P1 and NR1 plasmid replication during the cell cycle of Escherichia coli.

Replication patterns of the miniP1 plasmid pZC176, the miniNR1 plasmid pRR933, and the high-copy miniNR1 derivative pRR942 were examined during the Escherichia coli cell division cycle and compared to the cycle-specific replication pattern of a minichromosome and the cycle nonspecific pattern of pBR322. In E. coli cells growing with doubling times of 40 and 60 min, the miniP1 plasmid was found to replicate with a slight periodicity during the division cycle. The periodicity was not nearly as pronounced as that of the minichromosome, was not affected by the presence of a minichromosome, and was not evident in cells growing more rapidly with a doubling time of 25 min. Both miniNR1 plasmids, pRR933 and pRR942, replicated with patterns indistinguishable from that of pBR322 and clearly different from that of the minichromosome. It is concluded that both P1 and NR1 plasmids can replicate at all stages of the cell cycle but that P1 displays a slight periodicity in replication probability in the cycle of slower growing cells. This periodicity does not appear to be coupled to a specific age in the cycle, but could be associated with the achievement of a specific cell mass per plasmid. During temperature shifts of a dnaC(Ts) mutant, the miniP1 plasmid and pBR322 replicated with similar patterns that differed from that of the minichromosome, but were consistent with a brief eclipse between rounds of replication.

Initiation of eukaryotic DNA replication: conservative or liberal?

The mechanism for initiation of eukaryotic DNA replication is highly conserved: the proteins required to initiate replication, the sequence of events leading to initiation, and the regulation of initiation are remarkably similar throughout the eukaryotic kingdom. Nevertheless, there is a liberal attitude when it comes to selecting initiation sites. Differences appear to exist in the composition of replication origins and in the way proteins recognize these origins. In fact, some multicellular eukaryotes (the metazoans) can change the number and locations of initiation sites during animal development, revealing that selection of initiation sites depends on epigenetic as well as genetic parameters. Here we have attempted to summarize our understanding of this process, to identify the similarities and differences between single cell and multicellular eukaryotes, and to examine the extent to which origin recognition proteins and replication origins have been conserved among eukaryotes. Published 2000 Wiley-Liss, Inc.

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei.

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli.

Replication of the miniF plasmid pML31 was examined during the division cycle of Escherichia coli growing with doubling times between 40 and 90 min at 37 degrees C and compared to the replication of plasmid pBR322 and the minichromosome pAL70. The replication pattern of pML31 was indistinguishable from that of pBR322 at all growth rates and very different from the cell-cycle-specific replication of the minichromosome. It is concluded that both pML31 and pBR322 plasmids can replicate at all stages of the division cycle, with a probability of replication that increases gradually, but perhaps not exponentially, during the cycle. In contrast, the modes of segregation of pML31 and pBR322 plasmids into daughter cells at division appeared to differ, raising the possibility that pML31 may segregate in a nonrandom fashion similar to that of chromosomes and minichromosomes.

Gene transcription and chromosome replication in Escherichia coli.

Transcript levels of several Escherichia coli genes involved in chromosome replication and cell division were measured in dnaC2(Ts) mutants synchronized for chromosome replication by temperature shifts. Levels of transcripts from four of the genes, dam, nrdA, mukB, and seqA, were reduced at a certain stage during chromosome replication. The magnitudes of the decreases were similar to those reported previously ftsQ and ftsZ (P. Zhou and C. E. Helmstetter, J. Bacteriol. 176:6100-6106, 1994) but considerably less than those seen with dnaA, gidA, and mioC (P. W. Theisen, J. E. Grimwade, A. C. Leonard, J. A. Bogan, and C. E. Helmstetter, Mol. Microbiol. 10:575-584, 1993). The decreases in transcripts appeared to correlate with the estimated time at which the genes replicated. This same conclusion was reached in studies with synchronous cultures obtained with the baby machine in those instances in which periodicities in transcript levels were clearly evident. The transcriptional levels for two genes, minE and tus, did not fluctuate significantly, whereas the transcripts for one gene, iciA, appeared to increase transiently. The results support the idea that cell cycle timing in E. coli is not governed by timed bursts of gene expression, since the overall findings summarized in this report are generally consistent with cell cycle-dependent transient inhibitions of transcription rather than stimulations.

DNA sequestration and transcription in the oriC region of Escherichia coli.

In Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation. The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E. coli PC2 dnaC2(ts) aligned for initiation of chromosome replication. In both seqA- and dam- cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered. After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA+ dam+ cells than in seqA- or dam- cells, but before the release of oriC from sequestration. Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA. These findings, plus analyses of transcript levels in steady-state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC. Accordingly, a proposal for the sequence of events during the interinitiation interval in E. coli is presented based on the evident coupling of transcription to replication.

mioC transcription, initiation of replication, and the eclipse in Escherichia coli.

