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Copy-number control of the Escherichia coli chromosome: a plasmidologist's view.

The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number.

Cell Cycle↗

Evaluation of the dietary interaction of calcium and phosphorus in the high producing laying hen.

1. In a 6 x 7 factorial experiment using 2688 22-week-old laying hens of the Lohmann-SL strain kept in cages (4 birds/cage), diets containing six calcium (20, 25, 30, 35, 40, 45 g calcium/kg) and seven phosphorus concentrations (3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 16.2 g total phosphorus/kg (Pt)) were combined orthogonally. The resulting 42 treatments were replicated 8 times so that a replicate consisted of a double cage of 2 x 4 hens. The experiment lasted 40 weeks (10 x 28 days). 2. The experimental diets, based on maize and soyabean meals contained 11.5 MJ metabolisable energy/kg and 175 g/kg protein. Different dietary calcium and phosphorus contents were obtained by substituting oat hulls with limestone and dicalcium phosphate. 3. Mortality, egg production, egg weight, egg mass, food intake and food conversion efficiency were determined as well as the breaking strength, thickness of shells and the percentage of eggs with defective shells. 4. All responses measured were significantly influenced by the variance sources (calcium, phosphorus, interaction). Most of the production traits responded asymptotically to increasing dietary phosphorus concentration, the greatest increases or decreases generally being seen between 3.2 and 5.2 g Pt/kg. Further but weaker increases were seen between 5.2 and 8.2 or 16.2 g Pt/kg. 5. Increases in dietary calcium content always resulted in curvilinear responses. In all cases optimal effects were obtained with diets containing 25 g calcium/kg and the worst values at 45 g calcium/kg. The interaction between calcium and phosphorus was recognised by strong performance depressions and a high mortality at combinations of the lowest phosphorus concentration (3.2 g/kg) with high calcium contents (35 to 45 g/kg). These were largely offset by increasing dietary phosphorus. Thus, between 7.2 and 16.2 g Pt/kg and 25 and 45 g Ca/kg a plateau was formed where only small differences in egg production were observed. 6. From the three egg shell characteristics measured, breaking strength and shell thickness responded differently to the percentage of eggs with defective shells. While breaking strength and shell thickness were respectively negatively and positively influenced by increasing dietary phosphorus and calcium contents, both elements affected the proportion of eggs with defective shells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Ultraviolet mutagenesis and its repair in an Escherichia coli strain containing a nonsense codon.

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.

Base Sequence↗

The mammalian beta globin origin of DNA replication.

Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta globin initiation region, or IR). Mammalian beta globin loci replicate early during the S phase of the cell cycle in pre erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta globin IR.

Animals↗

alpha-Globulin sequences are located in a region of early-replicating DNA in murine erythroleukemia cells.

The time of replication in the S phase of regions of the mouse genome including the alpha-globin genes was determined in the murine erythroleukemia cell line transformed by Friend virus. Cells grown for short times in the presence of BrdUrd were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases, and fragments containing bromouracil (BrU-DNA) were isolated in density gradients of Cs2SO4. BrU-DNA fractions replicated during selected S-phase intervals were subjected to electrophoresis in agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to an alpha-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one alpha-globin gene demonstrated that the extent of hybridization provides an accurate measurement that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The alpha-globin fragments were detected primarily in the BrU-DNA replicated during early S phase (approximately the first quarter of S). This result was confirmed in other synchrony experiments and by Cot analysis. (Cot is the initial concentration of DNA in mol of nucleotide per liter multiplied by the time in sec.) The temporal replication of mouse satellite sequences, already known from previous studies, was used as an internal control for cell synchrony. To show that the globin sequences were not lost from the cells in late S phase during isolation of the DNA, we quantitated th alpha-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the alpha-globin gene regions in these cells are replicated during early S phase.

Animals↗

Carbon monoxide and human time discrimination: failure to replicate Beard-Wertheim experiments.

