p(1) values, and there were more solution failures by the RBF than by the 2CM. The solute generation rate (g) and solute distribution volume in the dry state (V(D)) increased with increases in p(1) in both models, but there were some differences between the two models. When g was normalized by V(D), it became relatively constant, irrespective of the p(1) value or model used (0.133 +/- 0.029 mg/min/l by the 2CM and 0.132 +/- 0.029 mg/min/l by the RBF for UN; 0.0200 +/- 0.0049 mg/min/l by the 2CM and 0.0198 +/- 0.0048 mg/min/l by the RBF for Cr). The intercompartmental mass transfer coefficient (K(c); liters/min) calculated by the 2CM decreased as p(1) increased (K(c) = -1.77.p(1) + 1.16, p < 0.0001, R = 0.999 for UN; K(c) = -0.847.p(1) + 0.556, p < 0.0001, R = 1.000 for Cr). The systemic blood flow (Q(sys); liters/min) calculated by the RBF also decreased as p(1) increased (Q(sys) = -11.1.p(1) + 6.21, p < 0.0005, R = 1.000 for UN; Q(sys) = -5.22.p(1) + 2.90, p < 0.001, R = 0.999 for Cr). Since the RBF more frequently failed to solve the solute kinetics and since there was a difference in its Q(sys) values for UN and Cr, the 2CM was considered to be a superior model. When p(1) was extremely low, the 2CM could be transformed into a modified variable-volume one-compartment model (1CM) which presented a similar g/V(D) (0.133 +/- 0.029 for UN; 0.0200 +/- 0.0048 for Cr). This modified 1CM was considered to satisfy appropriate conditions for clinical application, since it is simpler than the 2CM and provides useful information on the dialysis dose.

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon.

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Dynamic organization of chromosomal DNA in Escherichia coli.

We have revealed the subcellular localization of different DNA segments that are located at approximately 230-kb intervals on the Escherichia coli chromosome using fluorescence in situ hybridization (FISH). The series of chromosome segments is localized within the cell in the same order as the chromosome map. The large chromosome region including oriC shows similar localization patterns, which we call the Ori domain. In addition, the localization pattern of the large segment including dif is characteristic of the replication terminus region. The segment also shows similar localization patterns, which we call the Ter domain. In newborn cells, Ori and Ter domains of the chromosome are differentially localized near opposite cell poles. Subsequently, in the B period, the Ori domain moves toward mid-cell before the initiation of replication, and the Ter domain tends to relocate at mid-cell. An inversion mutant, in which the Ter domain is located close to oriC, shows abnormal subcellular localization of ori and dif segments, resulting in frequent production of anucleate cells. These studies thus suggest that the E. coli chromosome is organized to form a compacted ring structure with the Ori and Ter domains; these domains participate in the cell cycle-dependent localization of the chromosome.