The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated. When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling. Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation. Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription. This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse. Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation. It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely. It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature. Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of DnaA protein, but replication can initiate with the proper timing even when transcription is not shut down; i.e., mioC does not serve as a negative regulator of initiation. It is proposed, however, that the reappearance and subsequent disappearance of mioC transcription during a 10-min interval at the end of the eclipse serves as an index of the minimum time required for the establishment of active protein-DNA complexes at the DnaA boxes in the fully methylated origin region of the chromosome. On this basis, the eclipse constitutes the time for methylation of the newly formed DNA strands (15 to 20 min at 30 degrees C) followed by the time for DnaA protein to bind and activate oriC for replication (10 min).

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.

Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media.

Expression of type 1 fimbriae in Escherichia coli K-12 is phase variable and associated with the inversion of a short DNA element (switch). The fim switch requires either fimB (on-to-off or off-to-on switching) or fimE (on-to-off switching only) and is affected by the global regulators leucine-responsive regulatory protein (Lrp), integration host factor (IHF), and H-NS. Here it is shown that switching frequencies are regulated by both temperature and media and that these effects appear to be independent. fimE-promoted on-to-off switching occurs far more rapidly than previously estimated (0.3 per cell per generation in defined rich medium at 37 degrees C) and faster at lower than at higher temperatures. In direct contrast, fimB-promoted switching increases with temperature, with optima between 37 and 41 degrees C. Switching promoted by both fimB and fimE is stimulated by aliphatic amino acids (alanine, isoleucine, leucine, and valine), and this stimulation requires lrp. Furthermore, lrp appears to differentially regulate fimB- and fimE-promoted switching in different media.

The gene-protein database of Escherichia coli: edition 5.

The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.

Pharmacokinetic, biochemical and tolerance studies on carprofen in the horse.

Carprofen, a non-steroidal anti-inflammatory drug (NSAID) was administered to three Thoroughbred geldings and three Shetland ponies to determine its plasma disposition and tolerance. The main pharmacokinetic characteristics of carprofen in horses and ponies were a volume of distribution of 0.08 to 0.32 litres/kg (mean +/- se = 0.23 +/- 0.04) a systemic clearance of 26.4 to 78.5 ml/min (mean +/- se = 44.9 +/- 8.0) and a plasma elimination half-life of 14.5 to 31.4 h (mean +/- se = 21.9 +/- 2.3). There was no evidence of any accumulation of carprofen in plasma when the drug was given orally at a dose rate of 0.7 mg/kg for 14 consecutive days. Carprofen was well tolerated following intravenous (iv) and oral administration. Intramuscular (im) administration resulted in elevated levels of plasma creatine kinase suggesting muscle cell damage. According to the results of this study carprofen can be regarded as a long-acting NSAID in horses from a pharmacokinetic point of view. Either iv, im or the oral route of administration could be used to achieve high carprofen plasma concentrations.

Effect of meclofenamic acid on the response of parasite-naive lambs and adult sheep to Ostertagia circumcincta.

Meclofenamic acid was used to inhibit prostaglandin synthesis in lambs challenged with Ostertagia circumcincta. It lowered the number of parasites which established in treated animals but not significantly. In treated animals plasma pepsinogen values were elevated at the time of parasite emergence but had dropped below the values achieved in control lambs towards the end of the experiment when parasites were at the adult, lumenal dwelling stage. Meclofenamic acid administered to adult immune ewes during challenge with third stage O circumcincta larvae did not significantly affect the establishment of the parasites, nor did it affect the rise in pepsinogen concentration associated with the challenge.

Relay bioavailability and toxicity of isometamidium residues: a model for human risk assessment.

The bioavailability and potential toxicity of the residues of the antitrypanosomal drug isometamidium (ISMM) in bovine tissues were investigated in male Sprague-Dawley rats. They were allowed to feed for 7 and 21 days on a standard diet, to which were added lyophilized tissues from a calf treated im with a combination of 45 mg 14C-ISMM and 73 mg unlabelled ISMM (1 mg/kg bodyweight). Cumulative excretion of radioactivity of the residues in feces of the rats was on average 90% of the dose. No radioactivity was detectable in the tissues examined, including the kidney, liver, spleen, muscle, stomach and small intestine. No clinical effects were seen in any of the rats, and both gross and histopathological examinations did not reveal any lesions. In rats given 14C-ISMM (2.245 mg/kg bodyweight) by oral gavage, cumulative excretion of radioactivity in feces after 48 hr amounted to about 93% of the dose, and no radioactivity was detectable in tissues. Similarly, none of the rats showed any clinical effects and no gross lesions were seen at necropsy. This study shows that ISMM residues are not significantly bioavailable and exhibit no subchronic toxicity.