Beard and Wertheium described a dose-related deficit in human time perception during low level CO exposure. Two other laboratories were unable to replicate this finding, although methodological differences could explain these failures. This study more precisely repeated the original experiment, but failed again to obtain any CO-related deficit. The bulk of evidence, therefore, does not indicate any adverse effect of low level CO exposure on time perception in healthy young adults.

Acoustic Stimulation↗

A question of timing: emerging links between transcription and replication.

The coordination of transcription and timing of DNA replication during the S phase of the cell cycle has recently been studied chromosome-wide in high resolution. This revealed that in the complex genome of higher eukaryotes actively transcribed genes are more likely to replicate early in S phase. Dynamic changes in chromatin structure and nuclear organization appear to provide the underlying mechanism to link transcription and replication. A possible evolutionary benefit for this connection might result from differential replication fidelity during S phase, and comparisons of the human and chimpanzee genomes are compatible with this hypothesis.

Animals↗

Temporal order of gene replication in Chinese hamster ovary cells.

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

Animals↗

Replication dynamics of the yeast genome.

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

Algorithms↗

Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.

Animals↗

Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli.

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

Algal Proteins↗

The effect of pelleting, salt, and pentosanase on the viscosity of intestinal contents and the performance of broilers fed rye.

An experiment was conducted to determine the impact of pelleting of rye, dietary salt (.39 and .57%), and crude pentosanase supplementation (0 and .2%) on the viscosity of intestinal contents and the concomitant performance of broiler chicks fed rye-based diets. Each treatment was replicated six times with six birds per replicate. Test diets were fed from 1 day to 3 wk of age, at which time body weight, feed intake, intestinal viscosity, and molecular weight distribution of carbohydrate complexes were determined in fore and hind gut sections. Enzyme supplementation in all treatment combinations significantly increased weight gains and feed conversion efficiency (FCE). Pelleting and salt did not influence weight gain or FCE, although feed intake was increased through the addition of salt. The viscosity of fore and hind gut contents was significantly reduced with pentosanase supplementation, whereas the effects of salt or pelleting were not as clearly defined. Weight gain and FCE correlated with fore but not hind gut viscosity. The viscosity of gut samples was found to be best described by the concentration of carbohydrate complexes with an average molecular weight greater than 500,000 Da. Pentosanase treatment reduced lumenal concentration of these complexes, thereby reducing viscosity and stimulating improvements in growth and FCE.

Animal Feed↗

Age and interhemispheric transfer time: a failure to replicate.

In a recent study with the Poffenberger paradigm, Brizzolara et al. reported longer estimates of interhemispheric transfer time (IHTT) for children aged 7 years than for adults. They interpreted this finding as evidence for incomplete functional maturity of the corpus callosum in young children. The present study was we were unable to replicate the age effect reported by Brizzolara et al. A closer look at the original study revealed that only 80 observations per child had been collected, which makes it probable that the larger IHTTs in 7-year-olds were caused by stimulus-response compatibility rather than by the lower efficiency of the corpus callosum during childhood years.

Adult↗

Right place, right time, and only once: replication initiation in metazoans.

DNA replication is tightly regulated at the initiation step by both the cell cycle machinery and checkpoint pathways. Here, we discuss recent advances in understanding how replication is initiated in metazoans at the correct chromosome positions, at the appropriate time, and only once per cell cycle.

Animals↗

Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis.

In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (beta subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis.

Bacillus subtilis↗

Effects of betaine, pen space, and slaughter handling method on growth performance, carcass traits, and pork quality of finishing barrows.

