ABSTRACT
Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the "replicon model" (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329-348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmid-encoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE(*) (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/chemistry , F Factor/chemistry , Repressor Proteins/metabolism , Catalysis , DNA Primase/chemistry , DNA Replication , Dideoxynucleotides , Dimerization , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Hydroxyurea/pharmacology , In Vitro Techniques , Integration Host Factors/chemistry , Kinetics , Models, Genetic , Plasmids/metabolism , Protein Binding , Replication Origin , Sodium Dodecyl Sulfate/pharmacology , Thymine Nucleotides/chemistry , Time FactorsABSTRACT
We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori gamma of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein pi and on the host-encoded initiator DnaA. The wild type pi was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type pi-mediated replication in vivo requires IHF. Using a template that contained ori gamma flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.
Subject(s)
DNA Replication , Plasmids/metabolism , Bacterial Proteins/metabolism , DNA Primase/metabolism , DNA Primers/metabolism , DNA-Binding Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , In Vitro Techniques , Models, Genetic , Mutation , Origin Recognition Complex , Protein Binding , Ribonuclease H/metabolism , Time Factors , Viral Proteins/metabolismABSTRACT
The organophosphate pesticide, phorate, is an extremely hazardous insecticide. Not much experimental study is available on effects of phorate on different brain areas. We report in this study the alterations induced by phorate on enzyme profile of mouse olfactory bulb. Olfactory bulb, the first processing centre after the sensory cells in the olfactory pathway, has connections with the other higher centres of the brain like hippocampus and hypothalamus. Phorate was administered orally in the diet at the doses of 1.0 mg and 1.5 mg/kg body weight to adult albino mice. After 32 weeks of exposure animals were sacrificed and cryosections were processed for acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively) enzyme localization. Significant reduction occurs in AChE and BChE activity at higher dose level, whereas reduced BChE activity was found at both dose levels. Our results shows an obvious effect on cholinesterase enzyme profile of olfactory bulb of mice after systemic administration of low doses of phorate for long terms.