Flunixin pharmacokinetics and serum thromboxane inhibition in the dog.

Flunixin meglumine administered orally to beagle dogs at doses of 0.55, 1.10 or 1.65 mg/kg bodyweight was rapidly absorbed to produce maximum mean plasma concentrations of 2.40 +/- 0.70, 4.57 +/- 1.12 and 7.42 +/- 2.07 micrograms/ml, respectively. Thereafter, the plasma concentrations of flunixin fell rapidly to values less than 0.10 micrograms/ml from 24 hours after drug administration at all dosage levels. The maximum mean inhibition of serum thromboxane B2 was 91.5 per cent after the lowest dose of flunixin and 98.8 per cent for both the intermediate and high dose rates. At plasma concentrations of flunixin above 2 micrograms/ml there was more than 90 per cent inhibition of thromboxane.

Level of benzimidazole resistance in a strain of Ostertagia circumcincta studied over several infections in lambs.

A benzimidazole-resistant strain of Ostertagia circumcincta (HFRO) was used experimentally to infect lambs. The level of resistance, measured by an egg hatch assay, was studied throughout each infection and also after treatment of the lambs with fenbendazole. The HFRO strain was highly resistant to benzimidazoles. There was day-to-day variation in the level of resistance throughout a single infection with a high level of resistance in the early part of the infection, around Day 27 post-inoculation of infective larvae, falling to a lower level later in the infection. Egg hatch assays on the 3 days immediately post-treatment with fenbendazole showed the resistance level was high then resistance fell to the pre-treatment level after 7 days. Selection for benzimidazole resistance using fenbendazole treatment at the normal dose rate of 5 mg kg-1 over five passages of the HFRO strain in lambs failed to increase the resistance level. Storage of larvae over a 5-month period at 4 degrees C, prior to infection of lambs, did not produce any alteration in the resistance level. The possible reasons for the variations in resistance found with the HFRO strain are discussed along with the implications for sheep parasite control and further development of benzimidazole resistance.

Pharmacokinetics and efficacy of triclabendazole in goats with induced fascioliasis.

The pharmacokinetics of triclabendazole were evaluated in normal goats and in goats artificially infected with Fasciola hepatica. Triclabendazole and its metabolites were determined using a novel high performance liquid chromatographic method with fluorimetric detection after solid-phase extraction. In normal goats triclabendazole given orally was metabolized rapidly to its sulphoxide and sulphone derivatives. The maximum plasma concentrations for the sulphoxide and sulphone were similar ranging from 9 to 19 micrograms/ml and these were attained at an average 12.8 and 25.6 h, respectively, after administration. Both metabolites were eliminated slowly from plasma with elimination half-lives of 22.4 h for the sulphoxide and 19.4 h for the sulphone. They persisted at measurable concentrations in plasma for up to seven days. In milk, the two metabolites occurred in low concentrations and none of them was detectable (sulphoxide less than 0.04 microgram/ml, sulphone less than 0.02 microgram/ml) after seven days. The pharmacokinetic behaviour of triclabendazole was not altered in animals with fascioliasis. Efficacy of the drug against immature (six-week) F. hepatica was 100%.

The pharmacodynamics of ivermectin in sheep and cattle.

The concentrations of ivermectin in the gastrointestinal tract of sheep and cattle were determined after subcutaneous administration of ivermectin. Ivermectin was not detected (limit of detection 1 ng/ml) in abomasal and ruminal fluids either after a normal therapeutic dose of 200 micrograms/kg or even at an increased dose of 2000 micrograms/kg. It was also not detected in abomasal and ruminal fluids of a sheep infected with the abomasal parasite Ostertagia circumcincta. However, ivermectin was detectable at similar concentrations in abomasal mucus and in small intestinal mucus. Excretion of ivermectin was high in bile but the concentrations in small intestinal mucus, distal and proximal to the bile duct opening, were similar. It is hypothesized that the low efficacy of ivermectin against small intestinal nematodes compared with abomasal nematodes is not due to differences in ivermectin concentrations in the predilection sites but is probably due to tachyphylaxis in the nematodes allowing the small intestinal nematodes to re-establish before they have left their predilection site. Ivermectin was excreted in the milk of ewes at concentrations similar to those in plasma. Lambs suckling ivermectin-treated ewes received about 4% of a normal therapeutic dose (200 micrograms/kg) via the milk.

Production of strains of the sheep parasite Ostertagia circumcincta by implantation of adult parasites into the abomasum of lambs.

The implantation of a single pair of adult parasites of Ostertagia circumcincta via the abomasa of four lambs resulted in the production of 19, 53, 393 and 425 viable larvae after faecal culture using faeces collected from day 6 to day 20 after implantation. Implantation of a single female parasite into two lambs failed to produce larvae.