An experiment was conducted to determine the effects of betaine, pen space, and preslaughter handling method on growth, carcass traits, and pork quality of finishing barrows. For the growth trial, a 2 x 2 factorial arrangement of treatments was used: betaine (0 or 0.250%) and(or) pen space (m2/pig; adequate, 0.035 BW0.67 kg, or inadequate, 0.025 BW0.67 kg). Each treatment was replicated five times with four barrows per replicate. At trial termination, two barrows from each pen were selected to receive either minimal or normal preslaughter handling. Reducing pen space decreased (P < 0.05) overall ADG and gain:feed and tended (P = 0.12) to decrease overall ADFI. Betaine had no affect (P > 0.10) on overall ADG, ADFI, or gain:feed. Pigs fed betaine had decreased (P < 0.10) carcass length. Other carcass and ham measurements were not affected (P > 0.10) by betaine. Pigs with inadequate pen space had increased (P < 0.10) ultimate pH, subjective color, cooking loss (fresh and frozen chop), and shear force but decreased rectal temperature, loin muscle CIE L*, biceps femoris CIE b*, and drip loss. Pigs subjected to minimal preslaughter handling had decreased (P < 0.10) rectal temperature, plasma cortisol, loin muscle CIE b*, and fresh chop total loss (drip + cooking loss). Pigs fed betaine had increased (P < 0.01) initial pH and decreased (P < 0.10) drip loss (fresh chop). Cooking loss and total loss (frozen chop) were decreased in pigs fed betaine with adequate pen space but increased in pigs fed betaine with inadequate pen space (betaine x pen space, P < 0.01). Pigs fed betaine may have improved pork quality.

Animal Husbandry↗

Roles of the RAM signaling network in cell cycle progression in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Hym1p, Mob2p, Tao3p, Cbk1p, Sog2p and Kic1p proteins are thought to function together in the RAM signaling network, which controls polarized growth, cell separation and cell integrity. Whether these proteins also function as a network to affect cell proliferation is not clear. Here we examined cells lacking or over-expressing RAM components, and evaluated the timing of initiation of DNA replication in each case. Our results suggest opposing roles of RAM proteins, where only Hym1p can promote the transition from the G1 to S phase of the cell cycle. We also uncovered additive growth defects in strains lacking several pair-wise combinations of RAM proteins, possibly arguing for multiple roles of RAM components in the overall control of cell proliferation. Finally, our findings suggest that Hym1p requires the Dcr2p phosphatase to promote the G1/S transition, but it does not require the G1 cyclin Cln3p or the RAS pathway. Taken together, our results point to a complex regulation of cell proliferation by RAM proteins, in a non-uniform manner that was not previously anticipated.

Cell Cycle↗

Spatial and temporal predictability of the parasite community structure of a benthic marine fish along its distributional range.

The search for consistent patterns of organisation in parasite communities remains a central theme in parasite community ecology. However, to date, much evidence comes from studies without replication in both space and time; when replicate communities are examined, repeatable patterns are rarely observed. Here we determine, using nested subset analyses, whether the infracommunities of ectoparasites and endoparasites of a benthic marine fish (Sebastes capensis) show non-random structure. Then we examine the spatial repeatability of parasite community structure across the host's distribution in the southern Pacific, and the temporal repeatability of ectoparasite community structure from one locality. In total, 537 fish were captured from different latitudes (between 11 degrees S and 52 degrees S) along the Pacific coast of South America; a further 122 specimens were captured in two other years from one of the sampling localities, Valdivia (40 degrees S). In spite of variation in fish sizes among samples, fish size generally did not correlate with either ecto- or endoparasite species richness. The ecto- and endoparasite species richness of the component communities were also not correlated with fish sample size across the nine localities. Significant nested patterns were found in the ectoparasite communities of S. capensis at all eight localities, except at latitude 52 degrees S. Significant nested patterns were also found in the endoparasite infracommunities of S. capensis at seven of the nine localities, the exceptions being those from latitudes 11 degrees S and 20 degrees S. On a temporal scale, significant nestedness was observed in the ectoparasite infracommunities of S. capensis during each of the 3 years of sampling at Valdivia. In general, the same parasite species are responsible for the repeatability of nested patterns, though their importance varies among localities. The spatial and temporal predictability of the parasite community structure in S. capensis may be associated with the fish's benthic habitat and territorial behavior, suggesting that host biology may be a key determinant of the structure of parasite communities.

Animals↗