The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation.

The origin of replication of the P1 plasmid contains five direct, imperfect repeats (iterons) of a 19 bp sequence that binds the P1-encoded RepA initiator protein. RepA binding to these iterons triggers origin initiation and represses transcription from the repA promoter that is nested within the iterons. The origin iterons were replaced with ligated oligonucleotides that insert five perfect 19 bp repeats with identical spacer sequences. This eliminates the natural variation in the iteron and spacer sequences and removes the repA promoter. The reconstructed origin is functional, showing that the repA promoter is not essential for origin function. The method used to make the reconstructed origin allows substitution of identical iterons with altered sequence or spacer length. Single changes of conserved iteron bases gave reduced or non-existent origin activity, as did an increase in spacer length. Like the wild type, most of these mutant arrays retain avid primary binding activity for the RepA protein. However, although the wild-type arrays readily form a mature complex in which all iterons are saturated, the most replication-defective mutants were completely unable to do this, even at very high RepA concentrations. It appears that iteron spacing and contacts involving at least three of the conserved iteron bases play an important role in the assembly of the mature structure in which all sites are occupied. A model is presented in which an allosteric interaction between the DNA site and protein is needed for the saturated, mature complex required for initiation.

Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells.

The effect of nitric oxide (NO) exposure and sulfhydryl-reactive chemicals on L-arginine transport in pulmonary artery endothelial cells was evaluated. Exposure of pulmonary artery endothelial cells to 7.5 ppm (0.4 microM) NO for 4 h resulted in a significant (p < 0.05) reduction of Na(+)-dependent but not Na(+)-independent L-arginine transport. More prolonged exposure for 12-24 h reduced both Na(+)-dependent and Na(+)-independent transport of L-arginine with maximal loss of transport after 18 h of exposure (p < 0.02 for both). Similarly, incubation of cells in the presence of 50-200 microM S-nitroso-acetyl-penicillamine (SNAP) (but not 500 microM each of nitrate or nitrite) for 2 h also reduced both the Na(+)-dependent and Na(+)-independent transport of L-arginine (p < 0.05 for all concentrations). The SNAP-induced reduction of L-arginine transport was blocked by the NO scavenger oxyhemoglobin. When cell monolayers were exposed to varying concentrations of the sulfhydryl reactive chemicals N-ethylmaleimide (NEM) and acrolein, a dose-dependent reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes was observed. Na(+)-dependent L-arginine transport was more susceptible to inhibition by exposure to NO and to sulfhydryl reactive chemicals. Incubation of cells with 0.5 mM of the thiol-containing agent N-acetyl-L-cysteine prior to and during NEM or acrolein exposure blocked NEM and acrolein-induced reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes. Similarly, NO-induced reductions of Na(+)-dependent and Na(+)-independent L-arginine transport were reversed to control levels 24 h after termination of NO exposure. Treatment with the disulfide reducing agent dithiothreitol after exposure to NO resulted in partial reversal of the decreases in L-arginine transport. These results demonstrate that exposure to exogenous NO is responsible for reversible reductions of plasma membrane-dependent L-arginine transport mediated by both the Na(+)-dependent (system Bo,+) and the Na(+)-independent (system y+) transport processes. Modulation of the sulfhydryl status of plasma membrane proteins involved in L-arginine transport, such as L-arginine transporters and/or Na+/K(+)-ATPase, may be responsible, at least in part, for reductions in overall L-arginine transport in pulmonary artery endothelial cells.

Control of P1 plasmid replication by iterons.

The incA locus of plasmid P1 controls plasmid copy number by inhibiting the replication origin, oriR. Both loci contain repeat sequences (iterons) that bind the P1 RepA protein. Regulation appears to occur by contact of incA and oriR loci of daughter plasmids mediated by RepA-bound iterons. Synthetic incA iteron arrays were constructed with altered numbers, sequences or spacing of iterons. Using these in in vitro and in vivo assays, we examined two models: (i) that the origin and incA loci form a stable 1:1 complex in which multiple iterons of each locus are paired with those of the other, and (ii) that individual incA iterons act as freely diffusing nucleoprotein units that contact origin iterons in a random and dynamic fashion. The data presented here strongly favour the latter case. The origin, with its five iterons, acts as a target but not as an effector of regulation. We present a model for replication control based on random, dynamic contacts between incA iterons and the origin. This system would display randomness with respect to choice of templates and timing of initiation if multiple replicon copies were present, but would tend to act in a machine-like fashion in concert with the cell cycle if just two copies were present in a dividing cell.

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.

Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation.

A mutant mini-P1 plasmid with increased copy number can be established in Dam- strains of Escherichia coli, where mini-P1 plasmid replication is normally blocked. Comparison of this plasmid and a plasmid driven by the host oriC replication origin showed that both origins are subject to control by methylation at two different levels. First, both origins appear to be subject to negative regulation acting at the level of hemimethylation. This probably involves the sequestration of the hemimethylated DNA produced by replication, as has been previously described for oriC. Second, both origins show a positive requirement for adenine methylation for efficient function in vivo. This conclusion is supported by the behavior of the P1 origin in an improved in vitro replication system. In vitro, where sequestration of hemimethylated DNA is not expected to occur, the hemimethylated P1 origin DNA was fully functional as a template. However, the activity of fully unmethylated DNA was severely restricted in comparison with that of either of the methylated forms. This in vitro uncoupling of the two effects of origin methylation suggests that two separate mechanisms are involved.

