NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Polyadenylation of oop RNA in the regulation of bacteriophage lambda development.

We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains. Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants. However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host. On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation. Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant. We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain. Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant. Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host. The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript. Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection. Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.

Regulation of copy number and stability of phage lambda derived pTC lambda 1 plasmid in the light of the dimer/multimer catastrophe hypothesis.

The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.

A plasmid cloning vector with precisely regulatable copy number in Escherichia coli.

We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter. Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage.

Replication of lambda plasmid DNA in the Escherichia coli cell cycle.

The Cro repressor autoregulatory loop has long been considered the main regulatory process in controlling lambda plasmid replication initiation in Escherichia coli. However, we found recently that lambda plasmids can be maintained at a constant copy number in the absence of Cro function. Here we demonstrate that shortly after inactivation of the Cro repressor, the synthesis of lambda plasmid DNA increases significantly but is then stabilized at a level similar to that observed in the presence of the Cro function. We found that replication initiation of lambda plasmids carrying a functional cro gene proceeds randomly in the host cell cycle, but in the absence of Cro function the replication initiation of lambda plasmid DNA appears to be cell cycle dependent. The host DnaA protein appears to be at least one of the factors involved in the cell-cycle-specific control of lambda cro- plasmid replication. Therefore, it seems that the lambda cro- plasmid may serve as an amazingly simple model for studies on the regulation of DNA replication in the cell cycle.

Molecular mechanism of heat shock-provoked disassembly of the coliphage lambda replication complex.

We have found previously that, in contrast to the free O initiator protein of lambda phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the lambdaO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43 degrees C resulted in the decay of the lambdaO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of lambda plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from lambda DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroELS and ClpP/ClpX proteins. In contrast to lambda cro+, in lambda cro- plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the lambda crots plasmid-harboring cells, a Cro repressor-independent control of lambda DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to lambda DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the lambda cro- plasmid replication control.

Replication and maintenance of lambda plasmids devoid of the Cro repressor autoregulatory loop in Escherichia coli.

Plasmids derived from bacteriophage lambda are known as lambda plasmids. These plasmids contain the ori lambda region and lambda replication genes O and P. Typical lambda plasmids also contain the cro gene, the product of which is a repressor of the pR promoter when present at relatively high concentrations. These genes stably maintain the plasmid in Escherichia coli at copy numbers of 20 to 50 per cell. According to a generally accepted model, stable maintenance of lambda plasmids is possible due to the Cro repressor autoregulatory loop (the cro gene is under control of pR). Here we demonstrate that lambda plasmids devoid of the Cro autoregulatory loop can also be stably maintained in E. coli strains. We present data for two such plasmids: pTC lambda 1 in which the pR-cro region has been replaced by the ptetA promoter and the tetR gene (coding for the TetR repressor), and a standard lambda plasmid with inactivated cro gene (lambda cro-null plasmid). Thus, the presence of the Cro repressor autoregulatory loop does not appear to be essential to the maintenance of lambda plasmids in vivo.

DnaA-mediated regulation of phage lambda-derived replicons in the absence of pR and Cro function.

Bacteriophage lambda-derived replicons can replicate in Escherichia coli cells as plasmids. In the control of replication of these plasmids, an important role was ascribed to the lambda Cro repressor autoregulatory loop. However, the oR/pR-cro-tR-cII' region could be replaced by the ptetA promoter under the control of the TetR repressor, producing plasmid pTClambda. Here, we demonstrate that stable maintenance of pTClambda depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTClambda resulted in a decrease in the plasmid (pTClambda) copy number and poor maintenance of pTClambda in E. coli. Moreover, in contrast to the replication of the wild-type lambda plasmid, previously found to be positively regulated by DnaA (acting on a relaxed DnaA box situated immediately downstream of the pR promoter), the replication of pTC plasmids (devoid of pR) was found to be negatively regulated by DnaA. Contrary to wild-type lambda plasmids, in cells harboring lambda cro[temperature-sensitive (ts)] or pTClambda (but not pTClambda) plasmid, the lambda replication complex was heat shock resistant; this complex, however, disassembled after inactivation of DnaA function. This disassembly was blocked by DNA gyrase inhibitors. According to our model outlined previously, we propose that the heat shock resistance of the replication complex of lambdacro- plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound lambda replication complex. The replication complex-DnaA-lambda DNA structure may be directly related to the role of DnaA as the Cro-replacing negative regulator of lambdacro- plasmid replication.