Effect of phospholipid acyl chain modulation on vitamin E incorporation into pulmonary artery endothelial cell membranes.

Incorporation of vitamin E (alpha-tocopherol) was measured in total membranes of pulmonary artery endothelial cells (PAEC) following treatment with eight synthetic phosphatidylethanolamines (PE) (Palmitoyloleoyl, 16:0-18:1 PE1; distearoyl, 18:0-18:0 PE2; dioleoyl, 18:1-18:1 PE3; stearoyl- linoleoyl, 18:0-18:2 PE4; dilinoleoyl, 18:2-18:2 PE5; stearoyl-arachidonyl, 18:0-20:4 PE6; diarachidonyl, 20:4-20:4 PE7; and stearoyl-docosahexenoyl, 18:0-22:6 PE8). Endogenous PE content of native membranes was 0.88 +/- 0.01 nmol/mg protein. Incorporation of PE irrespective of fatty acid content significantly (P < 0.02) increased the PE content of total membranes. Vitamin E incorporation in control membranes was 63 +/- 9 nmol/mg protein. Incorporation of vitamin E in PE1- to PE7-treated cells were significantly (P < 0.05) increased compared to controls and were comparable to each other. Vitamin E incorporation into PE8-treated cells was threefold greater (P < 0.001) than controls and twofold greater (P < 0.001) than PE1- to PE7-treated cells. Increased PE content results in increased vitamin E incorporation into PAEC membranes irrespective of the fatty acids present on the acyl chain, and maximal incorporation of vitamin E in PE8-treated cells may relate to the increased carbon chain length rather than to the degree of unsaturation at the sn2 position.

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Critical sequences in the core of the P1 plasmid replication origin.

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.

Unique sequence requirements for the P1 plasmid replication origin.

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although five boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.

A single DnaA box is sufficient for initiation from the P1 plasmid origin.

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

Protein-DNA interactions in regulation of P1 plasmid replication.

The P1 RepA protein appears to play three roles in P1 plasmid replication: acting at the origin both as a specific initiator and as a repressor of transcription, and interacting with the copy-control locus incA to bring about a negative control of initiation. We have used the DNase I footprinting technique to show that RepA binds specifically to repeat units of a 19-base-pair consensus sequence present in both the origin and incA control regions. RNA polymerase was shown to bind to two specific regions within the origin repeats. One of these constitutes the known promoter sequence for the repA gene. We show evidence that the polymerase can be efficiently displaced from the promoter by subsequent RepA binding, thus providing a direct mechanism for RepA autoregulation. Under the conditions used, there were no obvious differences in the affinities of individual repeat sequences for the purified protein.

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA.

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.

Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region.

The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 X 10(3) base-pairs of DNA. The DNA sequence of the region revealed two large open reading frames that could encode proteins of Mr 44,000 and Mr 38,000. Both would be read in the same direction. The first open reading frame corresponds to the par A gene, the Mr 44,000 protein product of which was shown to be trans acting and essential for partition. The second open reading frame (parB) follows closely and may be cotranscribed with par A. The codon usage frequency for parB is consistent with its producing a protein product. The ParB protein was identified in cell extracts as a product with an apparent Mr of 45,000, suggesting that it behaves anomolously on gel electrophoresis. Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus. Subcloning of this site showed it to consist of a maximum of 174 base-pairs. The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat. Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis. The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes. It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Escherichia coli host.

P1 plasmid replication: multiple functions of RepA protein at the origin.

Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA). The origin region contains five 19-bp direct repeats. By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed by RepA. Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences. This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication. Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role.

Trans- and cis-acting elements for the replication of P1 miniplasmids.

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.

The partition functions of P1, P7, and F miniplasmids.

The partition regions of P1, P7, and F miniplasmids are discrete DNA sequences of about 3 kb in length that will promote accurate partition of hybrid plasmids independent of the source of replication functions or the position or orientation of the elements. Each of the par regions seems to be very similarly organized, with open reading frames for essential proteins and a terminal site which appears to be analogous to the centromere of eukaryotic cells. When cloned, these terminal sites exert incompatibility against their respective parent plasmids presumably because they can compete with the parent plasmids as substrates for partition. We have determined the complete DNA sequence of the P1 par region. In addition to the open reading frame for the essential parA protein (42-44 kd), the region contains a second open reading frame which could encode a 38-kd protein. The 2 large open reading frames appear to form an operon that is negatively regulated from a site adjacent to the promoter and responds to the par gene products in trans. Both this site and the downstream "centromere" site, incB, contain blocks of extremely AT-rich sequences, which are postulated to be binding sites for par proteins. The incB and upstream AT-rich regions both contain 20-bp imperfect inverted repeats. Further downstream from the minimal incB sequence (172 bp) lies an additional region which is essential for partition. The further analysis of the P1 par region should be greatly facilitated by the finding that it can function in cis to stabilize pBR322 vectors under conditions where the copy number of pBR322 is reduced.